Tubule Specific Notch1 Knock-Out Mice Do Not Protect from Fibrosis. (A) Experimental Scheme for Generating the Kspcre Notch1 Flox/Flox Mice

Supplemental Figure 1: Tubule specific Notch1 knock-out mice do not protect from fibrosis. (A) Experimental scheme for generating the Kspcre Notch1 flox/flox mice. Kidney injury was induced by FA injection. (B) Representative images of PAS-stained kidney sections from control and Kspcre Notch1flox/flox mice with or without FA injection. Scale bar: 10m. (C and D) Relative mRNA amount of Notch signaling (C) and fibrosis markers (D) in control and Kspcre Notch1flox/flox mice with or without FA injection. Data are represented as mean ± SEM. * P <0.05; ** P <0.01, *** P <0.001, **** P < 0.0001, by Two-way ANOVA with post-hoc Tukey test, n=5-7 per group.

Supplemental Figure 2: Notch3 knock-out mice do not protect from fibrosis. (A) Experimental scheme for generating the Notch3 knockout mice. Kidney injury was induced by FA injection. (B) Representative images of PAS-stained kidney sections from control and Notch3 knockout mice with or without FA injection. Scale bar: 10m. (C and D) Relative mRNA amount of Notch signaling (C) and fibrosis markers (D) in control and Notch3 null mice with or without FA injection. Data are represented as mean ± SEM. ** P <0.01, *** P <0.001, **** P < 0.0001, by Two-way ANOVA with post-hoc Tukey test, n=4-7 per group.

Supplemental Figure 3: Tfam plays an important role in kidney tubules. (A) Overlap of RBPJ ChIP-seq binding peaks with significant differential expressed genes in kidneys of Pax8rtTA/TREICNotch1 mice. (B) Experimental scheme for generating the Kspcre Tfamflox/flox mice. (C) Appearance of WT and Kspcre Tfamflox/flox pups at postnatal day 15. Scale bar: 1 cm.

Supplemental Figure 4: EGF did not rescue JAG1-induced metabolic defect and downstream profibrotic changes. (A) The mouse Egf locus and RBPJ ChIP-Seq from 6 and 24hr Notch activation and inhibition. (B) Correlation between interstitial fibrosis and EGF transcript level in 95 microdissected human kidney samples. (C-E) Normalized counts of Egf by RNA sequencing in whole- kidney lysates of folic acid-induced nephropathy group (C), sham and UUO group (D), and APOL1- G1/G2 mice (E). Data are represented as the mean ± SEM. *** P < 0.001, **** P < 0.0001, by 2-tailed Student’s t test, n=2-6 per group. (F) Relative mRNA expression of Acta2 and Snai1 in JAG1 co- culture system when treated with EGF. Data are represented as mean ± SEM. * P <0.05 by Two-way ANOVA with post-hoc Tukey test, n=4-8 per group.

Supplemental Figure 5: Tfam rescues JAG1-induced metabolic defect and downstream profibrotic changes. (A) Tfam expression level in JAG1 co-culture system. Data are represented as mean ± SEM. ** P <0.01, by 2-tailed Student’s t test, n=6 per group. (B) Oxygen consumption rate (OCR) in JAG1 co-culture system in the presence of GFP or Tfam plasmid. Where indicated, cells were incubated in palmitate (180 μM), etomoxir (40 μM), and oligomycin (1 μM). Data are represented as mean ± SEM. *P < 0.05, ** P <0.01, *** P <0.001 and **** P <0.0001 as compared to L cell group, #P < 0.05, ##P < 0.01, ### P <0.001 as compared to JAG1 cell group, by Two-way ANOVA with post-hoc Tukey test, n=3 per group. (C) ATP levels in JAG1 co-culture system in the presence of GFP or Tfam plasmid. Data are represented as mean ± SEM. * P <0.05, by One-way ANOVA with post-hoc Tukey test, n=6-8 per group. (D) Relative mRNA expression of transcripts related to fibrosis and proliferation in JAG1 co-culture system in the presence of GFP or Tfam plasmid. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by One-way analysis of variance with Tukey’s post hoc tests, n=8 per group. (E) Relative mRNA amount of Tfam in control and KspcreNotch1flox/flox mice with or without FA injection. Data are represented as mean ± SEM. ** P <0.01 by Two-way ANOVA with post-hoc Tukey test, n=5-7 per group. (F) Relative mRNA amount of Tfam in control and Notch3 knock-out mice with or without FA injection. Data are represented as mean ± SEM. * P <0.05 by Two-way ANOVA with post-hoc Tukey test, n=4-7 per group. 5

Supplemental Figure 6: PPAR agonist rescues JAG1 induced dedifferentiation. (A) Relative mRNA expression of Ppara and Ppargc1a in JAG1 co-culture system. Data are represented as mean ± SEM. *** P <0.01 by 2-tailed Student’s t test, n=6 per group. (B) Relative mRNA expression of Acta2 and Snai1 in JAG1 co-culture system when treated with fenofibrate (Feno). Data are represented as mean ± SEM. * P <0.05, *** P <0.001 by Two-way ANOVA with post-hoc Tukey test, n=4-8 per group.

Supplemental Table1: RBPJ binding sites overlap with upregulated genes in Pax8rtTA/TREICNotch1 mice Chromosome start end Associated Gene RBPJ binding chr12 105938345 105938945 Dicer1 Inducible chr2 125101167 125101767 Dut Inducible chr2 125105413 125106013 Dut Inducible chr14 35260679 35261279 Bmpr1a Inducible chr19 53631284 53631884 Dusp5 Inducible chr19 53629606 53630206 Dusp5 Inducible chr14 46149828 46150428 Fermt2 Constant chr9 63550165 63550765 Smad3 Inducible chr10 116874285 116874885 Cpsf6 Constant chr16 13354044 13354644 Mkl2 Inducible chr19 29703490 29704090 Ermp1 Inducible chr14 120772066 120772666 Mbnl2 Constant chr1 137533926 137534526 Nav1 Inducible chr1 178736644 178737244 Cep170 Constant chr13 93418835 93419435 Serinc5 Inducible chr4 84443853 84444453 Cntln Inducible chr14 61330968 61331568 Spata13 Inducible chr10 3156954 3157554 Cnksr3 Inducible chr10 3227813 3228413 Cnksr3 Inducible chr1 138455324 138455924 Kif14 Inducible chr11 16708806 16709406 Egfr Inducible chr17 73308955 73309555 Lbh Inducible chr4 47306423 47307023 Tgfbr1 Inducible chr12 112712876 112713476 Gm266 Inducible chr1 43502048 43502648 Nck2 Constant chr7 26496547 26497147 Tgfb1 Inducible chr19 11663565 11664165 Ms4a6d Inducible chr13 69724585 69725186 Srd5a1 Inducible chr3 8717326 8717926 Hey1 Inducible chr10 93105473 93106073 Ntn4 Inducible chr13 96315537 96316137 F2rl1 Inducible chr13 104061069 104061669 Mast4 Inducible chr16 30066154 30066754 Hes1 Inducible chr16 30063927 30064527 Hes1 Inducible chr16 30065039 30065639 Hes1 Inducible chr3 93341215 93341815 S100a11 Inducible chr16 92621330 92621930 Runx1 Inducible chr8 37217734 37218334 D8Ertd82e Inducible

7 chr18 20192618 20193218 Dsc2 Constant chr13 5347082 5347682 Klf6 Inducible chr3 95471701 95472301 Mcl1 Inducible chr5 97251538 97252138 Anxa3 Inducible chr2 128254807 128255407 Gm14005 Inducible chr2 128296053 128296653 Gm14005 Inducible chr2 128370195 128370795 Gm14005 Inducible chr11 20208861 20209461 Slc1a4 Inducible chr3 95491829 95492429 Adamtsl4 Inducible chr1 74324372 74324972 Gpbar1 Inducible chr2 26349755 26350355 Notch1 Inducible chr6 116594407 116595007 8430408G22Rik Inducible chr4 122904856 122905456 Heyl Inducible chr4 122916604 122917204 Heyl Inducible chr4 122905468 122906068 Heyl Inducible chr2 136875009 136875609 Jag1 Inducible chr2 25033045 25033645 Nrarp Inducible chr2 25028029 25028629 Nrarp Inducible

Supplemental Table2: RBPJ binding sites overlap with downregulated genes in Pax8rtTA/TREICNotch1 mice Chromosome start end Associated Gene RBPJ binding chr3 129389836 129390436 Egf Constant chr16 37615579 37616179 Hgd Inducible chr13 91903860 91904460 Acot12 Inducible chr3 59836361 59836961 Aadac Inducible chr6 117329822 117330422 Cxcl12 Inducible chr8 6608829 6609429 Slc10a2 Inducible chr5 45519151 45519751 Qdpr Inducible chr17 72481480 72482080 Clip4 Inducible chr6 101239295 101239895 Pdzrn3 Inducible chr3 96993376 96993976 Acp6 Inducible chr1 94344895 94345495 Ndufa10 Inducible chrX 16281500 16282100 Maoa Inducible chr8 94325366 94325966 Irx3 Constant chr17 74153736 74154336 Ehd3 Constant chr15 38655430 38656030 Atp6v1c1 Inducible chr2 27595697 27596297 Rxra Inducible chr15 36970843 36971443 Zfp706 Inducible chr1 158933051 158933651 Ralgps2 Inducible chr10 106642147 106642747 Lin7a Inducible chr11 113352046 113352646 Slc39a11 Inducible chr8 37249739 37250339 Lonrf1 Inducible chr17 87601850 87602450 Mcfd2 Inducible chr10 70700475 70701075 Tfam Inducible

Supplement Table 3. qPCR primer sequences Gene Forward Reverse m Notch1 ACAGTGCAACCCCCTGTATG TCTAGGCCATCCCACTCACA m Notch2 GGCATGTTGGGGAAAGCTAC GGACACAAAGCAGGGGTGAG m Notch3 GAGGACCTGGTTGAAGAATTGATC TGCAGTTTTTCCCCTTTTATCC m Jag1 ACACAGGGATTGCCCACTTC AGCCAAAGCCATAGTAGTGGTCAT m Hes1 GCTACCGGACCAAGGAAGTTC GAGCTAGACTGTTCTCAAAGTGAGTGA m HeyL TTCGGCATGAAGCGGCCCAG CTCCAGCTTGGAGGAGCCCTGT m Fibronectin 1 ACAAGGTTCGGGAAGAGGTT CCGTGTAAGGGTCAAAGCAT m Vimentin GATCGATGTGGACGTTTCCAA ATACTGCTGGCGCACATCAC m Col1a1 TGCCTGGACCTCCTGGCGAGCGT AGCAGGTCCGGGAGCACCACGTT m Col3a1 ACAGCTGGTGAACCTGGAAG ACCAGGAGATCCATCTCGAC m Col4a1 GACAGCCAGGTTTGACAGGT GGCAGCTCTCTCCTTTCTGA m Acta2 CTGACAGAGGCACCACTGAA AGAGGCATAGAGGGACAGCA m Snai1 TGGAAAGGCCTTCTCTAGGC GGAGAATGGCTTCTCACCAG m Snai2 GCTGGAACTTGTTTGCGCTTT GTGGTTATTTCTTTGGCCAGCA m Ccne1 GCGGACACAGCTTCGGGTCT GCGGACTGAAAGGTCGGAGCC m c-Myc CAACGACAGCAGCTCGCCCA CCGTGGGGAGGACTCGGAGG m Tfam GAGCAGCTAACTCCAAGTCAG GAGCCGAATCATCCTTTGCCT m Ubiquitin C GCCCAGTGTTACCACCAAGAAG GCTCTTTTTAGATACTGTGGTGAGGAA r Fibronectin CATGGCTTTAGGCGAACCA CATCTACATTCGGCAGGTATGG r Vimentin TTCCCTGAACCTGAGAGAAACTAAC TGTTCTTTTGGAGTGGGTGTCA r Col1a1 AATGGTGCTCCTGGTATTGC GGTTCACCACTGTTGCCTTT r Col4a1 TGCGCAAGTTCAGCACCAT GAGGCGAAGTTGCAGACGTT r Acta2 GGCTCCATCCTGGCTTCTCTAT GGCGTGACTTAGAAGCATTTGC r Snai1 TTAAGTCCAGGGACCTGTGG TGAATACTGAGGGGCAGGAG r Snai2 TCTGCAGACCCACTCTGATG AGCAGCCAGACTCCTCATGT r Ccna2 ACCCCGAAAAAGTGGCGCCAG GTCCTTAAGAGGCGCAACCCGC r Ccnb1 AGGTGTGGGCAGCCAGAGGT AGGCAGCAACTGCCACAGGC r Ccnd1 AGGGGATTCAGGACGACTCT GGGCAACCTTCCCAATAAAT r Ccnd2 GAACCTGGCCGCAGTCACCC GGCTGCTCCCACGCTTCCAG r Ccne1 ATGTCCAAGTGGCCTACGTC GCGAGGACACCATAAGGAAA r c-Myc TCTCGGCCGCTGCCAAACTG TGGGCGAGCTGCTGTCGTTG r Tfam GAAAGCACAAATCAAGAGGAG CTGCTTTTCATCATGAGACAG r Ppara CCTTTTTGTGGCTGCTAT TCCCTGCTCTCCTGTATG r Ppargc1a AGTGTGCTGCCCTGGTTGGTG GGAGGGTCATCGTTTGTGGTC r Cdh1 CGTGGATGTGGTAGACGTGAA TTCTCCGCAGGCACAAAAAT r Cdh2 CAGAGAGTCGCCAAATGTCA TTCACAAGTCTCGGCCTCTT r Ubiquitin C CACCAAGAAGGTCAAACAGGAA AAGACACCTCCCCATCAAACC