Fig. 2: DDR1 and ABL1/2 signal via ERK2-MYC in the control of autophagy.

a, b U2OS cells stably expressing GFP-WIPI1 were transfected with siRNAs targeting ABL1/2 (siABL1/2), DDR1 (siDDR1) or nontargeting control siRNAs (siControl) for 48 h as indicated. Cell extracts were analysed by immunoblotting against CRKL pTyr207, CRKL or tubulin. Representative immunoblots (left panels) and quantifications are presented (right panels). Welch’s t test, mean ± SD, n = 3. c SILAC-based quantitative phosphoproteomics comparing siControl, siABL1/2 and siDDR1 settings. Scatterplot of all phosphosites detected in phospho-SILAC upon downregulation of ABL1/2 (left) or DDR1 (right). Phosphorylation ratios of target siRNA/control siRNA (log(2) versus log(10)) signal intensity are plotted. Significant changes (p < 0.05) are highlighted in red. d Venn diagram of significantly changed phosphosites along with overlaps. e Changes in ERK2 phosphorylation following ABL1/2 knockdown were confirmed by Western blotting. U2OS cells were transfected with siABL1/2 or nontargeting siRNA (siControl) for 48 h, and cell extracts were analysed by immunoblotting against ERK1/2 p-Y204/Y187, ERK2 and tubulin. Quantification of protein abundance was conducted by Welch’s t test, mean ± SD, n = 3. f Confirmation of increased MAX p-Ser11 phosphorylation is displayed (n = 4, indicated as Exp 1 through Exp 4). g U2OS cells were transfected with siABL1/2 or siDDR1 and the corresponding nontarget control (siControl) for 48 h and then treated with 10 µM MG132 for 3 h to prevent proteasomal MYC degradation. Protein extracts were analysed by immunoblotting against MYC p-Ser62, MYC or tubulin. Welch’s t test, mean ± SD, n = 4. h U2OS cells were treated with siRNAs targeting ABL1/2 or DDR1 for 16 h prior to transfection with empty control plasmids or with plasmids encoding myc-tagged ERK2 or myc-tagged ERK2-MEK1 for 48 h. Total RNA was extracted, and relative FOS gene expression was analysed by qPCR. Two-way ANOVA with Tukey’s multiple comparison’s test, mean ± SD, n = 3 in triplicates. i Using CellProfiler-based single cell analysis of images acquired with automated confocal laser-scanning microscopy (LSM), the number of GFP-WIPI1 puncta per cell (threshold-based puncta segmentation) was determined in ABL1/2 KD (left panel) or DDR1 KD (right panel) U2OS cells overexpressing empty control plasmids or myc-tagged ERK2-MEK1 for 48 h in fed conditions. Welch’s t testing was performed (up to 1496 cells from n = 3 for each condition) and error bars show the mean ± SD deviation. Supplementary material is available (Supplementary Data 1). P values: *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant.