Fig. 1: KANSL1 is required for autophagy.
a Graphical representation of the siRNA screen output. The ‘Z-score’ conveys the distribution of autophagic activity per well across the entire library (see details in “Methods”). Dashed lines are screen-specific cutoffs for active siRNA reagents. Black dots represent positive control siRNAs (siULK1). b Western blot analysis of HeLa cells transfected with siRNAs after 12 h of EBSS treatment with the indicated antibodies. c Keima imaging in HeLa cells transfected with the indicated siRNAs after 12 h of EBSS treatment. The neutral Keima signal is excited at 458 nm (green) and the acid Keima signal is excited at 561 nm (red). Scale bar, 10 μm. d Quantification of Keima signal in (c). Values are normalized to the red/green signal in siCtrl with EBSS treatment group. Cells number was obtained from microscopy calculated for 45 to 157 from independent experiments. e Primary MEFs (tamoxifen treatment, 1 μM, 48 h) were cultured with EBSS treatment for 6 h. Cell extracts were immunoblotted with the indicated antibodies. The arrow indicated the KANSL1 protein band. f Quantitation of protein signal intensities from immunoblots in (e) showing the ratio of LC3B-II to LC3B-I (n = 6 Kansl1fl/fl mice and 4 Kansl1fl/fl/CAG-cre mice). g Representative immunofluorescence images of primary MEFs treated with 1 μM tamoxifen for 48 h and cultured with or without EBSS treatment for 12 h. LC3B was detected as green fluorescent signal and nuclei are labeled with DAPI (blue). Scale bar, 10 μm. h Quantification of the number of LC3B puncta per cell in (g). n = 5 Kansl1fl/fl mice and 3 Kansl1fl/fl/CAG-cre mice. 25–72 cells were collected in each mouse. i Electron microscopy images of primary MEFs treated with 1 μM tamoxifen for 48 h. N, nucleus. Arrows indicate autophagosomes. Scale bar, 500 nm. j Violin plot showing the number of autophagosomes per μm2 in (i). n = 33 Kansl1fl/fl cells and 26 Kansl1fl/fl/CAG-cre cells. Source data are provided as a Source data file. All data are means ± SEM. d, f One-way ANOVA with Dunnett’s multiple hoc test; h by one-way ANOVA with Tukey’s multiple hoc test; j by two-tailed Student’s t-tests.
