Decreased autophagic activity upon MARCH5 KD. Downregulation of central players during autophagy as demonstrated by (A) WB and (B) qPCR. (C) Decreased autophagosome formation in the MARCH5 KD group as determined by Cyto-ID staining. (D) RNA-seq and Gene Set Enrichment Analysis (GSEA) analysis. GSEA analysis of DEGs in MARCH5 KD vs scramble groups. (E) Central players in the initiation or autophagosome formation/maturation show significant downregulation in the RNA-seq analysis. Data are shown as mean ± standard error of the mean of 3 independent experiments. (F) ATF6, FOXO1, and FOXO3 are among 5 transcription factors involved in the positive regulation of autophagy, and their expression was significantly decreased in the MARCH5 KD group vs the scramble group in RNA-seq sample analysis, whereas HIF1a and TFEB, other positive autophagy regulators, were upregulated. (G) The observed expression patterns of transcription factors in the RNA-seq analysis were confirmed by qPCR. The data are shown as mean ± standard error of the mean of 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.