Supplementary Figure 1: Silibinin has no considerable radiosensitizer effect on radioresponsive prostate cancer 22RV1 cells. (A) Effect of IR and SB on 22RV1 cell proliferation after 48 h of treatment. Exponentially growing 22RV1 cells were treated either with 2.5 or 5 Gy of IR and/or 25 μM SB. After 48 h of these treatments, cells were collected and total cells were counted using a hemocytometer. (B) Clonogenic assay was done with 22RV1 cells treated with 2.5 Gy IR alone or in combination with 25 μM SB. The number of colonies with greater than 50 cells were counted in each group at the end of 10 days. SB, silibinin; n.s., not significant. Number of colonies > 50 cells Control 25 μM SB 2.5 Gy IR + SB * * * B n.s. Total Cell Number X 10 4 Control 25 μM SB 2.5 Gy 2.5 Gy + SB 5 Gy 5 Gy + SB * * * * * A n.s.

Supplementary Figure 2. Silibinin potentiates and prolongs G2/M arrest by IR exposure in DU145 cells. For cell cycle analysis, DU145 cells were exposed to IR (5 Gy) with or without silibinin (25  SB . After 24 and 48 h of treatments, cells were processed for saponin/PI staining. Representative histogram showing a prominent G2/M arrest in combination at 24 and 48 h in DU145 cells. The quantitative data is shown as figure 2A. Control 25  M SB 5 Gy IRIR+SB 24 h 48 h

IR+SB Control SB IR Control PC-3 DU145 Survivin PCNA B IR+SB SB IR IR+SB Control SB IR Control PC-3 DU145 IR+SB SB IR A Cyclin B1 Cdc-2 IR+SB Control SB IR GAPDH Cdc25C DU145 Supplementary Figure 3. Silibinin decreases the molecules regulating G2/M phase transition in PCa cells, but not in HEK-293 cells in response to IR exposure, and inhibits cell proliferation and survival. (A) mRNA expression levels of G2/M phase cell cycle regulatory molecules after 48 h of treatment with IR (5 Gy) and /or SB (25 μM) in DU145 and HEK-293 cells. GADPH was used as loading control. (B) Densitometric quantitation data showing the fold change in the protein levels of PCNA and survivin (from figure 2D) after indicated treatments in DU145 and PC-3 cells. GAPDH Cdc25C Cyclin B1 Chk-2 IR+SB Control SB IR HEK-293

48 h control 25 SB 5 Gy + 25 SB 5 Gy DU145 HEK-293 Counts ROS induced Fluorescence (FL1H) Counts 12 h24 h 48 h 72 h control SB IR+SB IR DCFH-DA – FL1 Channel Catalase GST SOD1 SOD2 GAPDH IR+ SB Control SB IR A control SB IR+SB IR control SB IR+SB IR control SB IR+SB IR B Supplementary Figure 4. Silibinin enhances IR-induced oxidative stress. (A) Representative overlap histograms showing ROS levels using DCF-DA treatment of DU145 cells with IR (5 Gy) and/or SB (25 μM silibinin) following h. (B) Representative microscopic pictures showing DCF positive cells (green) in different treatment groups. (C) RT-PCR analysis of antioxidant enzymes in response to IR and/or SB treatment after 48 h. (D) Histogram showing ROS levels in DU145 and HEK-293 cells after 24 h of treatment. C Control SB IR+SB IR Control SB IR IR+SB D

Control SB IR IR+ SB Sytox Green pEGFR(Y1068) Merge 3h Post-treatment - JB6 cells Supplementary Figure 5. Silibinin does not inhibit IR-induced nuclear translocation of EGFR in JB6 cells. Confocal microscopy showing distribution of EGFR (red) in JB6 cells in response to IR (5 Gy) and/or SB (25 μM) at 3 h of treatment. Sytox green (green) was used as nuclear counter stain.

IR SB IR+SB Control Mean GUT Weight (mg/mouse) GUT: Genitourinary Tract Mean Neutrophil Count (K/µL) Mean Monocyte Count (K/µL) Mean RBC Count (M/ul) Mean Hb Level (M/ul) # # $ $ A B C D E Supplementary Figure 6. Silibinin protects normal tissues against radiation injury in mice. (A) Representative pictures and graph showing discoloration, and shrinkage of the genitourinary tract (GUT) by radiation treatment which is being rescued by silibinin. (B- E) Effect of radiation and/or silibinin on (B) mean neutrophil number, (C) mean monocyte number, (D) mean Hb Levels and (E) mean RBC counts in athymic nude mice after cumulative 15 Gy (IR) and/or 200 mg/kg body weight of SB treatment.