RNAi-mediated knockdown of CAIX enhances the radiosensitivity of nasopharyngeal carcinoma cell line, CNE-2

Figures & data

Figure 1 CNE-2 cells successfully transfected with FAM-siRNA.

Notes: (A) Flow cytometric histogram profiles of CNE-2 cells and (B) quantification of CNE-2 cells after treatment with varying final concentrations of FAM-siRNA from 0 nM to 100 nM. (C) The green fluorescence emitted by FAM-siRNA at 50 nM was observed by laser scanning confocal microscope (The scale bar represents 20 μm; magnification ×400). **Denotes statistically significant differences (P<0.01) calculated by one-way ANOVA.
Abbreviations: FAM, fluorescent carboxyfluorescein; siRNA, small interfering ribonucleic acid; ANOVA, analysis of variance.

Figure 2 CAIX-siRNA treatment reduces CAIX mRNA and CAIX protein expression.

Notes: The expression level of CAIX determined by qRT-PCR (A) and Western blotting (B) in the in vitro transfection experiments. *Denotes statistically significant differences (P<0.05) calculated by two sample t-test.
Abbreviations: NC, negative control; CAIX, carbonic anhydrase IX; siRNA, small interfering ribonucleic acid; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 3 Knockdown of CAIX by siRNA inhibited cell growth of CEN-2 cells.

Notes: Cells were transfected with either CAIX-siRNA1, 2, or NC-siRNA. The effect of CAIX suppression on cell growth in CEN-2 cells was examined by MTT assay (n=3). ^#P<0.01 compared with the other three groups by one-way ANOVA.
Abbreviations: NC, negative control; CAIX, carbonic anhydrase IX; siRNA, small interfering ribonucleic acid; ANOVA, analysis of variance.

Figure 4 Knockdown of CAIX expression enhanced the radiosensitivity of CEN-2 cells.

Notes: (A) The abilities of CEN-2 cells in the control, NC, and CAIX-siRNA1 treatment group to form colonies after being treated with 0 Gy, 2 Gy, 4 Gy, and 6 Gy were assessed and (B) the clonogenic cell survival curves we created using multitarget single-hit model. The cell SF was calculated as: (Number of colonies formed following treatment/number of cells seeded) × inoculation efficiency.
Abbreviations: NC, negative control; CAIX, carbonic anhydrase IX; siRNA, small interfering ribonucleic acid; SF, survival fraction.

Figure 5 Knockdown of CAIX enhanced cell apoptosis after radiation treatment.

Notes: (A) Cell apoptosis without exposure to radiation; (B) cell apoptosis after exposure to 6 Gy radiation. Cells were irradiated with 6 Gy at 12 hours after transfection. The effect of CAIX suppression on cell apoptosis in CNE-2 cells was examined by flow cytometric analysis. **P<0.01 compared with the control group by two sample t-test.
Abbreviations: NC, negative control; CAIX, carbonic anhydrase IX; siRNA, small interfering ribonucleic acid.

Figure 6 Knockdown of CAIX arrested cells in the G₂/M phase induced by IR.

Notes: (A and C) Cell cycle distribution without exposure to radiation and (B and D) cell cycle distribution after exposure to 6 Gy radiation. *P<0.05 compared with the control group by two sample t-test.
Abbreviations: IR, ionizing radiation; NC, negative control; CAIX, carbonic anhydrase IX; siRNA, small interfering ribonucleic acid.