Figure 2. DSTYK is located in... | Rockefeller University Press

Figure 2.

$DSTYK is located in autophagosomes and regulates processes related to those vesicles. (A) Subcellular fractionation of LLC and H2009 cells. The following fractions were shown in the Western blot from left to right: total extract; nuclear extract (P3000); cytosolic extract (S3000); large organelle extract (P10000); cytosolic extract without large organelles (S10000); microsomal fraction (P100000); and soluble cytosolic extract (S100000). Different organelle markers were tested to control proper fractionation. (B) Top: Double IF of Flag-Dstyk LLC cells. Flag-DSTYK (magenta) showing co-localization with LC3 (blue). Scale bar: 20 µm. Bottom: Triple IF of Flag-Dstyk LLC cells. Detailed image of autophagosomes: Flag-DSTYK (magenta), LC3 (blue), and LAMP1 (green). Scale bars: 5 and 2 µm. Observed by super-resolution/Airyscan based/confocal microscopy. (C) PLA images of H2009 cells. Left: Red fluorescent dots result from DSTYK+LC3 proteins proximity (maximum distance: 40 nm). Right: Technical negative control. Scale bar: 20 µm. (D) Venn diagram picturing RNAseq analysis. Three RNAseq analyses were carried out to identify the differentially expressed transcripts: LLC comparing parental line vs. silenced (sh#) Dstyk (blue circle), UN-SCC679 line comparing parental vs. overexpression (O/E) Dstyk (green circle), and UN-SCC679 comparing parental vs. silenced (sh#) Dstyk (red circle). Once differentially expressed transcripts were selected (adjusted P value <0.05) in each contrast, all experiments were merged to verify the intersection of the transcripts based on name and the direction. 163 transcripts were finally selected. (E) Immunoprecipitation of Flag-DSTYK in the UN-SCC-679 cell line validating Western blots showing DSTYK binding to P62 protein. (F) Cropped images from the Western blot analysis of autophagy pathway proteins in LLC and H2009 parental and DSTYK-KO cells. (G) Cropped images from the Western blot analysis of lysosomal biogenesis pathway proteins in LLC and H2009 parental and DSTYK-KO cells in cytoplasmic/nuclear fractions. All Western blots show representative results from three independent experiments. (H) Lysosomal pH measurement in LLC and H2009 parental and DSTYK-KO cells. A t test was performed to compared both groups: LLC P value = 0.05; H2009 P value = 0.01. Images shown in A, B, F, G, and H show a representative experiment out of three similarly performed. *, P < 0.05; **, P < 0.01. Source data are available for this figure: SourceData F2.$

DSTYK is located in autophagosomes and regulates processes related to those vesicles. (A) Subcellular fractionation of LLC and H2009 cells. The following fractions were shown in the Western blot from left to right: total extract; nuclear extract (P3000); cytosolic extract (S3000); large organelle extract (P10000); cytosolic extract without large organelles (S10000); microsomal fraction (P100000); and soluble cytosolic extract (S100000). Different organelle markers were tested to control proper fractionation. (B) Top: Double IF of Flag-Dstyk LLC cells. Flag-DSTYK (magenta) showing co-localization with LC3 (blue). Scale bar: 20 µm. Bottom: Triple IF of Flag-Dstyk LLC cells. Detailed image of autophagosomes: Flag-DSTYK (magenta), LC3 (blue), and LAMP1 (green). Scale bars: 5 and 2 µm. Observed by super-resolution/Airyscan based/confocal microscopy. (C) PLA images of H2009 cells. Left: Red fluorescent dots result from DSTYK+LC3 proteins proximity (maximum distance: 40 nm). Right: Technical negative control. Scale bar: 20 µm. (D) Venn diagram picturing RNAseq analysis. Three RNAseq analyses were carried out to identify the differentially expressed transcripts: LLC comparing parental line vs. silenced (sh#) Dstyk (blue circle), UN-SCC679 line comparing parental vs. overexpression (O/E) Dstyk (green circle), and UN-SCC679 comparing parental vs. silenced (sh#) Dstyk (red circle). Once differentially expressed transcripts were selected (adjusted P value <0.05) in each contrast, all experiments were merged to verify the intersection of the transcripts based on name and the direction. 163 transcripts were finally selected. (E) Immunoprecipitation of Flag-DSTYK in the UN-SCC-679 cell line validating Western blots showing DSTYK binding to P62 protein. (F) Cropped images from the Western blot analysis of autophagy pathway proteins in LLC and H2009 parental and DSTYK-KO cells. (G) Cropped images from the Western blot analysis of lysosomal biogenesis pathway proteins in LLC and H2009 parental and DSTYK-KO cells in cytoplasmic/nuclear fractions. All Western blots show representative results from three independent experiments. (H) Lysosomal pH measurement in LLC and H2009 parental and DSTYK-KO cells. A t test was performed to compared both groups: LLC P value = 0.05; H2009 P value = 0.01. Images shown in A, B, F, G, and H show a representative experiment out of three similarly performed. *, P < 0.05; **, P < 0.01. Source data are available for this figure: SourceData F2.

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