Supplemental Figure 1. Mir-99 Family Induction Following IR, and Mir-99A and Sisnf2h Reduce

Supplemental Figure 1.miR-99 family induction following IR, and miR-99a and siSNF2H reduce BRCA1 foci by reducing SNF2H. (A)miR-99a, miR-99b, and miR-100 expression measured by qPCR, normalized to β-u6snRNA, 24 hours following IR treatment in MCF7 cells. Mean + SD (n=6)(B)miR-99a and miR-100 expression measured by qPCR, normalized to GAPDH, 24 hours following IR treatment in LNCaP cells. Mean + SD (n=3).(C) Immunofluorescence showing BRCA1 foci following 5 gy irradiation in C4-2 cells transfected with siGL2, miR-99a, or siSNF2H. (D) Immunofluorescence showing BRCA1 andγH2AX foci 1 hr following 0 gy (-IR) or 5 gy (+IR) irradiation in C4-2 cells infected with control PMSCV virus, or virus expressing the SNF2H ORF. The cells were transfected with siGL2, miR-99a or siRNA targeting the 3’ UTR of SNF2H.

Supplemental Figure 2.miR-100 reduces BRCA1 foci formation following IR. (A) Immunofluorescence showing BRCA1 foci following 5 gy irradiation in C4-2 cells transfected with miR-100 or siRNA targeting GL2. (B) Quantitation of panel A. At least 75 cells were counted per sample. Mean + SE (n=2).

Supplemental Figure 3.miR-99a and siRNA targeting SNF2H do not reduce foci containing MDC1 or conjugated ubiquitin. (A) Immunofluorescence showing MDC1 foci following 5 gy irradiation in C4-2 cells transfected with siRNA targeting GL2, SNF2H, or a double stranded mimic of miR-99a. (B) Immunofluorescence showing conjugated ubiquitin foci using FK2 antibody following 5 gy irradiation in C4-2 cells transfected with siRNA targeting GL2, SNF2H, or a double stranded mimic of miR-99a.

Supplemental Figure 4. The miR-99 family and siSNF2H reduce Rad51 foci formation following ionizing radiation.Immunofluorescence showing Rad51 foci formation 90 minutes following 0 gy or 5 gyirradiation in C4-2 cells transfected with siGL2, miR-99a, miR-100, or siSNF2H.

Supplemental Figure 5.miR-99a, miR-100 and SNF2H reduce the rate of DNA repair following ionizing radiation. (A) Neutral comet assays of control PMSCV infected C4-2 cells transfected with siGL2, miR-99a, or siRNA targeting the 3’UTR of SNF2H. Cells were treated with 10 gy IR and collected at the indicated time points. (B) Neutral comet assays of C4-2 cells infected with PMSCV virus expressing the SNF2H ORF and transfected with siGL2, miR-99a, or siRNA targeting the 3’UTR of SNF2H. Cells were treated with 10 gy IR and collected at the indicated time points.(C)miR-100 slows DSB repair in C4-2 cells. Quantitation of comet assays performed in C4-2 cells transfected with siRNA targeting GL2 or a double stranded mimic of miR-100. X axis indicates time following treatment with 10 gy of IR.

Supplemental Figure 6.Induction of miR-99 family following irradiation impairs DNA repair foci formation when facing subsequent DNA damage. (A) Immunofluorescence showing BRCA1 and pH2AX foci formation in C4-2 cells treated with 2’-O-methyl anti-GL2 or 2’-O-methyl anti-99 when unirradiated, treated with 2 gy, or treated with 2 gy, incubated for 24 hours and treated once more with 2 gy IR.(B)Reduction in miR-99a and miR-100 by 2’-O-methyl anti-99. qPCR analysis of miR-99a and miR-100 levels in LNCaP cells following transfection with 2’-O-methyl anti-miR-99. Mean + SD of 3 measurements.(C)qRT-PCR of SNF2H expression normalized to GAPDH in LnCAP cells either left untreated(no pre), or pretreated with 2 gy IR and incubated with 2’-O-methyl antisense oligonucleotides targeting GL2 negative control(anti-GL2) or miR-99a and miR-100(anti-99) for 24 hours prior to subsequent treatment with 2 gy IR.