Figure 2. Specific knockdown of IL-17RC expression in 4T1 cells promotes tumor proliferation and tumor invasiveness in vitro and in vivo despite increased stress-induced apoptosis.

4T1 cells were transduced with retroviral vectors containing shRNAs against IL-17RC or random sequences. (a-b) IL-17RA and RC expression from a representative IL-17RCKD clone (RCKD4.8) and the pSMP control of 4T1 cells were examined by RT-PCR and flow cytometry. The threshold of gene expression for selecting the knockdown clones is shown as a red line. (c) CXCL1 production upon IL-17A stimulation was determined by ELISA. (d-e) Cell growth was measured by direct cell counting and MTT assay with serum-free starvation treatment. (f-g) Tumor volume, weight and lung metastasis of 4T1-IL-17RCKD and 4T1-pSMP control in Balb/c mice were determined. (h-i) RCKD and pSMP control subclones of B16 and 4T1 cells were starved in serum-free medium for 14 hours and recovered in complete medium (CM) for different periods of time. The rates of apoptosis were determined by Annexin V staining 1 hour following CM (h). Whole-cell extracts were harvested and immunoblotted with antibodies to detect pro- and cleaved-caspase-3. GAPDH was used as a loading control (i). (j) Representative images and quantitative results of cleaved-caspase-3 protein levels observed from day 18 in 4T1 tumors by immunohistochemistry. All values are presented as the mean ± SEM of 3-5 independent experiments for in vitro studies (a-e, h), or the mean ± SEM of 5-10 mice per group per time point for in vivo studies (f-g, j). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; statistical analysis was compared with the pSMP control.