Supplementary Information s49

Supplementary Information

Figure S1 Ingenuity Pathway Analysis (IPA) summary. To investigate possible interactions of differently regulated genes, datasets representing 182 genes with altered expression profile obtained from the illumina microarray were imported into the Ingenuity Pathway Analysis Tool and the following data is illustrated: (a) The top five toxicology functions as calculated by IPA based on differentially expressed genes are highlighted along with their respective p-value. (b)

X-axis represents the top canonical pathways identified by IPA based on differentially expressed genes whereas y-axis (log value) represents the number of genes from the dataset that map to the pathway divided by the number of all genes ascribed to the pathway. The yellow line represents the threshold of p<0.05 as calculated by Fischer's test. (c) Taqman assay for mature miR-128 in

HEK293T cells showing overexpression of miR-128-2 in cells transfected with 4g p(128). n = 3

± SEM; *p < 0.05. U = Untransfected, p(128) = cells transfected with 4μg p(128), N= negative control. (d) Annexin V-staining shows that miR-128 induces apoptosis in HEK293T cells.

Representative of three independent experiments has been shown with similar results (n=3,

P<0.05). (e) Schematic of Cholesterol Homeostasis. Cholesterol homeostasis is governed by complex processes of cholesterol uptake and synthesis and cholesterol efflux. Two key transcription factors SREBPs and LXRs regulate these complex processes. SREBP1 target genes involved in fatty acid metabolism and SREBP2 is specific to cholesterol synthesis. Under low cholesterol conditions, SREBP2 activates cholesterogenic genes. During cholesterol accumulation, LXR activates its target genes involved in cholesterol efflux after heterodimerization with RXR.

Figure S2 MiR-128-2 regulates SREBPs. (a-b) Real-time PCR analysis of SREBP2 (a) SREBP1

(b) in p(128) transfected HepG2, MCF7 and HEK293T cells. 18s rRNA was used for the normalization. (c-f) Western blot analysis for cleaved SREBP2 and SREBP1 expression after overexpression of p(128) (c-d), after overexpression of SIRT1 siRNA (e) and after overexpression of SIRT1ORF (f) in HEK293T cells . (g-h) Western blot analysis for SIRT1 in the presence of SIRT1 siRNA (at 100 nM dose) / scrambled siRNA/ SIRT1 ORF / negative control (N) in HepG2, MCF7 and HEK293T cells. Tubulin was used as a loading control and numbers represent normalized densitometric values. U = Untransfected, p(128) = cells transfected with 4μg p(128), N= negative control. n = 3 ± SEM; *p < 0.05 for all experiments.

Figure S3 MiR-128-2 regulates genes of cholesterol/lipid homeostasis. (a-h) Real-time PCR analysis of HMGCS1 (a), LDLR (b), HMGCR (c), LXR (d), LXR (e), RXR (f), ABCA1

(g) and ABCG1 (h) mRNAs in p(128) )/ SIRT1 siRNA/ SIRT1ORF transfected HepG2, MCF7 and HEK293T cells. 18s rRNA was used for the normalization. U = Untransfected, p(128) = cells transfected with 4μg p(128), N= negative control. n = 3 ± SEM; *p < 0.05 for all experiments.

Figure S4 Fatty acid synthesis genes are regulated by miR-128-2. (a-c) Real-time PCR analysis of ACACA, FASN, SCD1 after overexpression of p(128) in HEK293T cells. 18s rRNA was used for the normalization. U = Untransfected, p(128) = cells transfected with 4μg p(128), N= negative control. n = 3 ± SEM; *p < 0.05 for all experiments.

Figure S5 Conservation of binding sites. The binding site of miR-128-2 in ABCA1 3’UTR (a) and ABCG1 (b) is well conserved among different species (Hsa, human; Ptr, chimpanzee; Rno, rat; Cpo, guinea pig; Mmu, mouse; Dno, armadillo; Eca, horse; Cfa, dog; Eeu, hedgehog; Ocu, rabbit, Mml, rhesus; Oga, bushbaby). (c-d) Real-time PCR analysis of ABCA1 (c) and ABCG1

(d) after overexpression of p(128)/ anti-miR in HEK293T cells. 18s rRNA was used for the normalization. U = Untransfected, p(128) = cells transfected with 4μg p(128), N= negative control, AM=antimiR, scrambled anti-miR (SAM), n = 3 ± SEM; *p < 0.05 for all experiments.

(e) Evolutionary conservation of the predicted miR-128-2 binding site in the 3’ UTR of RXR.

Figure S6 (a-b, e-f) Western blot analyses of ABCA1 (a-b) and ABCG1 (e-f) protein expression in MCF7 cells after 24 h of transfection of p(128) or anti-miR-128 in a dose dependent manner.

Tubulin served as a loading control. Bar diagram represents the integrated densitometric values normalized to Tubulin. U = Untransfected, 2 = cells transfected with 2μg p(128), 4 = cells transfected with 4μg p(128), N= negative control, AM 100 = cells transfected with 100nM anti- miR-128, AM 200 = 200nM anti-miR-128, SAM = Scrambled anti-miR. (c-d,g-h) Real-time

PCR analysis analyses ABCA1 (c-d) and ABCG1 (g-h) mRNA in p(128) or anti-miR-128 transfected MCF7 cells. 18s rRNA was used for the normalization. n = 3 ± SEM; *p < 0.05 .

Figure S7 (a) Evolutionary conservation of mature-miR-128-2 among different species. (b)

Bioinformatic analyses show presence of SREBP transcription factor in the promoter region of

ARPP21 gene encoding miR-128-2. (c) Real time PCR of SREBP2 in simvastatin (10M) treated HepG2 cells at different time points (at 0, 3, 6, 12, 24 h). 18s rRNA was used for the normalization.

Supplementary Table S1: Sequence of primers of different metabolic genes of human used for real time PCR in a 5’-3’ direction. (FP-Forward Primer, RP-Reverse primer).

LXRα FP GTTATAACCGGGAAGACTTTGCCA

LXRα RP GCCTCTCTACCTGGAGCTGGT

LXRβ FP CGTGGACTTCGCTAAGCAAGTG

LXRβ RP GGTGGAAGTCGTCCTTGCTGTAGG

ABCA1 FP GCACTGAGGAAGATGCTGAAA

ABCA1 RP AGTTCCTGGAAGGTCTTGTTCAC

ABCG1 FP CAGGAAGATTAGACACTGTGG

ABCG1 RP GAAAGGGGAATGGAGAGAAGA

PPARα FP GGCGAGGATAGTTCTGGAAGC

PPARα RP CACAGGATAAGTCACCGAGGAG

PGC-1α FP TGCCCT GGATTGTTGACATGA

PGC-1α RP TTTGTCAGGCTGGGGGTAGG

APOE FP CTGCTCAGCTCCCAGGTC

APOE RP TTGTTCCTCCAGTTCCGATT

SREBP2 FP AGGAGAACATGGTGCTGA

SREBP2 RP TAAAGGAGAGGCACAGGA

SREBP1 FP GCAAGGCCATCGACTACATT

SREBP1 RP GGTCAGTGTGTCCTCCACCT

ABCG5 FP ACCCAAAGCAAGGAACGGGAA

ABCG5 RP CAGCGTTCAGCATGCCTGTGT

LDLR FP GTCTTGGCACTGGAACTCGT

LDLR RP CTGGAAATTGCGCTGGAC Supplementary Table S2: Sequence of primers of different metabolic genes of mouse used for real time PCR in a 5’-3’ direction. (FP-Forward Primer, RP-Reverse primer).

ABCA1 FP GGTTTGGAGATGGTTATACAATAGTTGT

ABCA1 RP CCCGGAAACGCAAGTCC

ABCG1 FP TCACCCAGTTCTGCATCCTCTT

ABCG1 RP GCAGATGTGTCAGGACCGAGT

GAPDH FP AACTTTGGCATTGTGGAAGG

GAPDH RP ACACATTGGGGGTAGGAACA

SREBP2 FP GCGTTCTGGAGACCATGGA

SREBP2 RP ACAAAGTTGCTCTGAAAACAAATCA

ARPP21 FP CCCTACCTCAACCTCACAGC

ARPP21 RP GACTGGCTGGTAACCTGCTT