Figure 2.

Knockdown of calreticulin suppressed VEGF-A mRNA expression and protein secretion in bladder cancer cell. Details on CRT-knockdown human bladder cancer cell lines (control and CRT-RNAi) were described previously [36]. (a) Real-Time PCR was used to detect VEGF-A mRNA levels in J82 control and CRT-knockdown cells. Total RNA was isolated by the TRIzol reagent following the manufacturer's instructions. Reverse transcription PCR was carried out using ReverTra Ace reverse transcriptase. Real-Time PCR was performed using the iCycler iQ Real-Time detection system (Bio-Rad, Hercules, CA) with the DNA double-strand specific SYBR Green I dye for detection. RNA expression was normalized to the internal control, GAPDH. (b) VEGF-A secretion levels were detected by enzyme-linked immunosorbent assay (ELISA) in conditioned media of J82 control and CRT-knockdown cells. Cells were plated at 5 × 10⁵ cells/well in six-well plates. Conditioned media were collected and analyzed using an ELISA kit specific for human VEGF (BioSource, Camarillo, CA, USA). Statistical differences were compared to the control level (^* P < 0.05, ^*** P < 0.001).