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. 2016 Jun 21;7(32):51965–51980. doi: 10.18632/oncotarget.10207

Figure 4. Effects of miR-99a and 100 on DNA repair processes are regulated by SMARCA5 and SMARCD1.

Figure 4

A. Representative western blot analysis of SMARCA5 and SMARCD1 expression in miR-inhibitor transfected malignant CB cells. B. Immunofluorescence staining for nuclear SMARCA5 and SMARCD1 in miR-99a and 100-inhibitor transfected CB cells, 5 minutes after exposure to 5-Gy radiation (n=5 PCa). Scale bar: 100 uM. Right panel shows the quantification of SMARCA5 and SMARCD1 positive CB cells, >250 cells/sample counted and SMARCA5 and SMARCD1 fluorescence quantification using Velocity quantitation software. C. Colony forming experiments of CB cells transfected simultaneously with miR-99a inhibitor and SMARCA5 or SMARCD1 endoribonuclease-prepared siRNA (esiRNAs) following 5-Gy radiation show a rescue of the effects mediated by miR-99a inhibitor alone (n=5 PCa, each sample is in triplicate). D. Quantification of nuclear pan-histone 3-acetylation immunofluorescence staining by Velocity Quantitation software in miR-99a inhibitor and SMARCA5 or SMARCD1 esiRNAs transfected CB cells. Immunofluorescence staining was performed 30 minutes after exposure to 5-Gy radiation (n=3 PCa, each sample in triplicate). n indicates total number of cells included in quantification analysis. E. Quantification of nuclear BRCA1 and RAD51 in CB cells simultaneously transfected miR-99a inhibitor and SMARCA5 or SMARCD1 esiRNA. Immunofluorescence staining was performed 120 minutes after exposure to 5-Gy radiation (n=3 PCa, each sample in triplicate), >250 cells/sample were counted. F. Quantification of nuclear SMARCA5 and SMARCD1 in CB cells simultaneously transfected with miR-99a inhibitor and treated with or without the PARP1 inhibitor nicotinamide. Immunofluorescence staining was performed 120 minutes after exposure to 5-Gy radiation (n=5 PCa, each sample in triplicate), >250 cells/sample were counted. G. Quantification of nuclear SMARCA5 and SMARCD1 in CB cells simultaneously transfected with miR-99a inhibitor and PARP1 esiRNA. Immunofluorescence staining was performed 120 minutes after exposure to 5-Gy radiation (n=5 PCa, each sample in triplicate), >250 cells/sample were counted. H. Colony forming experiments of CB cells transfected with SCRMBL or PARP1 esiRNA or treated with nicotinamide following by 5-Gy radiation showing a rescue of the effects mediated by miR-99a inhibitor alone (n=5 PCa, each sample in triplicate). Data are expressed as mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's ttest).