miR-210 and myogenic differentiation

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Table S1 PCR primers for mRNA quantification

Gapdh FWD 5’- TGCCAAGTATGATGACATCAAGAAG - 3’ Gapdh REV 5’- GGTCCTCAGTGTAGCCCAAGAT - 3’ Rpl13 FWD 5’- CTCGGCCGTTCTCCTGTAT - 3’ Rpl13 REV 5’- GTGGAAGTGGGGCTTCAGTA - 3’ Myod FWD 5’- ATGCTGTCTTTTCCCCAAA - 3’ Myod REV 5’- AACAAGCATGCCAGGATACA - 3’ Myog FWD 5’- CCTTGCTCAGCTCCCTCA - 3’ Myog REV 5’- TGGGAGTTGCATTCACTGG - 3’ Myh2 FWD 5’- AGAGCAAAGATGCAGGGAAA - 3’ Myh2 REV 5’- TAAGGGTTGACGGTGACACA - 3’ Des FWD 5’- GCCACCTACCGGAAGCTACT - 3’ Des REV 5’- GCAGAGAAGGTCTGGATAGGAA - 3’ Table S2

PCR primers for ChIP assay miR-210 promoter fragment containing HRE3 and 4 FWD 5’- CACAGCCAGATGGACACACATC - 3’ REV 5’- GCTCCCCTTCGGGAACTCATA – 5’ miR-210 promoter fragment containing HRE1 and 2 FWD 5’- CAGGGAGACACTGTCCAGCG -3’ REV 5’- CTGAAGGGCTGCAGCCAGTG -3’ Table S3

PCR primers for miR-210 promoter luciferase plasmids generation Universal REV

5’- CCCAAGCTTGGGCTCATCTCCTCTAACTTG - 3’

Construct A FWD

5’- GAAGATCTTCAGAGAAGTGAGTGAGCTGGG - 3

Construct B FWD

5’- GAAGATCTTCAGGATCTCCAGGCAAGTA - 3’

Construct C FWD

5’- GAAGATCTTCATGGTGTCCCTGGCCT - 3’

Construct D FWD

5’- GAAGATCTTC CAGGGAGACACTGTCCAG - 3’

Construct E FWD

5’- GAAGATCTTCTCGCCCGCGAAGGGTTGC - 3’

Construct F FWD

5’- GAAGATCTTCCCGCAGCTGAAGTTGGGC - 3’

Construct Myod mut1

5’- GAAGATCTTCAGCCCCGCCCAGTGGGTGGCGGCCCCT - 3’

Construct Myod mut2

5’- GAAGATCTTCAGCCCCGCCCAGCGGGAGGCGGCCCCT - 3’

Mutated sequences are underlined

miR-210 and myogenic differentiation

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Table S4

PCR primers for miR-210 promoter methylation study FWD 1 5’ – GAGGAAAAGGTATGTTTTGGGTTGTA – 3’ REV 1 5’ - CCCCCACCCTCATCTCCTCTAACTT – 3’ FWD 2 5’ - GAGGAAAAGGTATGTTTTGGGTTGTA – 3’ REV 2 5’ -CCCCATATACAAATAATCTATCTT – 3’ FWD 3 5’ - AAGATAGATTATTTGTATATGGGG – 3’ REV 3 5’ - CCCCCACCCTCATCTCCTCTAACTT – 3’

miR-210 and myogenic differentiation

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SUPPLEMENTARY FIGURE LEGENDS Figure S1. miR-210 expression is induced upon rat and quail myoblast differentiation. L6C5 rat myoblasts were cultured in GM and then switched to DM for the indicated time. Then, RNA was extracted and miR-210 (A) and miR-1 (B) levels were measured by qPCR (n=4; # p< 0.02; ** p<0.01; *** p<0.001). Polyclonal populations of quail myoblasts transformed by the temperature-sensitive mutant of the Rous Sarcoma Virus LA29 were grown in GM conditions (high serum and 35°C allowing src activity) and moved to DM conditions (low serum and 41°C, inactivating src) for 48 hrs. Then, RNA was extracted and miR-210 (C) and miR-1 (D) levels were measured by qPCR (n=3; *** p<0.001). Figure S2. A specific siRNA knocks-down Hif1a efficiently and does not affect skeletal muscle markers. C2C12 were transfected with a Hif1a specific siRNA (Hif1a siRNA) or with a control scramble (SCR) sequence (n=8). Then, cells were allowed to differentiate in DM for 24 and 48 hrs, and the indicated mRNA and miRNA levels were measured by qPCR. Average values are expressed as fold change in a log2 scale. Green and red colors indicate down- or up-regulation, respectively. Hif1a silencing significantly decreased Hif1a transcript levels at both differentiation timepoints (p<0.01). Differentiation was monitored detecting the following markers by qPCR: miR-1, myogenin (Myog), Myod and Myosin Heavy Chain (Myh2). Differences observed between control and Hif1a silenced cells, at each timepoint, were not statistically significant. The inset shows miR-1 levels using a different scale. Figure S3. A specific shRNA knocks-down Hif1a efficiently, attenuates miR-210 hypoxic induction and does not affect miR-1. C2C12 myoblasts were infected with lentiviral vectors expressing either a Hif1a specific shRNA (Hif1 shRNA) or a scramble sequence (SCR) and, after puromycin selection, RNA was extracted. A) Hif1a mRNA levels were analyzed by qPCR. (n=4; * p<0.05). Hif1a knockdown significantly decreased Hif1a transcript levels. B) miR-210 levels were measured in both normoxic (black bars) and hypoxic (grey bars) myoblasts. Hif1a silencing significantly decreased miR-210 levels (n=4; ** p<0.01). C) miR-210 and miR-1 (D) levels were measured in C2C12 myoblasts (black bars) and in cells switched to DM for 48 hrs (white bars). Hif1a shRNA significantly decreased miR-210 levels, while miR-1 was unaffected (n=4; ** p<0.01; ns = not significant). Figure S4. miR-210 promoter mutational analysis. A) miR-210 promoter fragments were cloned into the pGL2-luciferase vector. Serial deletion constructs (identified by the letters A–F) are schematically represented above the bar graph. Potential HREs are shown. Lower panel: GM C2C12 cells were cotransfected with reporter vectors and a plasmid encoding Hif1a or vector alone. The relative promoter activities are displayed. Empty pGL2-basic vector was used to monitor basal activity of the luciferase reporter gene (basic). Luciferase activity is expressed as firefly/renilla luciferase ratio. Constructs A-D display similar expression levels in the presence of Hif1a and activity drops significantly in E and F constructs (n=3; ***p<0.001). B) Upper panel: The two proximal HREs were mutated either alone (D mut 1 and D mut 2), or in combination (D mut 1+2). Lower panel: Promoter activity was detected by luciferase assays in GM C2C12 cells cultured either in normoxia or hypoxia for 24 hrs. HRE2 mutation significantly decreased D construct activity in hypoxia, while HRE1 mutation was ineffective (n=3; ** p<0.01; # p< 0.02). C). o different constructs were generated to mutate a potential Myod binding site 523 nucleotides upstream of mature miR-210 (Myod mut1 and Myod mut2). Luciferase assays were performed in C2C12 cells cultured either in GM or in DM for 24 hrs. Results show that these mutations did not affect miR-210 promoter expression, either in GM or in DM (n=6; ns= not significant). D) Bioinformatic analysis of the ≈2.4 kbases upstream of mouse miR-210 revealed the presence of a potential CpG island from -548 bp to -280 bp upstream mature miR-210. Figure S5. miR-210 blockade does not affect myogenic differentiation. C2C12 were transfected with anti-miR-210 or a control scramble sequence (SCR) in GM and then switched to DM for the indicated time (n=8). Next, total RNA was extracted and the expression of miR-210, -1, 133a and -133b, myogenin (Myog), Myod, and desmin (Des) was measured by qPCR. Average values are expressed as fold change in a log2 scale. Green and

miR-210 and myogenic differentiation

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red colors indicate down- or up-regulation, respectively. Insets show miR-1 and miR-133a levels using a different scale. miR-210 blockade significantly decreased miR-210 levels at all differentiation timepoints (p<0.001). Differences in the expression levels of all myogenesis markers tested in C2C12 cells treated with anti-miR-210 compared with SCR are not statistically significant. Figure S6. miR-210 blockade does not affect myoblast fusion. C2C12 were transfected with anti-miR-210 or a control scramble sequence (SCR) in GM and then switched to DM for 48 hrs (n=5). Cells were stained for MHC and the number of nuclei/myofiber was evaluated (A). B) The bar graph shows myoblast fusion index. Differences between SCR and anti-miR-210 are not statistically different. Figure S7. No evidence that miR-210 is necessary for skeletal muscle regeneration. Anti-miR-210 or a control scramble LNA sequence (SCR) were injected in the tail vein of CD1 mice. Two days after LNA administration, tibialis anterior muscle injury was induced by CTX. A) miR-210 levels in both CTX treated and contralateral muscles 2 days after CTX injection. Anti-miR-210 decreased miR-210 levels efficiently (n=3; *** p< 0.001). B) CTX induced damage measured by EBD assay. CTX induced damage was similar in anti-miR-210 and SCR mice (n=3; ns= not significant). C) The number of centrally nucleated regenerating myofibers was assayed by histological analysis of H&E stained sections 7 and 14 days after CTX injection. A similar number of regenerating myofibers was observed in anti-miR-210 and SCR mice, at both timepoints (n=6; ns= not significant). D) The indicated mRNA and miRNA levels were measured by qPCR (n=6). Average values are expressed as fold change in a log2 scale. Green and red colors indicate down- or up-regulation, respectively. Anti-miR-210 significantly decreased miR-210 levels both at 7 and 14 days timepoints (p<0.005). Differences in miR-206 and myogenin regeneration markers observed between anti-miR-210 and SCR CTX-treated muscles were not statistically significant. Figure S8. miR-210 increases myotube survival upon antimycin A and rotenone treatment. C2C12 myoblasts transfected with anti-miR-210 or a control scramble sLNA equence (SCR) were cultured for 24 hrs in DM and then treated with 6µM antimycin A (A and B) or 2µM rotenone (C and D). After 8, 16 and 24 hrs of treatment, MHC immunofluorescence was performed (A and C) and myotubes were counted (B and D). The bar graphs show that miR-210 blockade significantly decreased myotube survival when mitochondrial function was impaired (n=3; * p<0.05; ** p<0.01; *** p<0.001). Calibration bar=30µM.

miR-210 and myogenic differentiation

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Figure S1

miR-210 and myogenic differentiation

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Figure S2

miR-210 and myogenic differentiation

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Figure S3

miR-210 and myogenic differentiation

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Figure S4

miR-210 and myogenic differentiation

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Figure S5

miR-210 and myogenic differentiation

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Figure S6

miR-210 and myogenic differentiation

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Figure S7

miR-210 and myogenic differentiation

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Figure S8