Complete Account of the Selection Process

Complete account of the selection process

Andreas Hoppe. October 17, 2012

Contents

1 Principles 1 1.1 Selection of functions ...... 1 1.2 Selection of genes ...... 2

2 Selection in detail 3 2.1 Collagens ...... 4 2.2 Ethanol degradation ...... 5 2.3 Sphinganine-1P synthesis ...... 6 2.4 Tyrosine degradation ...... 7 2.5 Panthetheine synthesis ...... 8 2.6 Inositol synthesis ...... 9 2.7 Urea synthesis ...... 9 2.8 Bilirubin conjugation ...... 10 2.9 Cholesterol synthesis ...... 10 2.10 Bile acid synthesis and excretion ...... 12 2.11 β-hydroxybutyrate ...... 13 2.12 Glycolysis ...... 13 2.13 Triglycerides ...... 14

1 Principles 1.1 Selection of functions The selection process was based on the result of the ModeScore computation of the most regulated metabolic functions, in particular (with decreasing order of importance)

• the comparison of the control and the TGFβ series at the 24h time point (C/T 24h), • the comparison of the 1h to the 24h time point in the control series (C1h/24h),

• the comparison of the 1h to the 24h time point in the TGFβ series (T1h/24h), • the comparison of the 1h to the 6h time point in the control series (C1h/6h) in conjunction with the comparison of the 6h to the 24h time point in the control series (C6h/24h), and

• the comparison of the control and the TGFβ series at the 6h time point (C/T 6h), • the comparison of the 1h to the 6h time point in the control series (T1h/24h) in conjunction with the comparison of the 6h to the 24h time point in the TGFβ series (T6h/24h) — not shown in the diagrams below.

1 For each the top and the bottom 20 of the list have been used. For each of this functions, a detailed reaction chart was produced. These charts have been manually analyzed to select the most relevant functions with the following criteria: 1. central, well-known liver functions are favored to more peripheral functions (synthesis or degradation of of low-amount metabolites)

2. if a function can be seen as a subfunction of another, more central function, check if the superfunction is also a good candidate (e.g. the synthesis of homogentisate from tyrosine can be seen as a subfunction of tyrosine degradation, the more important function) 3. prefer functions which deviates in the amount the regulation from the average (see bar graphs in the main manuscript) — up-regulation or strong down-regulation 4. prefer functions which are top/bottom scorer in more than one of the comparisons 5. prefer functions which show a plausible pattern of genes regulated in a consistent manner.

6. prefer functions which have already been noted as speciﬁcally sensitive to TGFβ or the monolayer cell culture 7. prefer functions which contain genes with highly remarkable regulation amplitude or pattern 8. prefer functions whose pattern is not based on a single gene regulation It must be noted that this selection process takes into account the available domain knowledge.

1.2 Selection of genes Once the functions have been selected, the list of the relevant genes which led to the amplitude estimation of the whole function are selected. This is also a manual process which takes into account several information sources and is guided by the following criteria: • prefer genes with large transcript value changes, • prefer genes with large absolute values, • prefer genes associated to reactions which are adjacent to reactions whose genes have already been selected, • prefer genes of highly speciﬁc enzymes or transporters, • prefer genes of reactions which are most important for the function under regard and not for any more important function,

• close gaps in an otherwise preferable reaction path. Mostly, a connected reaction path (as a subset for the whole flux distribution) appears to be responsible for the regulation. Transporters are also integrated if consistent with the regulation of the enzymes. This is not often the case but some cases it seems to be highly relevant. This aspect is noteworthy in comparison to the PathRanker concept [1] which ignores transporters altogether. Another important aspect in comparison to PathRanker (based on the KEGG MODULE reaction paths [2]) is that the definition of the relevant paths is not predefined (and fixed) but flexible and dependent on the actual expression values of the experiment. Often, they are in accordance with the well-known reaction paths but in some cases there is a unique and noteworthy different selection of genes forming a functional unit.

2 2 Selection in detail

Function C/T 24h C1h/24h T1h/24h C1h/6h C6h/24h C/T 6h T1h/24h T6h/24h pos neg pos neg pos neg pos neg pos neg pos neg pos neg pos neg Tyrosine degr 854 134 978 10 980 8 955 33 982 6 505 483 795 193 979 9 Phenylalanine degr 792 196 975 13 979 9 959 29 980 8 479 509 850 138 980 8 Tyrosine 986 2 915 73 977 11 373 615 882 106 221 767 900 88 982 6 Collagen CORA1 c synthesis 2 986 5 983 1 987 347 641 4 984 3 985 1 987 4 984 Collagen COIA1 c synthesis 922 66 546 442 859 129 326 662 725 263 322 666 145 843 901 87 Collagen COFA1 c synthesis 1 987 418 570 2 986 718 270 279 709 4 984 5 983 19 969 Collagen CO4A5 c synthesis 872 116 4 984 18 970 290 698 1 987 388 600 130 858 28 960 Ethanol degr 987 1 597 391 954 34 271 717 679 309 984 4 801 187 974 14 Bilirubin conjugation 851 137 545 443 900 88 10 978 807 181 423 565 225 763 922 66 Urea from alanine 970 18 655 333 928 60 801 187 985 3 493 495 937 51 908 80 Cholesterol 525 463 829 159 760 228 904 84 629 359 894 94 945 43 605 383 (R)-Mevalonate 13 975 777 211 761 227 919 69 442 546 532 456 870 118 550 438 Farnesyl-PP 722 266 672 316 662 326 923 65 397 591 366 622 857 131 523 465 Creatine 944 44 19 969 556 432 416 572 921 67 335 653 318 670 578 410 Glycogen glucose release 249 739 973 15 960 28 329 659 954 34 102 886 982 6 955 33 (R)-3-Hydroxybutanoate 747 241 863 125 77 911 796 192 822 166 234 754 4 984 701 287 Glutamine 859 129 513 475 472 516 570 418 459 529 509 479 602 386 450 538 Inositol 192 796 3 985 4 984 33 955 3 985 350 638 49 939 2 986 PI 201 787 416 572 695 293 283 705 581 407 355 633 56 932 777 211 Sphinganine-1P 3 985 223 765 9 979 540 448 156 832 13 975 37 951 9 979 4-Hydroxyphenylpyruvate 983 5 974 14 978 10 986 2 957 31 560 428 987 1 953 35 Saccharopine 984 4 940 48 951 37 982 6 869 119 550 438 986 2 947 41 Tyrosine 986 2 915 73 977 11 373 615 882 106 221 767 900 88 982 6 Urea from NH3 971 17 656 332 929 59 802 186 986 2 494 494 938 50 909 79 Pantetheine 979 9 1 987 204 784 909 79 175 813 10 978 8 980 36 952 Glucose-6P 985 3 18 970 984 4 60 928 26 962 983 5 983 5 978 10 Alanine 968 20 25 963 939 49 12 976 728 260 468 520 46 942 928 60 Proline degr 628 360 984 4 507 481 729 259 369 619 521 467 840 148 421 567 beta-Alanine degr 778 210 986 2 985 3 970 18 193 795 308 680 926 62 526 462 Aspartate degr 866 122 987 1 987 1 972 16 987 1 300 688 950 38 987 1 PEP 274 714 983 5 950 38 987 1 951 37 6 982 985 3 949 39 Lysine degr 700 288 926 62 652 336 976 12 883 105 293 695 321 667 658 330 Proline 478 510 460 528 363 625 721 267 291 697 720 268 392 596 311 677 CoA 305 683 166 822 584 404 90 898 148 840 619 369 503 485 606 382 Anaerobic rephosph of ATP 323 665 741 247 637 351 797 191 736 252 970 18 294 694 667 321 Proline 478 510 460 528 363 625 721 267 291 697 720 268 392 596 311 677 Triacylglycerol 723 265 419 569 667 321 223 765 587 401 288 700 67 921 738 250 Choloyl-CoA 559 429 791 197 798 190 856 132 701 287 882 106 885 103 672 316 Gly-CD-cholate(b) 781 207 797 191 784 204 870 118 633 355 853 135 935 53 673 315

Table 1: Ranks of selected functions. The respective ﬁrst number is the rank from above (up-regulation) or below (down- regulation). At the blank ranking space, no score could be computed.

For the following functions, the respective ranks are summarized in Table 1. Note that not all functions have been selected upon the rank but on other reasons described below. Other high-ranked functions have been omitted as they are obviously subfunctions of other selected functions.

3 2.1 Collagens

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h

9 C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 9 8 8 7 7 6 6 log2 expression log2 expression 5 5 4 4 p<0.87 p<0.43 p<1 p<0.45 p<0.9 p<0.98 p<0.39 p<0.85 p<0.39 p<0.26 p<0.82 p<0.052 p<0.081 p<0.5 p<0.011 p<0.13 p<0.52 p<0.12 p<0.05 p<0.64 p<0.8 p<0.021 p<0.095 p<0.05 p<0.43 p<0.0063 p<0.09 p<0.56 p<0.42 p<0.0018 p<0.46 p<0.091 p<0.0005 p<0.46 p<0.7 p<0.34 p<0.18 p<0.21 p<0.29 p<0.07 p<0.54 p<0.23 p<0.0009 p<0.0037 p<0.016 p<0.12 p<0.0091 p<0.044 p<0.01 p<0.36 p<0.12 p<0.073 p<0.011 p<1e−05 p<0.0002 p<0.016 p<0.12 p<0.0091 p<0.044 p<0.01 p<0.36 p<0.12 p<0.073 p<0.011 p<1e−05 p<0.0002

Col1a1 Col4a2 Col4a3bp Col4a5 Col5a2 Col6a1 Col6a3 Col7a1 Col12a1 Col15a1 Col27a1 Col1a1 Col4a2 Col4a3bp Col4a5 Col5a2 Col6a1 Col6a3 Col7a1 Col12a1 Col15a1 Col27a1

Figure 1: Collagen up-regulation genes, A full time period, B 1h/6h and 6h/24h comparisons.

The top ranks of the several function lists are dominated by some collagens. These are dominated by a single gene (the coding gene of itself) and therefore not in the focus of a pathway-oriented analysis. They are dealt with in a separate section in the main manuscript. Most of the collagens are up-regulated but to complement the section also the collagen with the highest down-regulation is used.

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 10 10 8 8 log2 expression log2 expression 6 6 4 4

p<0.55 p<0.0057 p<0.39 p<0.8 p<0.28 p<0.075 p<0.55 p<0.64 p<0.37 p<0.0002 p<0.047 p<0.054 p<0.46 p<0.34 p<0.0086 p<0.043 p<0.33 p<0.23 p<0.69 p<0.058 p<0.013 p<0.65 p<0.011 p<0.66 p<0.027 p<0.013 p<0.65 p<0.011 p<0.66 p<0.027

Cd36 Col4a1 Col12a1 Col13a1 Col18a1 Cd36 Col4a1 Col12a1 Col13a1 Col18a1

Figure 2: Collagens down-regulation genes, A full time period, B 1h/6h and 6h/24h comparisons.

It has been checked if other metabolic genes involved in the formation of collagens are suitable to be combined with the collagen synthesis but that does not seem to be the case. The genes coding for amino acid

4 transport are quite constant in their expression and are furthermore at a relatively low rate. This might be due to the fact that the dominant share of the amino acids required for the protein synthesis are recruited from protein degradation.

A B

C1h/24h C/T 24h C1h/24h C/T 24h C1h/24h C/T 24h C1h/6h C1h/6h C1h/6h C6h/24h C6h/24h C6h/24h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h 8.0 8.0 7.5 7.5 log2 expression log2 expression 7.0 7.0 6.5

6.5 p<0.04 p<0.48 p<0.14 p<0.17 p<0.49 p<0.014 p<0.016 p<0.06 p<0.0048 p<0.024 p<0.0096 p<0.98 p<0.0013 p<0.013 p<0.12 p<0.0096 p<0.0013 p<0.12

Aldh18a1 Aldh4a1 Prodh Aldh18a1 Aldh4a1 Prodh

Figure 3: Proline synthesis genes, A full time period, B 1h/6h and 6h/24h comparisons.

As most ﬁbrogenic collagens are immensely proline-rich, it has also been checked if the transamination paths to proline are up-regulated, however, they appear to be quite constantly expressed. As the only useful addition to the otherwise single-gene functions Sox9 [3] was selected as a collagen promoter (which is not based on the ModeScore analysis as Sox9 is not a metabolic gene).

2.2 Ethanol degradation

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 14 C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 12 12 10 10 8 8 log2 expression log2 expression 6 6 4

4 p<0.89 p<0.0041 p<0.078 p<0.15 p<0.76 p<0.83 p<0.031 p<0.15 p<0.031 p<2e−05 p<0.063 p<0.017 p<0.054 p<0.58 p<0.073 p<0.27 p<0.047 p<9e−06 p<0.19 p<0.025 p<0.046 p<0.5 p<0.0007 p<0.0087 p<0.015 p<0.53 p<0.002 p<0.063 p<0.79 p<0.3 p<0.2 p<0.52 p<0.02 p<0.63 p<0.0098 p<0.0008 p<0.0003 p<0.24 p<0.013 p<0.0038 p<0.02 p<0.63 p<0.0098 p<0.0008 p<0.0003 p<0.24 p<0.013 p<0.0038

Adh1 Cyp2e1 Aldh1a1 Aldh3a2 Aldh7a1 Aldh9a1 Acss2 Ces1d Adh1 Cyp2e1 Aldh1a1 Aldh3a2 Aldh7a1 Aldh9a1 Acss2 Ces1d

5 Figure 4: Ethanol degradation genes, A full time period, B 1h/6h and 6h/24h comparisons.

Ethanol degradation is the negative top scorer of C/T 24h and is selected as it has already been observed that there is a strong relation to TGFβ [4] and is therefore selected for the main manuscript.

2.3 Sphinganine-1P synthesis

A B

C1h/24h C/T 24h C1h/24h C/T 24h C1h/24h C/T 24h C1h/6h C1h/6h C1h/6h

10 C6h/24h C6h/24h C6h/24h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h 10 9 9 8 8 7 7 log2 expression log2 expression 6 6 5

5 p<0.014 p<0.8 p<0.14 p<0.0092 p<0.08 p<0.019

4 p<0.081 p<0.017 p<0.0018 p<0.0061 p<0.64 p<0.12 p<0.024 p<0.58 p<0.0027 p<0.64 p<0.024 p<0.0027

Sptlc2 Kdsr Sphk1 Sptlc2 Kdsr Sphk1

Figure 5: Sphinganine-1P synthesis genes, A full time period, B 1h/6h and 6h/24h comparisons.

The positive top scorer in C/T 24h not considering collagens is the synthesis of Sphinganine-1P. However, the gene coding sphingosine kinase is the only one up-regulated in the TGFβ series. As this is remarkable anyway it has been added to the solitude genes section.

6 2.4 Tyrosine degradation

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 11 10 10 9 8 8 log2 expression log2 expression 7 6 6

5 p<0.0052 p<0.014 p<0.016 p<0.99 p<0.38 p<0.0061 p<0.0097 p<0.0003 p<9e−05 p<0.0016 p<0.014 p<0.0012 p<0.038 p<0.0059 p<0.0014 p<0.0021 p<0.027 p<0.0054 p<0.96 p<0.2 p<0.85 p<0.6 p<0.83 p<0.79 p<0.037 p<0.0022 p<0.21 p<0.017 p<0.0082 p<0.094 p<0.037 p<0.0022 p<0.21 p<0.017 p<0.0082 p<0.094

Pah Tat Hpd Hgd Gstz1 Fah Pah Tat Hpd Hgd Gstz1 Fah

Figure 6: Tyrosine degradation genes, A full time period, B 1h/6h and 6h/24h comparisons.

Among the negative top 10 of C/T 24h, T1h/24h, and C6h/24h. Additionally, several functions can be found which are in fact subfunctions of the Phenylalanine/Tyrosine degradation pathway: synthesis 4- hydroxyphenylpyruvate, homogentisate, 4-maleoylacetoacetate, fumarylacetoacetate, several transamination functions from tyrosine and phenylalanine. Indeed, each of the 6 involved enzymes in the degradation pathway (starting from phenylalanine) is regulated in a similar way: heavily down-regulated in control and even more so in the TGFβ treated experiment. The amount the down-regulation and the consistent pattern throughout a complete reaction path makes it the top result in the ModeScore ranking. See Supplementary File 4 for a full acount of rankings. In Table 1.1 (comparing TGFβ treated and control at 24h), column “bottom up”, ranks 2,5,7,8,14,19 are occupied by functions related to phenylalanine degradation. In Table 1.2 (comparing 1h and 24h in the control) the ranks are 7,10,11,12,13,14,16-25. In Table 1.3 (comparing 1h and 24h in the treated cells) the ranks are 6,8-23.

7 2.5 Panthetheine synthesis

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 12 12 10 10 log2 expression log2 expression 8 8 6 6

p<0.0084 p<0.44 p<0.18 p<0.036 p<0.049 p<0.019 p<0.24 p<0.0009 p<0.054 p<0.19 p<0.0087 p<0.092 p<0.64 p<0.88 p<3e−05 p<0.39 p<0.075 p<0.011 p<0.48 p<0.0093 p<0.22 p<0.16 p<0.017 p<0.088 p<0.033 p<0.046 p<0.06 p<0.66 p<0.11 p<0.48 p<0.95 p<0.028 p<0.77 p<0.081 p<0.093 p<0.11 p<0.48 p<0.95 p<0.028 p<0.77 p<0.081 p<0.093

Cdo1 Csad Ado Vnn1 Pank2 Pank3 Coasy Cdo1 Csad Ado Vnn1 Pank2 Pank3 Coasy

Figure 7: Panthetheine synthesis genes, A full time period, B 1h/6h and 6h/24h comparisons.

Top positive scorer in C1h/24h and rank 10 of negative scorers in list C/T 24h is Panthetheine synthesis. Only Vanin 1 (Vnn1) which is directly involved in the CoA synthesis pathway, shows this intriguing pattern. Cysteine dioxygenase 1 (Cdo1) which is also involved in this flux distribution shows an opposite pattern, down-regulated in time, aggravated by TGFβ, a more average pattern. The other genes show a constant and rather low expression. In fact, cysteine dioxygenase is also involved in cysteine degradation. As this is presumably the process with a higher flux rate, its regulation should not be interpreted with respect to CoA synthesis. The full flux distribution of CoA synthesis (see Supplementary File 4) shows that other enzymes involved in CoA pathway post panthetheine are remarkably constant. Thus, Vanin 1, will be kept as a solitude gene.

8 2.6 Inositol synthesis

A B

C1h/24h C/T 24h C1h/24h C/T 24h C1h/24h C/T 24h C1h/6h C1h/6h C1h/6h C6h/24h C6h/24h C6h/24h

11 C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h 10 10 9 9 log2 expression log2 expression 8 8

7 p<0.061 p<0.11 p<0.35 p<0.0007 p<0.16 p<0.015 p<0.37 p<0.018 p<0.83

7 p<0.0003 p<0.4 p<0.19 p<0.48 p<0.076 p<0.18 p<0.4 p<0.48 p<0.18

Isyna1 Impa1 Cdipt Isyna1 Impa1 Cdipt

Figure 8: Inositol synthesis genes, A full time period, B 1h/6h and 6h/24h comparisons.

Inositol synthesis is the top positive 2 ranker in list C1h/24h and C6h/24h. Apparently, the only gene which accentuates this behavior is Inositol-1P synthase (Isyna1). Presumably, the up-regulation shows a larger activity in phosphoinositol in later time points, but Isyna1 is the only information in the transcripts on which this hypothesis can be observed. Also the gene coding the enzyme to the binding of inositol into the CDP-diacylglycerol (Cdipt) is expressed constantly. Thus, it is also kept as a solitude gene.

2.7 Urea synthesis

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 12 12 10 10 8 8 log2 expression log2 expression 6 6

p<0.035 p<0.085 p<0.071 p<0.45 p<0.0065 p<0.15 p<0.65 p<0.0046 p<0.013 p<0.2 p<0.75 p<0.091 4 p<0.0015 p<0.01 p<0.014 p<0.11 p<0.0094 p<0.002 p<0.28 p<0.85 p<0.079 p<0.024 p<0.045 p<0.21 p<0.0011 p<0.002 p<0.0025 p<0.0033 p<0.0057 p<0.011 p<0.18 p<0.0092 p<0.011 p<0.019 p<0.13 p<0.0052 p<0.51 p<0.84 p<0.58 p<0.098 p<0.8 p<0.49 p<0.64 p<0.074 p<0.85 p<0.067 p<0.48 p<0.084 4 p<0.0089 p<0.05 p<0.47 p<0.0026 p<0.76 p<0.45 p<0.015 p<0.012 p<0.0064 p<0.0057 p<0.27 p<0.0043 p<0.0089 p<0.05 p<0.47 p<0.0026 p<0.76 p<0.45 p<0.015 p<0.012 p<0.0064 p<0.0057 p<0.27 p<0.0043

Cps1 Otc Ass1 Asl Arg1 Got1 Gpt Gpt2 Glud1 Gls2 Pdha1 Pdhb Cps1 Otc Ass1 Asl Arg1 Got1 Gpt Gpt2 Glud1 Gls2 Pdha1 Pdhb

9 Figure 9: Urea synthesis genes, A full time period, B 1h/6h and 6h/24h comparisons.

Urea synthesis appears at rank 3 of negative scorers in C6h/24h and is also among the negative top 20 in C/T 24h. Subfunctions such as ornithine synthesis, aspartate degradation, and alanine degradation also appear as high negative scorers. It is also selected because it is such a central hepatocyte function. One involved gene, arginase 1, is the top down-regulated gene in the comparison C1h/24h and also the top down-regulated metabolic gene in T1h/24h (just short of cytochrome p450 2b10). It can be observed that the diﬀerent enzymes in the urea cycle and further supporting enzymes are regulated in a rather irregular pattern.

2.8 Bilirubin conjugation

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h 13 C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 12 12 11 11 xpression log2 e log2 expression 10 10 9 9

p<0.82 p<0.36 p<0.12 p<0.63 p<0.11 p<0.12 p<0.0029 p<0.0085 p<0.0001 p<0.8 p<0.011 p<0.0034 p<0.029 p<0.01 p<0.073 p<0.6 p<0.4 p<0.16 p<0.51 p<0.65 p<0.032 p<0.036 p<0.0002 p<0.017 p<0.012 p<0.032 p<0.036 p<0.0002 p<0.017 p<0.012

Pgm2 Ugp2 Ugdh Ugt1a1 Abcc3 bile export UTP Bilirubin Pgm2 Ugp2 Ugdh Ugt1a1 Abcc3 Glc-6P UDP-glucuronate Bilirubin-bisgluc.

Figure 10: Bilirubin conjugation genes, A full time period, B 1h/6h and 6h/24h comparisons.

Bilirubin conjugation is an important clinical indicator of liver status and is regarded here even though the function’s ModeScore evaluation is not as spectacular. However, the supply of UDP-glucuronate is considerably down-regulated in comparison to the glucuronate transfer to bilirubin. There is one issue, however, which complicates the evaluation of this function. On the Affymetrix chip Mouse430_2 there is no way to uniquely identify each of the different isoforms of glucuronosyltransferase 1, the probes do not allow to distinguish between them. Thus, the probeset allows an average assessment for all isoforms which could blur a more specific regulation.

2.9 Cholesterol synthesis Cholesterol is synthesized from acetyl-CoA in a rather long chain of reactions which are considered in several blocks, mainly because certain intermediates of this chain are also used as building blocks for other cellular constituents, namely mevalonate and farnesyl-diphosphate.

10 A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h 11 C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h

11 C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 10 10 9 9 8 8 log2 expression log2 expression 7 7 6 6 p<0.72 p<0.13 p<0.049 p<0.049 p<0.048 p<0.24 p<0.018 p<0.1 p<0.1 p<0.16 p<0.46 p<0.0051 p<4e−07 p<4e−07 p<0.029 p<0.0068 p<0.038 p<0.14 p<0.14 p<0.96 p<0.14 p<0.12 p<0.011 p<0.011 p<0.0006 p<0.14 p<0.12 p<0.011 p<0.011 p<0.0006 5

Acat1 Acat2 Hmgcs1 Hmgcs1/2 Hmgcr Acat1 Acat2 Hmgcs1 Hmgcs1/2 Hmgcr

Figure 11: Mevalonate from acetyl-CoA genes, A full time period, B 1h/6h and 6h/24h comparisons.

The reactions up to mevalonate are regulated inconclusively. Worth noting is that the Acetoacetyl- CoA synthase (Acat1) and the HMG-CoA-Reductase are up-regulated in the treated group, in the case of Acat1even as an early response (6h). HMG-CoA-synthase appears twice as two transcripts are deﬁned for the gene, however, the probes on the chip are not suﬃcient to distinguish between them, so the actual values are identical.

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C6h/24h C6h/24h C6h/24h C6h/24h C/T 24h C/T 24h C/T 24h C/T 24h 12 C/T 6h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h C/T 24h 11 11 10 10 9 9 8 log2 expression log2 expression 8 7 7 6 p<0.033 p<0.15 p<0.024 p<0.063 6 p<0.13 p<0.031 p<0.04 p<0.73 p<0.0018 p<0.027 p<0.0006 p<0.062 p<0.14 p<0.094 p<0.052 p<0.4 p<0.12 p<0.054 p<0.0017 p<0.0009 p<0.12 p<0.054 p<0.0017 p<0.0009

Mvk Mvd Idi1 Fdps Mvk Mvd Idi1 Fdps

Figure 12: Farnesyl-diphosphate from mevalonate genes, A full time period, B 1h/6h and 6h/24h comparisons.

The rest of the cholesterol synthesis pathway seems to be regulated in a very consistent way: down- regulated in the control group, aggravated by TGFβ. This is meaningful as this down-regulation occurs at a higher amplitude than the average gene. An interesting feature in the reactions towards farnesyl-diphosphate is that Idi1 and Fdps show an early

11 down-regulation, see Figure 12B. For most other genes the largest amplitude of change occurs between 6h and 24h and the change between 1h and 6h is low.

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h

11 C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h

11 C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 10 10 9 9 8 8 log2 expression log2 expression 7 7 6 6

p<0.086 p<0.078 p<0.0041 p<0.019 p<0.018 p<0.021 p<0.013 p<0.0048 p<0.021 p<0.57 p<0.85 p<0.058

5 p<0.14 p<0.015 p<0.43 p<0.01 p<0.0009 p<0.025 p<0.041 p<9e−05 p<0.0023 p<0.001 p<0.6 p<0.11 p<0.053 p<0.0035 p<0.0012 p<0.0076 p<0.0002 p<0.002 p<0.0061 p<7e−05 p<0.0012 p<0.034 p<0.56 p<0.0037 p<0.023 p<0.0044 p<0.42 p<0.36 p<0.089 p<0.027 p<0.72 p<0.0079 p<0.19 p<0.012 p<0.061 p<0.12

5 p<0.0065 p<0.0039 p<0.011 p<0.0093 p<0.16 p<0.035 p<0.1 p<0.0009 p<0.014 p<0.0038 p<0.063 p<0.031 p<0.0065 p<0.0039 p<0.011 p<0.0093 p<0.16 p<0.035 p<0.1 p<0.0009 p<0.014 p<0.0038 p<0.063 p<0.031

Fdft1 Sqle Lss Cyp51 Tm7sf2 Sc4mol Nsdhl Hsd17b7 Ebp Dhcr24 Sc5d Dhcr7 Fdft1 Sqle Lss Cyp51 Tm7sf2 Sc4mol Nsdhl Hsd17b7 Ebp Dhcr24 Sc5d Dhcr7

Figure 13: Cholesterol from farnesyl-diphosphate genes, A full time period, B 1h/6h and 6h/24h comparisons.

The pathway is selected for the main manuscript and dealt with in more detail there.

2.10 Bile acid synthesis and excretion

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h

C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 14 C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 12 12 10 10 8 8 log2 expression log2 expression 6 6 4 4

p<0.066 p<0.43 p<0.0081 p<0.47 p<0.0002 p<0.89 p<0.078 p<0.16 p<0.0089 p<0.071 p<0.077 p<0.12 p<0.36 p<0.025 p<0.0024 p<0.035 p<0.0013 p<0.031 p<0.063 p<0.026 p<0.0002 p<0.46 p<0.0012 p<0.0019 p<0.028 p<0.12 p<0.0004 p<0.07 p<0.0002 p<0.047 p<0.19 p<0.19 p<1e−05 p<0.31 p<0.0003 p<0.0033 p<0.43 p<0.037 p<0.62 p<0.08 p<0.59 p<0.015 p<0.002 p<0.76 p<0.67 p<0.09 p<0.11 p<0.13 p<0.61 p<3e−05 p<0.27 p<0.019 p<0.0085 p<0.02 p<0.0098 p<0.0092 p<0.084 p<0.0076 p<0.002 p<0.013 p<0.61 p<3e−05 p<0.27 p<0.019 p<0.0085 p<0.02 p<0.0098 p<0.0092 p<0.084 p<0.0076 p<0.002 p<0.013

Cyp7a1 Hsd3b7 Akr1d1 Akr1c14 Cyp27a1 Adh1 Aldh1a1 Abcd3 Slc27a5 Acaa1b Baat Abcb11 Cyp7a1 Hsd3b7 Akr1d1 Akr1c14 Cyp27a1 Adh1 Aldh1a1 Abcd3 Slc27a5 Acaa1b Baat Abcb11

Figure 14: Glycochenodeoxycholate from cholesterol genes, A full time period, B 1h/6h and 6h/24h comparisons.

Several of genes show down-regulation with high amplitude only in the treated group, some other are strongly down-regulated also in the control group and thus are not sensitive to TGFβ treatment. Which of

12 the groups of genes dominates could only be decided upon the knowledge which are the rate-limiting steps in the pathway.

2.11 β-hydroxybutyrate

A B

C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h

11 C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h

11 C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 10 10 9 9 8 8 xpression log2 e log2 expression 7 7 6 6 5

5 p<0.72 p<0.13 p<0.28 p<0.098 p<0.041 p<0.3 p<0.46 p<0.0051 p<0.049 p<0.0037 p<0.0088 p<0.0024 p<0.24 p<0.018 p<0.081 p<0.023 p<0.078 p<0.0018 p<0.0068 p<0.038 p<0.0001 p<0.92 p<0.55 p<0.26 p<0.14 p<0.12 p<0.0074 p<0.051 p<0.016 p<0.0096 p<0.14 p<0.12 p<0.0074 p<0.051 p<0.016 p<0.0096

Acat1 Acat2 Hmgcs2 Hmgcl Bdh1 Bdh2 Acat1 Acat2 Hmgcs2 Hmgcl Bdh1 Bdh2 Ac-CoA Ac-CoA Acac-CoA HMG-CoA Acac BHB

Figure 15: β-hydroxybutyratefrom Acetyl-CoA genes, A full time period, B 1h/6h and 6h/24h comparisons.

This pathway is selected as there is an intriguing early up-regulation in the mitochondrial HMG-CoA synthase.

2.12 Glycolysis

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h 14 C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 14 12 12 10 10 log2 expression log2 expression 8 8 6 6

p<0.037 p<0.016 p<0.46 p<0.064 p<0.2 p<0.44 p<0.031 p<0.15 p<0.17 p<0.11 p<0.063 p<0.078 p<0.12 p<0.31 p<0.02 p<0.029 p<0.052 p<0.045 p<0.21 p<0.058 p<0.0077 p<0.019 p<0.024 p<0.0073 p<0.11 p<0.064 p<0.029 p<0.95 p<0.008 p<0.0061 p<0.0056 p<0.0097 p<0.061 p<0.13 p<0.003 p<0.0067 p<0.0086 p<0.0044 p<0.025 p<0.029 p<0.0039 p<0.2 p<0.27 p<0.51 p<0.46 p<0.81 p<0.94 p<0.5 p<0.56 p<0.43 p<0.12 p<0.7 p<0.5 p<0.72 p<0.5 p<0.59 p<0.0038 p<0.02 p<0.16 p<0.057 p<0.21 p<0.44 p<0.044 p<0.032 p<0.11 p<0.017 p<0.13 p<0.072 p<0.042 p<0.23 4 p<0.0038 p<0.02 p<0.16 p<0.057 p<0.21 p<0.44 p<0.044 p<0.032 p<0.11 p<0.017 p<0.13 p<0.072 p<0.042 p<0.23

Slc2a2 Hk2 Hk3 Gpi1 Pfkl Aldoa Aldob Tpi1 Gapdh Pgk1 Pgam1 Eno1 Pklr Pkm2 Slc2a2 Hk2 Hk3 Gpi1 Pfkl Aldoa Aldob Tpi1 Gapdh Pgk1 Pgam1 Eno1 Pklr Pkm2

13 Figure 16: Glycolysis genes, A full time period, B 1h/6h and 6h/24h comparisons.

The function called “Anaerobic rephosph of ATP” in Table 1 does not show any particularly strong regulation and is dealt with only because its cellular importance. The strongest down-regulation shows the glucose transporter gene. As the same protein is integrated in the plasma membrane and the membrane of the endoplasmatic reticulum (ER), it is not possible to assign a unique purpose for this regulation. There is a straightforward interpretation of the regulation of hexokinases. Apparently, the hexokinase 2 with a broad substrate specificity is up-regulated, allowing the use of imported sugars for the own energy supply. The glucokinase which has a high affinity to glucose is related to the homeostatic task of the liver to reduce the blood glucose higher than a threshold very efficiently. This enzyme is down-regulated, in other words, the hexokinase activity is switched from the Glc to Hk2, indicating a loss of specific liver function of the hepatocytes.

2.13 Triglycerides

A B

C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/24h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C1h/6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 12 C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C6h/24h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 6h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h C/T 24h 11 11 10 10 9 9 log2 expression log2 expression 8 8 7 7 p<0.96 p<0.32 p<0.17 p<0.17 p<0.21 p<0.56 p<0.0086 p<0.0089 p<0.12 p<0.0022 p<0.42 p<0.014 p<0.88 p<0.68 p<0.56 p<0.13 p<0.0005 p<6e−05 p<0.0059 p<0.068 p<0.0042 p<0.21 p<0.26 p<0.78 p<0.03 p<5e−05 p<0.0032 p<0.63 p<0.34 p<0.32 p<0.38 p<0.081 p<0.049 p<0.036 p<0.14 p<1 p<0.029 p<0.017 p<0.14 p<0.0095 p<0.83 p<0.015 p<0.085 p<0.14 p<0.045 p<0.029 p<0.017 p<0.14 p<0.0095 p<0.83 p<0.015 p<0.085 p<0.14 p<0.045

Slc27a2 Agpat6 Agpat9 Acsl5 Agpat2 Ppap2b Ppap2c Dgat1 Dgat2 Slc27a2 Agpat6 Agpat9 Acsl5 Agpat2 Ppap2b Ppap2c Dgat1 Dgat2

Figure 17: Triacylglycerol synthesis genes, A full time period, B 1h/6h and 6h/24h comparisons.

This function also does not show any particularly strong regulation and is dealt with because there is an experimental indication that the cells lose their internal triglycerides in the culture. The strongest down-regulation, aggravated by TGFβ can be found in the fatty acid transporter (Slc27a2), 1-acylglycerol- 3-phosphate acyltransferase (Agpat9), and diacylglycerol acyltransferase (Dgat2). The other isozyme diacylglycerol acyltransferase (Dgat1) is up-regulated, independently of treatment. Thus, a redistribution of isozyme can be hypothesized.

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