Table I. Primers used in quantitative real-time PCR for detecting gene expressions in mouse liver tissues

Transcription

1 Table I. Primers used in quantitative real-time PCR for detecting gene expressions in mouse liver tissues Gene name Forward primer 5' to 3' Gene name Reverse primer 5' to 3' CC_F TGCCCTGTTGGGGTTG CC_R TGTGGCGGGGCTTG CC_F CTTGGGCCCGCTGTC CC_R TGCCCTTTGCCTCCG lbumin_f GTGCGGGCGGCTTG lbumin_r GCCCTTCCTGGTCCTC FSN_F GGCTCTTGGGCCTCCTT FSN_R GCTGCGCCGCCTCTCT GPDH_F CTTTGGCTTGTGGGG GPDH_R GGTGCGGGTGTGTTCT HMGCR_F CTTTCGCGCTGTGCTCC HMGCR_R CTGTGGGTGTGGCTGT HMGCS1_F TTTGTGCGCTGTTTGGG HMGCS1_R CCCCTGTGGTCTGGCTT HMGCS2_F CCCCTGGGTTCCG HMGCS2_R TGCTCTCTCCCTCGTTC HNF1a_F GCCCGGCCCCGTGCC HNF1a_R GGCTTCCCCTCGCTCCCG HNRNP-D_F GTCGGGGTGTGTGGTC HNRNP-D_R GGCCCTTTTGGTCTGCTT HNRNP-I_F GCCTGGCGTGC HNRNP-I_R GCGCCCCGTGTTGTGT KSRP_F CTGGGCCCTGGTCTGT KSRP_R CGTTGTCGTGCTGTCCT LDLR_F CCTGCCGCCTGTGTTC LDLR_R GCGTCTGTTCCGGTCC Luc2_F CGCCTTCGGGTGGC Luc2_R GCGCTTTCTCGCTGCC PCSK9_F TTGCGCGCTGGGCTT PCSK9_R CCGCTGTGTGCCTCTGG SREP1c_F CGGCCTCGCTCTCCG SREP1c_R CCCCTTCGGGTTTCTGC SREP2_ F CCGGGGGGGCGG SREP2_ R CGCCGCTTGTGCTCTTG

2 Table II. mirn microarray analysis to identify R regulated micro RNs in HepG2 cells with target sites in LDLR 3 UTR. Fold change (R 4 h vs. DMSO 4 h) Fold change (R 8 h vs. DMSO 8 h) Fold change (R 24 h vs. DMSO 24 h ) Column # Column ID mml-mir-19a hsa-mir-130b hsa-mir-615-5p hsa-mir-671-5p hsa-mir hsa-mir has-mir-1244* hsa-mir-1979* hsa-mir crm-mir * mirn target site is not present in LDLR 3 UTR

3 Supplemental Figure I Relative Luc ctivity WT * * RE1-mu RE2-mu RE3-mu RE1+2-mu RE1+2+3-mu R fold induction WT RE1-mu C RE2-mu Normalized Luc ctivity RE3-mu pluc RE1+2-mu pluc-ldlrutr1 RE1+2+3-mu pluc-re1 pluc-re2 pluc-re3 Supplemental Figure I: Identification of three destabilizing RE motifs in mouse LDLR 3 UTR. () HepG2 cells were transfected with individual luciferase-utr reporter plasmid along with prl-sv40 as the normalizing transfection vector for 48 hours. R was added to cells for 8 hours before cell lysis. The stimulatory activity of R on the wild-type UTR reporter was defined as %, and activities of R on mutated vectors were plotted relative to that value. The data shown are mean ± SEM of three separate transfections in which triplicate wells were used for each reporter. () Comparison of human and mouse LDLR mrn 3 UTR sequences. Segments of RE containing sequences are shown. Mouse nucleotide sequences that differ from those of the human sequence are boxed. The sequences of REs are in red color and the UUU motifs are underlined. (C) LDLR3 UTR reporter plasmids or control vector pluc were cotransfected with prl-sv40 into Hepa 1-6 cells. Two days post-transfection, cell lysates were prepared. Dual luciferase activities were measured and expressed as normalized luciferase activity. Each value represents the mean ± SEM of four wells per condition. p < 01 as compared to pluc. The graph shown is representative of three separate experiments.

4 Supplemental Figure II p42/40 Liver MPH Hepa 1-6 HNRNPD /actin: /actin: shrn: CGGGGTGTGTGGTC human HNRN PD V1: TCCGGGCGTCGGGGTTTTGGCTTTGTGCTTTTGTCGGGGTGTGTGGTCTGGTCG mouse HNR N PD V1: TCCGGGCGTCGGGGTTTTGGCTTTGTGCTTTTGGTCGGGGTGTGTGGTCTGGTCGG mouse HNR NPD V2: TCCGGGCGTCGGGGTTTTGGCTTTGTGCTTTTGGTCGGGGTGTGTGGTCTGGTCGG mouse HNR N PD V3: TCCGGGCGTCGGGGTTTTGGCTTTGTGCTTTTGGTCGGGGTGTGTGGTCTGGTCGG mouse HNR N PD V4: TCCGGGCGTCGGGGTTTTGGCTTTGTGCTTTTGGTCGGGGTGTGTGGTCTGGTCGG Supplemental Figure II: Characterization of mouse hnrnp D protein isoforms and hnrnpd shrn sequence alignment with hnrnpd cdns. () Western blot analysis of hnrnpd isoforms in mouse liver tissue, mouse primary hepatocytes (MPH) and hepatoma cells. Protein abundances of hnrnp D / were quantified using the lpha View Software with normalization by signals of. Values are average of two samples. The relative signal intensity of hnrnp D in liver is expressed as 1. () hnrnpd shrn shows % alignment with all mouse hnrnp D isoforms and human hnrnp D V1.

5 Supplemental Figure III Normalized Luc ctivity 1.4 Hepa HepG mock p40 p42 C Normalized Luc ctivity (Fold of DMSO) mock p40 p42 mock HNRNPD DMSO R (40uM) p40 control CMV-Flag (mock) Flag- Flag-p40 Flag-p42 Flag- p42/40 Supplemental Figure III: Exogenous expression of hnrnp D isoforms reduced LDLR3 UTR reporter activity in hepatic cells. () Hepa 1-6 and HepG2 cells were cotransfected with LDLR3 UTR luciferase reporter and indicated hnrnp D isoform expression plasmids or the control vector (mock). prl-sv40 was cotransfected in each condition for normalization of transfection efficiency. In each cell line, the normalized luciferase activity in mock-transfected cells is expressed as 1. p<01 as compared to the mock transfection. The graph is representative of two independent transfection experiments. () Huh7 cells were transfected with flag-tagged hnrnp D isoforms or the empty vector. Cell lysates from transfected or untransfected control cells were analyzed for isoform expression using anti-flag antibody. (C) HepG2 cells were cotransfected with LDLR3 UTR luciferase reporter and indicated hnrnp D isoform expression plasmids or the control vector (mock). One day post transfection, cells were treated with R or DMSO for 24 h before cell lysis. prl-sv40 was cotransfected in each condition for normalization of transfection efficiency. The normalized luciferase activity in mock-transfected cells without R treatment is expressed as 1. The graph is representative of two independent transfection experiments in which six wells were used for each condition.

6 Supplemental Figure IV Relative LDLRmRN levels C h Relative LDLR mrn levels 4h DMSO h R control anti-mir-130b anti-mir h DMSO 8h R 24h DMSO anti-mir-615-5p anti-mir-671-5p anti-mir-760 anti-mir-1201 anti-mir-1826 anti-mir-223 anti-mir h R D Normalized luciferase activity anti-mir control anti-mir-130b LDLR LDLR pluc pluc-ldlr3'utr anti-mir-1979 anti-mir-615-5p anti-mir-671-5p anti-mir-760 anti-mir-1201 anti-mir-1826 anti-mir-223 anti-mir-1244 anti-mir control anti-mir-130b anti-mir-1979 anti-mir-615-5p anti-mir-671-5p anti-mir-760 anti-mir-1201 anti-mir-1826 anti-mir-223 anti-mir-1244 SSY #1 SSY #2 Supplemental Figure IV: Evaluation of mirns in LDLR3 UTR reporter activity, LDLR mrn and protein expression in HepG2 cells () HepG2 cells were treated with R for indicated times. Total RN was isolated for qpcr analysis of LDLR and GPDH mrn expressions. fter normalization with GPDH mrn levels, the relative levels are presented, and the results are means ± SE of triplicate measurements for each cdn sample. p < 01 compared to DMSO. () HepG2 cells were cotransfected with pluc control vector or LDLR3 UTR luciferase reporter and indicated anti-mirn oligos or the control oligo (mock). prl-sv40 was cotransfected in each condition for normalization of transfection efficiency. The graph is representative of two independent transfection experiments in which six wells were used for each condition. (C-D) HepG2 cells were transfected with indicated anti-mirn synthetic oligonucleotides for three days before isolation of total RN or toaly cell lysates for analyses of LDLR mrn and protein expression levels.

7 Supplemental Figure V HepG2 Hepa1-6 H9C2 7R5 Lading Y1 MPH Supplemental Figure V: Measurement of cyclin D1 mrn decay rates in various cell lines and mouse primary hepatocytes (MPH). Cells were treated with actinomycin D (5 µg/ml) for different intervals. Total RN was isolated and analyzed for the amounts of cyclin D1 mrns by qrt-pcr. The cyclin D1 mrn levels were plotted as the percentage of the mrn remaining. Decay curves were plotted versus time.