Figure S2.
DSTYK is involved in autophagic molecular pathways. (A) Left: Double IF of Flag-Dstyk LLC cells. DSTYK (magenta) showing co-localization with LC3 (blue). Scale bar: 20 µm. Middle: Single channel images correspondent to triple IF of Flag-Dstyk LLC cells. DSTYK (red), LC3 (blue), LAMP1 (green), and nuclei (white). Scale bars: 20 µm. Right: Technical negative control. Scale bars: 10 µm. Observed by super-resolution/Airyscan based/confocal microscopy. (B) Double IF of Flag-Dstyk LLC cells. DSTYK (red) do not co-localize with cellular vesicles markers (blue). Scale bar: 20 µm. (C) Scheme of differentially expressed genes from the RNAseq analysis enriched in the following categories: lysosome, autophagy, mitochondria, oxidative stress, and cytoskeleton. (D) Validation of mRNA expression of candidate genes from the RNAseq analysis in LLC and H2009 parental and CRISPRed cells by quantitative PCR. Analysis was assessed by t test. P values for parental vs. KO comparation were the following: Dstyk in LLC, P < 0.0001 and DSTYK in H2009, P < 0.0001; Lamp1 in LLC, P = 0.001 and LAMP1 in H2009, P < 0.0001; Atp6v0a in LLC, P < 0.0001 and ATP6V0A in H2009, P < 0.03; Nqo1 in LLC, P < 0.0001 and NQO1 in H2009, P < 0.0001; Tomm20 in LLC, P < 0.001 and TOMM20 in H2009, P < 0.03. Three experimental replicates were performed for each experimental condition. (E) Scheme of DSTYK interactor proteins from immunoprecipitation analysis enriched in the following categories: lysosome, autophagy, mitochondria, oxidative stress, and cytoskeleton. (F) Cropped images from Western blot analysis of autophagy pathway proteins in LLC and H2009 parental, DSTYK-KO, and KO treated with rapamycin cells. A representative experiment out of two similarly performed is shown. (G) Glucose levels (mg/dl) in cell culture supernatants of parental H2009 and KO cells. Analysis by t test rendered P = 0.001. Three experimental replicates were performed for each experimental condition. (H) Cropped images from Western blot analysis of autophagy pathway proteins in LLC and H2009 parental, parental treated with chloroquine, and DSTYK-KO cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. A representative experiment out of two similarly performed is shown. Source data are available for this figure: SourceData FS2.

DSTYK is involved in autophagic molecular pathways . (A) Left: Double IF of Flag-Dstyk LLC cells. DSTYK (magenta) showing co-localization with LC3 (blue). Scale bar: 20 µm. Middle: Single channel images correspondent to triple IF of Flag-Dstyk LLC cells. DSTYK (red), LC3 (blue), LAMP1 (green), and nuclei (white). Scale bars: 20 µm. Right: Technical negative control. Scale bars: 10 µm. Observed by super-resolution/Airyscan based/confocal microscopy. (B) Double IF of Flag-Dstyk LLC cells. DSTYK (red) do not co-localize with cellular vesicles markers (blue). Scale bar: 20 µm. (C) Scheme of differentially expressed genes from the RNAseq analysis enriched in the following categories: lysosome, autophagy, mitochondria, oxidative stress, and cytoskeleton. (D) Validation of mRNA expression of candidate genes from the RNAseq analysis in LLC and H2009 parental and CRISPRed cells by quantitative PCR. Analysis was assessed by t test. P values for parental vs. KO comparation were the following: Dstyk in LLC, P < 0.0001 and DSTYK in H2009, P < 0.0001; Lamp1 in LLC, P = 0.001 and LAMP1 in H2009, P < 0.0001; Atp6v0a in LLC, P < 0.0001 and ATP6V0A in H2009, P < 0.03; Nqo1 in LLC, P < 0.0001 and NQO1 in H2009, P < 0.0001; Tomm20 in LLC, P < 0.001 and TOMM20 in H2009, P < 0.03. Three experimental replicates were performed for each experimental condition. (E) Scheme of DSTYK interactor proteins from immunoprecipitation analysis enriched in the following categories: lysosome, autophagy, mitochondria, oxidative stress, and cytoskeleton. (F) Cropped images from Western blot analysis of autophagy pathway proteins in LLC and H2009 parental, DSTYK-KO, and KO treated with rapamycin cells. A representative experiment out of two similarly performed is shown. (G) Glucose levels (mg/dl) in cell culture supernatants of parental H2009 and KO cells. Analysis by t test rendered P = 0.001. Three experimental replicates were performed for each experimental condition. (H) Cropped images from Western blot analysis of autophagy pathway proteins in LLC and H2009 parental, parental treated with chloroquine, and DSTYK-KO cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. A representative experiment out of two similarly performed is shown. Source data are available for this figure: SourceData FS2.

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