Fig. 3: Targeting HMMR inhibits PCa cell growth and survival.

Knockdown of HMMR with sMARTpool siRNA (siHMMR) reduced proliferation of LNCaP (a) and MR49F (b) cells, as determined by CyQuant assay. Cell proliferation was evaluated by measure of fluorescence intensity on days 3-, 4-, 5- and 6 post-transfection. Data are presented as mean ± SD of 5 wells and represent three independent experiments. Data were statistically evaluated using two-way ANOVA with Tukey’s multiple comparison test (****p < 0.0001). c HMMR knockdown induced apoptosis of LNCaP and MR49F cells, as determined using flow cytometry-based 7-AAD/Annexin V assays at 3 days post-transfection. Data are presented as mean ± SD of 3 biological replicates and represent three independent experiments. Dead cell proportions upon HMMR knockdown were compared to the vehicle using ANOVA and Dunnett’s multiple comparison tests (^ap<0.0001, ^bp < 0.001, ^cp < 0.01). HMMR knockdown with siHMMR inhibits colony formation of LNCaP (d) and MR49F (e) cells. Formalin-fixed cells were stained with 1% crystal violet then colonies containing >50 cells were manually counted. Data are presented as mean ± SD of 3 wells and represent three independent experiments. Data were statistically analysed using unpaired student’s T-test (**p < 0.01, ****p < 0.0001). (f) Pharmacological inhibition of HMMR with 4-MU inhibits LNCaP cell viability. Viable and dead cells were determined by Trypan blue exclusion assay after 3- and 6- days of treatment. Data are presented as mean ± SD of triplicate wells, are representative of two independent experiments, and were analysed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, ****p < 0.0001). g 4-MU dose-dependently suppresses colony formation in LNCaP cells. Data are presented as mean ± SD of 3 wells, are representative of two independent experiments, and were analysed using one-way ANOVA with Dunnett’s test. (**p < 0.01, ***p < 0.001, ***p < 0.0001). h 4-MU dose-dependently suppresses cell proliferation in V16D and MR49F PCa cells. Cells were counted using the Trypan blue dye exclusion method after 3- and 6- days of treatment. Data are presented as mean ± SD of triplicate wells and represent two independent experiments. Statistical analysis was performed using one-way ANOVA with Turkey’s multiple comparison test. (****p < 0.0001). i 4-MU treatment dose-dependently induces apoptosis in MR49F cells three days post-treatment. Data are presented as mean ± SD of 3 wells, represent two independent experiments and were analysed by two-way ANOVA with Turkey’s multiple comparison test (^ap < 0.0001, ^cp < 0.01, ^dp < 0.05. j 4-MU inhibits proliferation in patient-derived prostate cancer explants (PDEs). PDEs (n = 7) were treated for 48 h with an increasing dose of 4-MU, then paraffin-embedded and formalin-fixed prior to immunohistochemistry (IHC) with proliferation marker Ki67. Digital images were manually counted. Data represents mean ± SEM and were statistically analysed using one-way ANOVA with Dunnett’s test (*p < 0.05, **p < 0.01). Quantification of Ki67 staining on the left and representative IHC images on the right.