Fig. 2
circCacna1c inhibits H2O2-induced necroptosis. A H9c2 cells were transfected with the circCacna1c expression vector, and the expression level of circCacna1c was analyzed by qRT-PCR. The empty vector was used as a NC (empty vector). ***P < 0.001. n = 3. B, C The impact of circCacna1c on necroptosis in H9c2 cells was assessed through experiments detecting the rate of PI-positive cells and the activity of LDH. B A representative image is displayed on the left side, while the calculated rates of necroptosis from three independent experiments are shown on the right side. Red indicates PI-positive nuclei, while blue represents DAPI stained nuclei. Scale bars, 50 μm. **P < 0.01, ***P < 0.001. n = 3. C The level of LDH in the cell supernatant was measured. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3. D The protein levels of RIPK1 and RIPK3 were quantified in H9c2 cells, with GAPDH selected as a reference. n = 3. E, F The mRNA levels of RIPK1 and RIPK3 were also quantified in H9c2 cells. *P < 0.05, **P < 0.01, ns > 0.05. n = 3. G Following transfection of circCacna1c siRNA (si-circCacna1c) into H9c2 cells, the expression level of circCacna1c was assessed using qRT-PCR. si-NC: negative control siRNA. ***P < 0.001 versus si-NC. n = 3. H, I Subsequent experiments were conducted to evaluate the impact of si-circCacna1con necroptosis in H9c2 cells through detection of the rate of PI positive cells and LDH activity. H The level of LDH in the cell supernatant was measured. *P < 0.05, ***P < 0.001. n = 3. I A representative image is displayed on the left side, while the calculated rates of necroptosis from three independent experiments are shown on the right side. Red indicates PI-positive nuclei, while blue represents DAPI stained nuclei. Scale bars, 50 μm. *P < 0.05, ***P < 0.001. n = 3