Additional file 1: Figure S1. of ING3 promotes prostate cancer growth by activating the androgen receptor

The four panels show representative images of a line of C4-2 cells stably infected with inducible lentiviral shCtrl or shING3, with or without Dox. The western blots show the efficiency of ING3 knockdown in the presence or absence of Dox. Figure S2. (A) Lysates from three AR-positive prostate cancer cell lines were subject to western blotting with antibodies against ING3, GAPDH, and actin. (B) mRNA levels of ING3 were normalized to actin in three prostate cancer cell lines. (C) LNCaP, C4-2, and VCaP cells were grown in media with charcoal stripped serum (CSS) for 48 h and treated with mibolerone (MB) or bicalutamide (Bic). Protein levels of ING3 and AR were visualized by western blotting with actin used as a loading control. (D) qRT-PCR study of ING3 in LNCaP cells after treatment with increasing concentrations of MB. The left graph shows mRNA levels of ING3 in response to MB. The right graph shows mRNA levels of seven androgen-regulated genes in response to MB. (E) A cycloheximide experiment using LNCaP cells grown in the presence or absence of MB to estimate ING3 half-life. Figure S3. (A) HEK293T cells were co-transfected with 1 μg of Myc-tagged AR and 1 μg of either empty vector or HA-tagged ING3 +/– 10 nM MB. ING3 was pulled down with HA-affinity beads, and precipitates were blotted with α-AR and α-HA. (B) To determine the effects of DNA on the interaction, co-immunoprecipitations were repeated with addition of ethidium bromide (EtBr). ING3 was precipitated using HA-affinity beads. Figure S4. LNCaP cells were infected with shCtrl or shING3 lentiviral particles for 72h under androgen deprived conditions and stained with anti-Ki67. Arrows indicate infected (RFP-positive) cells with associated Ki67 staining. RFP-positive and Ki67-positive cells were counted and percentages are shown in the table. Figure S5. ING3 affects PC migration. (A) LNCaP, PC3, and DU145 cells were transfected with either siCtrl or siING3 and, in case of LNCaP, treated with 1 nM MB for the times indicated. Transwell migration assays were performed at the indicated time points. Representative images are shown. (B) Images were taken from six random fields for each condition and counted manually on a computer using a blind experimental protocol (t test * < 0.05, ** < 0.01). (C) Wounds were made in monolayers of C4-2 cells stably expressing either shCtrl or shING3 in the presence of 10 nM MB and Dox to induce shRNA expression. Wounds were then allowed to heal during a course of 4 days. Images were taken from the same fields for each condition. Percentage of healed wound was then calculated based on pixels observed in each condition. (D) LNCaP cells were transfected with siCtrl or siING3 and treated with 1 nM MB for 72 h, then fixed and stained with Texas Red-conjugated phalloidin. Arrows highlight actin projections along cell axes consistent with filopodia formation. (E) The numbers of actin projections per cell were counted in a blind experimental protocol from a total of 50 cells, and the mean number of filopodia/cell was plotted (t test *** < 0.001). Figure S6. Representative images of prostate cancer samples showing low and high expression of AR as determined by immunohistochemistry. Samples are from the prostate cancer patient cohort used in this study. Figure S7. Kaplan-Meier survival curves using Gleason score as a known prognostic marker in the derivation and validation datasets. Figure S8. Kaplan-Meier survival curves in our prostate cancer patient cohort with low levels of AR, in the derivation and validation datasets. (PPT 4506 kb