Panel A shows a vertical bar graph of relative SIRT1 messenger RNA expression in LNCaP cells containing control short hairpin RNA or silenced for SIRT1 using two independent short hairpin RNAs, shSIRT1 number 1 and shSIRT1 number 2. The y-axis represents relative SIRT1 messenger RNA expression, and the x-axis shows Day 0 and Day 7 conditions. Panel B shows a vertical bar graph of relative SIRT1 messenger RNA expression in 22RV1 CRISPR activation cells containing single guide control or two independent single guides activating SIRT1, sgSIRT1 number 1 and sgSIRT1 number 2. The y-axis represents relative SIRT1 messenger RNA expression, and the x-axis shows different experimental conditions. Panel C shows a line graph of cell proliferation measured by methyl thiazolyl tetrazolium absorbance over time from 0 to 80 hours. The y-axis represents optical density at 560 nanometers, and the x-axis represents time in hours. Panel D shows representative images of 22RV1 prostate tumors with immunostaining for SIRT1, insulinoma-associated protein 1, synaptophysin, and androgen receptor. Panel E shows crystal violet stained images of colony formation assays with single guide control, sgSIRT1 number 1, or sgSIRT1 number 2. Panel F shows a bar graph of the relative number of colonies. The y-axis represents the relative number of colonies, and the x-axis shows different experimental conditions. Panel G shows a schematic diagram illustrating induction of neuroendocrine prostate cancer in LNCaP cells using dibutyryl cyclic adenosine monophosphate and isobutylmethylxanthine treatment. Panel H shows images of relative lysine acetylation levels following treatment of LNCaP cells with 5 or 10 micromolar Selisistat. Panel I shows heatmaps representing relative expression levels of the SIRT1 regulon and markers associated with neuroendocrine prostate cancer or androgen signaling.
Additional analyses of validation studies in human cells (related to Figs. 6 and 8 ). (A and B) Real-time PCR showing relative expression of SIRT1 in human prostate cancer cell lines. Data are normalized using GAPDH as internal control. (A) LNCaP cells with control shRNA or silenced for SIRT1 with two independent shRNA (shSIRT1#1 or shSIRT1#2). (B) 22RV1 CRISPRa cell line with sgControl or two independent sgs to activate SIRT1 (sgSIRT1#1 or sgSIRT1#2). (C–E) Analyses of activation of SIRT1 in 22RV1 CRISPRa cells in vitro (C and E) and in vivo (D). (C) Quantification of cell proliferation from 0 to 72 h of growth measured by detection of MTT absorbance. OD, optical density. (D) Representative images of 22RV1 prostate tumors (n = 6/group). Shown is immunostaining for SIRT1, INSM1, synaptophysin (SYP), and AR. Scale bars represent 20 µm. (E and F) Crystal violet staining of colony formation assay showing 22RV1 CRISPRa cell lines treated for 12 days with sgControl, sgSIRT1 #1 or #2 (E), and quantification of the number of colonies (F). (G–I) Pharmacological inhibition of SIRT1 in LNCaP cells. (G) Strategy for induction of NEPC in LNCaP cells by treatment with db-cAMP and IBMX (abbreviated +cAMP). Treatment with the SIRT1 inhibitor Selisistat is predicted to inhibit this transition. (H) Relative levels of lysine acetylation following treatment of LNCaP cells with 5 or 10 µM Selisistat. Scale bars represent 20 µm. (I) Heatmap representation of relative expression levels of the SIRT1 regulon (top) and known markers of NEPC or androgen signaling (bottom). Experiments were done in triplicate with two independent biological replicates.