Fig 1.
Expression of CL-STK4, LR-STK4, and NL-STK4 protein in Tet-inducible C4-2 prostate cancer cells.
(A) Time-dependent expression of CL-STK4, LR-STK4, and NL-STK4 protein in the engineered cells that were exposed to doxycycline (Dox) for 24 and 48h. Levels of ectopic STK4 proteins were assessed by Western blotting (WB) using the HA-tagged antibody. (B-D) Analysis of CL-STK4, LR-STK4, and NL-STK4 protein in cytoplasmic, lipid raft, and nuclear fractions, respectively. Expression of HA-STK4 protein was evaluated by WB with the HA-tag antibody at 48h after treatment with and without doxycycline (Dox, 4 μg/ml). Lam (lamin) A/C was used as a nuclear marker. Giα2 was used as a lipid raft marker. (E-G) Immunofluorescence (IF) analysis of CL-STK4, LR-STK4, and NL-STK4 protein in the C4-2/CL-STK4, C4-2/LR-STK4, and C4-2/NL-STK4 cells, respectively. IF was performed at 24h post Dox (4 μg/ml) treatment. Micrographs are the representation of two independent experiments. CTxB-FITC labeled lipid raft in C4-2/LR-STK4 cells. For both experiments, cells were grown in Tet-approved serum conditions. CL: Cytoplasmic localization, LR: Lipid raft; NL: nuclear localization, HA: Hemagglutinin.
Fig 2.
Regulation of C4-2 cell growth by STK4 signaling in all three subcellular locations.
(A) Representative bright field images of C4-2/Vector, C4-2/CL-STK4, C4-2/LR-STK4, and C4-2/NL-STK4 cells. Cell images were captured at 72h post to Dox treatment (4 μg/ml). (B) Growth of C4-2/Vector, C4-2/CL-STK4, C4-2/LR-STK4, and C4-2/NL-STK4 cells in vitro. Cell growth was determined by MTS assay at 72h post Dox exposure. Data (±SD) are the representation of two independent experiments in triplicates, *, **, ***P < 0.007. (C) Prostate tumor xenografts in mice (n = 10 per conditions). C4-2/Vector, C4-2/LR-STK4, and C4-2/NL-STK4 cells were subcutaneously inoculated into the intact nude, immunocompromised male mice. Animals were treated with Dox (0.5 mg/ml) for 6 weeks in drinking water. Tumor sizes were measured weekly for 5 weeks. Tumor growth (volumes) was presented as a function of time, *, **P < 0.01. (D) Growth of C4-2/LR-STK4 cells treated with and without Dox and epidermal growth factor (EGF). Cell growth was determined by MTS assay at 72h post Dox and/or EGF treatment, *, **P < 0.001. Data (±SD) are the representation of two independent experiments in triplicates.
Fig 3.
Overview of the RNAseq data and the validation.
(A) Volcano plots of DE genes from the DESeq2 analysis. (B) Heatmap of DE genes using fold-change values in all three STK4 conditions (C) Venn diagram of DE genes in C4-2/CL-STK4, C4-2/LR-STK4, and C4-2/NL-STK4 cells. (D) Quantitative RT-PCR analysis of select DE genes, *P < 0.01. Data are representation of the two independent experiments in duplicates.
Table 1.
Top 100 DE genes regulated by the enrichment of STK4 in three subcellular locations (FDR ≤ 0.01).
Numbers are in log2 fold change. Minus signs indicate downregulation with respect to the vector control.
Table 2.
Common DE genes regulated by the enrichment of STK4 in three subcellular locations (FDR ≤ 0.01).
Numbers are in log2 fold change. Minus signs indicate downregulation with respect to the vector control.
Table 3.
Fold change values of AR targets in response to STK4 enrichment in all three subcellular compartments (FDR ≤ 0.01).
Fold change values are in log2. Minus signs indicate downregulation with respect to the vector control.
Fig 4.
STK4 selectively regulates the expression of putative oncogenes and tumor suppressors.
Log2 fold change values for a list of putative oncogenes (black) and tumor suppressors (orange) in CL-STK4, LR-STK4, and NL-STK4 cells.
Fig 5.
Top 20 KEGG pathways and GO biological process terms enriched by DE genes in all three STK4 cells.
Each term in the bar chart appears in the top 20 (A) KEGG pathways (B) GO biological process terms enriched by DE genes in at least one condition. Red, blue, and green bars refer to the log2 enrichment FDR values in CL-STK4, LR-STK4, and NL-STK4, respectively. The terms with missing FDR values for CL-STK4 indicate that the DE genes in CL-STK4 were not significantly enriched for those terms.
Fig 6.
Each gene is colored by gene expression fold change in (A) CL-STK4 (B) NL-STK4 conditions using Cytoscape. Red: Upregulated, green: downregulated, white: not differentially expressed. Edge labels “e”: expression interaction, “+p”: phosphorylation, “-p”: dephosphorylation.
Fig 7.
Network enrichment annotation of DE genes in NL-STK4 cells.
DE genes in NL-STK4 cells that have known functional interactions were clustered based on their connectivity in the network using Reactome FI plugin in Cytoscape. Each clustered module was annotated to a representative significantly-enriched GO term. Each edge represents a known interaction between two genes (i.e., nodes) in the network.
Fig 8.
Network enrichment annotation of DE genes in LR-STK4 cells.
DE genes in LR-STK4 cells that have known functional interactions were clustered based on their connectivity in the network using Reactome FI plugin in Cytoscape. Each clustered module was annotated to a representative significantly-enriched GO term. Each edge represents a known interaction between two genes (i.e., nodes) in the network.
Fig 9.
Schematic representation of the current study.
TFs: Transcription factors. DE: Differentially expressed.