Fig 2: Gli2 knockdown inhibits the growth of castration-resistant tumors in vivo. (A) LNCaP Gli2shR or LNCaP Ctl cells were inoculated subcutaneously into the right flank of male SCID mice (n=20 mice per group). After 2-3 weeks, mice with a tumor burden >100 mm3 in volume (volume=0.5 × length × width2) were recruited into the study groups (n=10 mice per group). (B) Average tumor volume in the Ctl-DOX, Ctl+DOX, Gli2shR-DOX and Gli2shR+DOX subgroups. Data is presented as the mean ± SEM of tumors from five mice per group. *P<0.05, **P<0.01 and ****P<0.0001, GLI2-DOX vs. GLI2+DOX. (C) Final tumor weight was measured 60 days after castration when the mice were euthanized and their tumors were excised. Each dot represents one mouse. *P<0.05 and **P<0.01. (D) Mouse body weight was monitored following the initiation of DOX treatment. Percentage change in body weight relative to the value obtained on day 0 is presented. Data is presented as the mean ± SEM from five mice. *P<0.05 and **P<0.01, GLI2-DOX vs. GLI2+DOX. (E) Gli2 mRNA expression in tumors samples isolated from mice in each subgroup was measured using reverse transcription-quantitative PCR. Each dot represents the quantified value from one tumor sample. *P<0.05. (F) Representative images showing Gli2 immunohistochemical staining in tumor tissues isolated from the mice in each of the four groups. Scale bar, 50 µm. (G) Representative images showing Ki67 immunohistochemical staining in tumor tissues isolated from the mice in each of the four groups. Scale bar, 100 µm. (H) Tumor volume following DOX withdrawal. The percentage tumor volume relative to the value obtained one day prior to DOX treatment for each tumor was plotted in the figure. Data is presented as the mean ± SEM of tumor volumes obtained from three mice per group. *P<0.05 between day 40 and 62 after castration. ns, not significant; SCID, severe combined immunodeficient; DOX, doxycycline; Gli2, Glioma-associated oncogene family zinc finger 2; Ctl, control vector not encoding the shRNA; Gli2shR, vector encoding Gli2 short hairpin RNA.

Fig 3: Gli2 knockdown exerts additive inhibitory effects with Casodex in the anchorage-dependent and -independent growth of prostate cancer cells. (A) MTT cell viability assay was performed with LNCaP Ctl, LNCaP Gli2shR, 22RV1 Ctl and 22RV1 Gli2shR cells treated with DOX (0.1 µM or 1.0 µM) and/or 30 µM Casodex or equivalent control vehicle, DMSO. Cell viability was measured on days 2, 4, 6 and 8 by measuring absorbance at 595 nm. Data is presented as the mean ± SEM of absorbance of 3 wells per group. ****P<0.0001, Casodex vs. Casodex +0.1 DOX. Soft agar assay was performed with LNCaP Gli2shR, LNCaP Ctl, 22RV1 Ctl and 22RV1 Gli2shR cells following treatment with DOX (0.1 µM or 1.0 µM) and/or 30 µM Casodex. The number of colonies formed were then counted 15-20 days later. (B) Representative images of colonies formed by LNCaP Gli2shR and LNCaP Ctl cells. (C) Quantified data of the images shown in (B). (D) Representative images of colonies formed by 22RV1 Gli2shR and 22RV1 Ctl cells. (E) Quantified data of the images shown in (D). Data is presented as the mean ± SEM of three experimental replicates. *P<0.05, ***P<0.001 and ****P<0.0001. ns, not significant; OD, optical density; CAS, Casodex; DOX, doxycycline; Gli2, Glioma-associated oncogene family zinc finger 2; Ctl, control vector not encoding the shRNA; Gli2shR, vector encoding Gli2 short hairpin RNA.

Fig 4: Gli2 serves a significant role in cell cycle regulation. (A) Heatmap showing the significant DEGs following Gli2 knockdown in LNCaP cells. Whole transcriptome sequencing data of cell culture samples from the LNCaP GLI2shR + DOX group were compared against control samples of the other three experimental groups, with each condition performed in duplicate. In the color bar on the left, red indicates genes that are upregulated whereas blue indicates genes that are downregulated upon Gli2 knockdown. In the color bar on the top, the six control samples are marked red whereas the two knockdown samples are marked purple. Expressions of the genes were scaled so that red indicates high expression, whereas blue indicates low expression. (B) Gene Ontology analysis of the DEGs using Database for Annotation, Visualization and Integrated Discovery, which identified the top biological processes that were enriched following Gli2 knockdown. Each circle represents one significantly enriched GO term. The size of each circle is proportional to the number of genes that were enriched for the GO term. (C) Log2 FC of significant DEGs involved in cell cycle. The genes were identified using Qiagen's Ingenuity® Pathway Analysis and ranked by log2FC such that upregulated DEGs were on the left and downregulated genes on the right. FC, fold-change; DEGs, differentially expressed genes; Gli2, Glioma-associated oncogene family zinc finger 2; DOX, doxycycline; Ctl, control vector not encoding the shRNA; Gli2shR, vector encoding Gli2 short hairpin RNA; GO, Gene Ontology.

Fig 5: Gli2 knockdown reduces prostate cancer cell viability and induces cell cycle arrest at the G0/G1 stage. (A) LNCaP Gli2shR, LNCaP Ctl, 22RV1 Gli2shR and 22RV1 Ctl cells were treated without or with 1 µM DOX, following which cells were harvested for the measurement of Gli2 mRNA expression using reverse transcription-quantitative PCR and (B) protein expression using western blotting 48 h after treatment. (C) MTT cell viability assay was performed in LNCaP Ctl, LNCaP Gli2shR, 22RV1 Ctl and 22RV1 Gli2shR cells following treatment with or without 1 µM DOX for five days. (D) Cell cycle analysis was performed with LNCaP Gli2shR and LNCaP Ctl cells following treatment with or without 1 µM DOX for 48 h by propidium iodide staining. Representative flow cytometry histograms for the plotted data are shown in Fig. S3. Data are presented as the mean ± SEM from three experimental repeats. **P<0.01 and ****P<0.0001. ns, not significant; Gli2, Glioma-associated oncogene family zinc finger 2; DOX, doxycycline; Ctl, control vector not encoding the shRNA; Gli2shR, vector encoding Gli2 short hairpin RNA; OD, optical density.