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. 2020 Aug 18;11:1281. doi: 10.3389/fphar.2020.01281

Figure 3.

Figure 3

Effects of NLRC5 inhibition on autophagy in EESCs (n = 7). (A, B) Representative western blotting and qRT-PCR results showing inhibition of NLRC5 by transfection with siRNA-NLRC5 significantly inhibited NLRC5, LC3 Beclin1 expressions, and promoted p62 expression when compared with those from the scrambled-RNAi group (**P < 0.01 vs. scrambled-RNAi group). (C) Representative images showing LC3 staining in EESCs infected with GFP-RFP-LC3 adenovirus; inhibition of NLRC5 by siRNA-NLRC5 transfection significantly inhibited yellow puncta when compared with scrambled-RNAi group. Photographs were taken at magnifications of 1600×, quantification of mean yellow puncta of 10-15 cells per condition is shown (**P < 0.01 vs. scrambled-RNAi group). (D) Representative TEM image showing inhibition of NLRC5 by siRNA-NLRC5 transfection significantly inhibited autophagosomes formation when compared with scrambled-RNAi group, autophagosomes were highlighted by red arrows(left scale bar: 1μm; right scale bar: 2μm; **P < 0.01 vs. scrambled-RNAi group). The expression levels of mRNA were normalized with respect to β-actin and were calculated using the 2-ΔΔCt method. The protein expression levels were quantified by Image J software and normalized to β-actin protein levels. The results are represented as the mean ± SEM from at least three independent experiments.