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. 2020 Jun 15;10:9653. doi: 10.1038/s41598-020-66377-z

Figure 2.

Figure 2

Expression of ZKSCAN3 in the Drosophila fat body prevents induction of autophagy due to loss of M1BP function. (A) Upon loss of M1BP expression (red) through expression of M1BP RNAi in L3F fat body cells using the fat body-specific cgGal4 driver, autophagy induction is widespread, as seen by the upregulation and cytoplasmic location of the Atg8a autophagy marker (green). Autophagy induction is largely prevented by the co-expression of myc-tagged vertebrate ZKSCAN3 (bottom left) but not by ZKSCAN4 (bottom right). Contrasts of individual channels are shown and scale bar represents 50 µm. G: Gonad (B) Clonal loss, RNAi-expressing cells are GFP-identified and autophagy monitored using mCherry::Atg8a confirming that co-expression of myc-tagged ZKSCAN3 can prevent autophagy induction through M1BP knockdown, whereas ZKSCAN4 expression does not. Contrasts of individual channels are shown and scale bars represent 20 µm. (C) Western blots of wild type, M1BP RNAi, ZKSCAN3, and M1BP RNAi;ZKSCAN3 expressing whole fat body protein preparations confirm autophagy induction due to M1BP RNAi, through the upregulation of Atg8a expression and the presence of major phosphatidylethanolamine-modified forms of Atg8a (arrowhead), which are largely prevented through ZKSCAN3 co-expression. The blots were reprobed with anti-tubulin antibodies for loading control. Quantification of the ratio of unmodified Atg8a (Atg8a-I) to phosphatidylethanolamine-modified Atg8a (Atg8a-II) is presented below the blots. Note, quantification is of this single unrepeated blot and thus does not incorporate error bars. (D) RT-qPCR analyses confirm significant upregulation of autophagy-related Atg gene expression upon M1BP knockdown through M1BP RNAi in the fat body using the cgGal4 driver. Co-expression of ZKSCAN3 prevents Atg gene overexpression without modifying M1BP knockdown. (E) Transmission electron micrographs of Drosophila fat body cells display widespread autophagy induction upon M1BP RNAi through the presence of numerous autophagosome vesicles (lower panels) which are largely not observed in wild type cells or cells co-expressing ZKSCAN3 with M1BP RNAi (upper panels). Autophagosomes from all stages of maturity from early phagophore onset (green outline), autophagosomes containing large quantities of cellular material (cyan outline) and late-stage autophagosomes/autolysosomes (red outline) can be observed in M1BP RNAi expressing cells. LD: lipid droplets. Scale bars 10 µm in main micrographs and 1 µm in enlarged area (bottom right).