Journal: Communications Biology

Article Title: The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension

doi: 10.1038/s42003-023-05236-9

Figure Lengend Snippet: 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 ). P values: * p < 0.05; ** p < 0.01; *** p < 0.001; ns not significant.

Article Snippet: The following phospho-specific primary antibodies were used: phospho-CrkL (Tyr207) (Cell Signaling Technologies, 3181S; WB: 1:1000), phospho-p44/42 ERK1/ERK2 (Y204/Y187) (Cell Signaling Technologies, 5726; WB: 1:1000), phospho-MAX (S11) (Thermo Fisher, PA5-97346; WB: 1:500), phospho-c-Myc (Ser62) (Cell Signaling Technologies, 13748; WB: 1:500), phospho-S6K (Cell Signaling Technologies, 9234, WB: 1:500-1:1000), phospho-TFEB (Ser211) (Cell Signaling Technologies, 37681, WB: 1:500) and phospho-ULK1 (Ser757) (Cell Signaling Technologies, 6888, WB: 1:500-1:1000).

Techniques: Stable Transfection, Expressing, Transfection, Control, Western Blot, Multiplex sample analysis, Phospho-proteomics, Knockdown, Quantitative Proteomics, Gene Expression, Single-cell Analysis, Confocal Laser Scanning Microscopy

Journal: Communications Biology

Article Title: The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension

doi: 10.1038/s42003-023-05236-9

Figure Lengend Snippet: a MAX, MYC or both were transiently downregulated using siRNAs in U2OS cells for 72 h, and total RNA was extracted, followed by assessing WIPI1 mRNA levels by RT‒qPCR. One-way ANOVA with Holm-Sidak post-hoc testing, mean ± SD, n = 3. b U2OS cells were treated with 10 µM AZD0364 for 48 h, and during the last 3 h, 10 µM MG132 was added to prevent proteasomal MYC degradation. Cell extracts were analysed by immunoblotting against MYC p-Ser62, MYC or tubulin, (left panel: representative Western blot, right panel: quantification). Welch’s t test, mean ± SD, n = 4. c , d U2OS cells were treated with or without 10 µM AZD0364 for 0, 3, 24 or 48 h. Total RNA was extracted, and relative WIPI1 ( c ) or FOS ( d ) gene expression was analysed by RT‒qPCR. Two-way ANOVA with Tukey’s post hoc testing, mean ± SD, n = 5. e Canonical E-box sequence used to identify potential E-boxes in the human WIPI1 promoter (left upper panel) using ConTra v3. Clustal Omega-based multiple nucleotide sequence alignment using a canonical E-box sequence along with putative E-boxes (M + 1, M11, M19) in the human WIPI1 promoter (upper right panel, indicated are the positions relative to the transcription start site (+1) according to Ensembl Release 109 (Feb 2023), transcript ID ENST00000262139.10 WIPI1-201). A schematic WIPI1 promoter overview with putative E-boxes was created with BioRender.com. Published binding sites for PU.1 are additionally indicated. f – h U2OS cells were crosslinked, and chromatin immunoprecipitation against G9A, MAX, MYC or negative control IgG was performed. qPCR against M11 ( f ), M19 ( g ) or M + 1 ( h ) binding sites was conducted, the %input was calculated and analysed with one-way ANOVA followed by Dunnett’s post-hoc testing (mean ± SD, n = 5) (upper panels). Alternatively, chromatin immunoprecipitation was performed against H2K9me2 (lower panels). Welch’s t test, mean ± SD, n = 5. i Dual-luciferase reporter assays were conducted using U2OS co-transfected for 24 h with pGL4.73[hRluc/SV40] expressing Renilla luciferase together with plasmids with the potential to drive firefly luciferase from a minimal promotor: empty pGL4.23[luc2/minP] plasmid (Luc2/minP), pGL4.23-WIPI1promotor (WIPI1 promotor, upper panel, n = 6) or pGL4.23-5xE-box (5x canonical E-box, lower panel, n = 4). Firefly- and Renilla luciferase activities were measured via luminosity and firefly luminosity normalized to Renilla luminosity for each sample. For statistical analysis, an unpaired t -test with Welch’s correction was performed. Error bars show the mean ± Standard deviation. Supplementary material is available (Supplementary Fig. ; Supplementary Data ). P values: * p < 0.05; ** p < 0.01; *** p < 0.001; ns not significant.

Article Snippet: The following phospho-specific primary antibodies were used: phospho-CrkL (Tyr207) (Cell Signaling Technologies, 3181S; WB: 1:1000), phospho-p44/42 ERK1/ERK2 (Y204/Y187) (Cell Signaling Technologies, 5726; WB: 1:1000), phospho-MAX (S11) (Thermo Fisher, PA5-97346; WB: 1:500), phospho-c-Myc (Ser62) (Cell Signaling Technologies, 13748; WB: 1:500), phospho-S6K (Cell Signaling Technologies, 9234, WB: 1:500-1:1000), phospho-TFEB (Ser211) (Cell Signaling Technologies, 37681, WB: 1:500) and phospho-ULK1 (Ser757) (Cell Signaling Technologies, 6888, WB: 1:500-1:1000).

Techniques: Western Blot, Gene Expression, Sequencing, Binding Assay, Chromatin Immunoprecipitation, Negative Control, Luciferase, Transfection, Expressing, Plasmid Preparation, Standard Deviation