Supplementary Figure 7: Evaluation of the phenotypic effect of ARLNC1 in vitro.

(a) Knockdown efficacy of three independent siRNAs targeting ARLNC1 in MDA-PCa-2b cells. Mean ± s.e.m. are shown, n = 3. ***P = 0.0001 determined by ANOVA with Dunnett correction. (b) ARLNC1 siRNA transfection has no effect on cell proliferation in AR-negative prostate cancer cells, PC3. This serves as an additional negative control for Figure 8a. Mean ± s.e.m. are shown, n = 6. P value not significant (ns, P = 0.7207) compared to si-NT treated cells, by one way ANOVA analysis. (c) Increased apoptosis observed in MDA-PCa-2b and LNCaP cells 48 hours after transfected with ARLNC1 siRNAs. ARLNC1-negative PNT2 cells serve as negative control. Fold change of apoptosis was calculated relative to si-NT treated samples. n = 3 independent cell cultures. Mean ± s.e.m. are shown. P values determined by two-tailed Student’s t-test. (d) Positions of ARLNC1 ASO-targeting sites (1 to 6) is indicated on the schematic representation of the ARLNC1 transcript. (e) MDA-PCa-2b cells were transfected with six independent ASOs targeting ARLNC1. Knockdown efficacy was evaluated by qPCR analysis. Mean ± s.e.m. are shown, n = 3. ***P = 0.0001 determined by ANOVA with Dunnett correction. (f) Correlation analysis of siRNA-mediated knockdown and ASO-mediated knockdown of ARLNC1 among replicated microarray experiments in MDA-PCa-2b cells (n = 2 biological replicates per ASO treatment group and n = 3 biological replicates per siRNA treatment group). (g) Free-uptake efficacy of ARLNC1 ASOs was examined in MDA-PCa-2B cells 72 hours post ASO addition to the culture medium (10 μM). ARLNC1 expression was evaluated by qPCR analysis. Mean ± s.e.m. are shown, n = 3. ***P = 0.0001, **P = 0.0032; determined by ANOVA with Dunnett correction. (h) Free-uptake treatment of ASOs targeting ARLNC1 resulted in retarded growth of MDA-PCa-2b cells in vitro. ARLNC1-negative prostate cell line PNT2 served as a negative control. Mean ± s.e.m. are shown, n = 6. P values were determined by two-tailed Student’s t-test (ns: not significant). (i,j) ARLNC1 ASOs inhibit MDA-PCa-2b cell proliferation in 3D sphere models. Cells were harvested at the end of the experiment and ARLNC1 expression was evaluated by qPCR analysis. Mean ± s.d. are shown, n = 6. ***P < 0.0001 compared to control ASO treated cells, by two-tailed Student’s t-test.