ac220  (MedChemExpress)

Journal: EMBO Molecular Medicine

Article Title: Chaperone-mediated autophagy regulates the metastatic state of mesenchymal tumors

doi: 10.1038/s44321-025-00210-w

Figure Lengend Snippet: ( A ) Cellular fractions isolated from ES2 cells by differential large-scale multi-layered density gradient centrifugations and analyzed by immunoblotting using antibodies against lysosomal membrane protein LAMP1, lysosomal matrix protein cathepsin D (CTSD), mitochondrial protein translocase of outer mitochondrial membrane 40 (TOMM40), cytosolic protein Lactate dehydrogenase A (LDHA), the Hsc70 chaperone protein and macroautophagy marker LC3B. The red square indicates the low percentage gradient fraction, enriched in lysosomes, and chosen for further analysis. ( B ) Cellular lysate and lysosomal fractions from control (DMSO), 8 and 16 h CMA activated (AC220+Spautin1) and 16 h CMA+si LAMP-2A conditions analyzed by immunoblotting using LAMP-2A antibody. Total protein loading is visualized by Ponceau S Red staining. ( C ) Score plot of principle component analysis (PCA) in 2D for control, 16 h CMA, and 16 h CMA+si LAMP-2A treated sample sets (n exp = 4). ( D ) Pathway enrichment analysis by Gene ontology (GO) of biological processes comparing control vs 16 h CMA treated sample sets (Over Representation Analysis). ( E ) Pie graphs of the experimentally validated LAMP-2A-dependent lysosome proteome showing the percentage of proteins with indicated types of KFERQ-like motifs. ( F ) GO analysis comparing 16 h CMA vs CMA+si LAMP-2A treated sample sets. Dot plots of top 10 enriched pathways are indicated as the ratio of the differentially expressed gene number to the total gene number for a certain annotation. The size and color of the dots represent the gene ratio and the range of adjusted P values, respectively (Over Representation Analysis). .

Article Snippet: AC220 , Selleckchem , S1526.

Techniques: Isolation, Western Blot, Membrane, Marker, Control, Staining

Journal: EMBO Molecular Medicine

Article Title: Chaperone-mediated autophagy regulates the metastatic state of mesenchymal tumors

doi: 10.1038/s44321-025-00210-w

Figure Lengend Snippet: ( A ) Heatmap presentation of EMT-related quantitative proteomics data obtained from multiplexed (TMT) mass spectrometry analysis detected in isolated lysosome fractions from ES2 cells untreated (control), 16 h CMA (AC220+Spautin1) or 16 h CMA following 48 h siRNA-mediated LAMP-2A knockdown conditions (n exp = 4). The heatmap represents the relative expressions for each gene on each row. The row max and row min are shown for the overall heatmap. ( B ) Box plots depicting the fold TMT intensity of the lysosomal enrichment of TGFβ1 and Vimentin proteins in isolated lysosomes from ES2 cells treated as in A. Data presented as (n exp = 4) indicated by the data points. Bars represent mean ± sd. P values refer to ** P < 0.01, *** P < 0.001 (TGFβ1: P Ctrl vs CMA = 0.0055, P CMA vs CMA+siLAMP-2A = 0.0017; Vimentin: P Ctrl vs CMA < 0.0001, P CMA vs CMA+siLAMP-2A = 0.0015; ANOVA). The boxes extend from the 25th to 75th percentile, the middle line shows the median, whiskers extend to the most extreme data. ( C ) Schematics depicting selection criteria of CMA motif search on the total human EMT-promoting genes. Illustration ( D ) and prevalence/percentages ( E ) of EMT-inducer, EMT-transcription factors (TFs), and mesenchymal marker proteins containing canonical CMA (KFERQ)-motifs marked with star (red). Putative CMA motifs can be generated by post-translational modifications that change the biophysical properties of amino acids. .

Article Snippet: AC220 , Selleckchem , S1526.

Techniques: Quantitative Proteomics, Mass Spectrometry, Isolation, Control, Knockdown, Selection, Marker, Generated

Journal: EMBO Molecular Medicine

Article Title: Chaperone-mediated autophagy regulates the metastatic state of mesenchymal tumors

doi: 10.1038/s44321-025-00210-w

Figure Lengend Snippet: ( A ) CRISPR/Cas9 genome editing and genotyping by agarose gel electrophoresis/PCR in regular (+), wild-type (WT), and LAMP-2A knockout (KO) A549 and HT1080 cells. Target amplicon in human LAMP2 gene is detected by the fragment of ~800 bp. M: marker. ( B ) RT–qPCR detection of LAMP1 , LAMP-2A , - 2B , and - 2C expression in WT and LAMP-2A KO A459 and HT1080 cell lines. Bars represent mean ± sd (n exp = 3). P values refer to *** P < 0.001 (A549: P LAMP-2A = 0.0001; HT1080: P LAMP-2A < 0.0001 Student’s t -test). ( C ) Immunoblot detection of LAMP-2A and LAMP1 in WT and LAMP-2A KO A549 and HT1080 cells. β -Actin: loading control. ( D ) Representative immunofluorescence confocal images of WT and LAMP-2A KO A549 and HT1080 cell lines stained with anti-LAMP-2A (green), anti-LAMP1 (red), and DAPI (blue) for nuclei highlighted with (circular insets) magnification. Scale bars: 20 μm. ( E ) CMA activity measured in WT and LAMP-2A KO A549 and HT1080 cells expressing PAmCherry-KFERQ CMA reporter. Cells were treated with AC220+Spautin1 to activate CMA for 16 h. Confocal images (upper panel) of merged red (PAmCherry-KFERQ) and blue (DAPI) for nuclei highlighted with (circular insets) magnification. Scale bars: 40 μm. Quantification (lower panel) of CMA activity as number of fluorescent puncta per cell. For each replicate (n exp = 3), at least four images were analyzed. Each dot in the graph represents the average number of puncta per cell. P values refer to *** P < 0.001, ns: non-significant (A549: P WT Ctrl vs CMA = 0.0008, P KO Ctrl vs CMA = 0.28; HT1080: P WT Ctrl vs CMA = 0,0013, P KO Ctrl vs CMA = 0,75; Student’s t -test). .

Article Snippet: AC220 , Selleckchem , S1526.

Techniques: CRISPR, Agarose Gel Electrophoresis, Knock-Out, Amplification, Marker, Quantitative RT-PCR, Expressing, Western Blot, Control, Immunofluorescence, Staining, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: Chaperone-mediated autophagy regulates the metastatic state of mesenchymal tumors

doi: 10.1038/s44321-025-00210-w

Figure Lengend Snippet: ( A ) Immunoblot detection of indicated LAMP, EMT markers, and TGFβ signaling proteins in an ovarian carcinoma cancer cell line panel spanning from epithelial to mesenchymal states. β -actin: loading control. ( B ) RT-qPCR detection of LAMP-2A expression in the indicated cell line panel as in A ( P < 0.0001; ANOVA). ( C ) RT-qPCR detection of VIM (Vimentin), CDH1 (E-cad), and LAMP-2A expression in untreated and TGFβ ligand treated OVPA8 cells for 48 h to induce EMT ( P VIM < 0.0001, P CDH1 = 0.0099, P LAMP-2A = 0.0012; Student’s t -test). ( D ) RT-qPCR detection of LAMP-2A expression following CMA activation (AC220+Spautin1 16 h) or TGFβ ligand treatment for 48 h in OVPA8 cells ( P Ctrl vs CMA < 0.0001, P CMA vs CMA+TGFβ = 0.0002; ANOVA). ( E ) RT-qPCR detection of LAMP-2A expression (left panel; P Ctrl vs Tranilast < 0.0001; P Ctrl vs CMA < 0.0001; ANOVA) and immunoblot detection (right panel) of LAMP-2A protein level in FUOV1 cells treated with the TGFβ inhibitor (Tranilast) or following CMA activation (AC220+Spautin1) for 16 h. β -actin: loading control. ( F ) RT-qPCR detection of TGFβR2 and LAMP-2A expression following siRNA-mediated TGFβR2 knockdown (KD) in ES2 (TGFβR2: P si#1 < 0.0001, P si#2 < 0.0001; LAMP-2A: P si#1 < 0.0001, P si#2 < 0.0001; ANOVA) and FUOV1 (TGFβR2: P si#1 < 0.0001, P si#2 < 0.0001; LAMP-2A: P si#1 = 0.0069, P si#2 = 0.083; ANOVA) cells. The KD efficiency is indicated by TGFβR2 expression. All RT-qPCR graphs (n exp = 3) as indicated by the data points. Bars represent mean ± sd. P values refer to * P < 0.05, ** P < 0.01, *** P < 0.001. ( G ) Immunoblot detection of LAMP-2A protein levels following siRNA-mediated TGFβR2 KD in FUOV1 cells. β -actin: loading control. All immunoblot data are presented with their quantification (n exp = 3) indicated by the average signal intensity for each protein band. .

Article Snippet: AC220 , Selleckchem , S1526.

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Activation Assay, Knockdown

Journal: EMBO Molecular Medicine

Article Title: Chaperone-mediated autophagy regulates the metastatic state of mesenchymal tumors

doi: 10.1038/s44321-025-00210-w

Figure Lengend Snippet: ( A ) CMA activity measured in FUOV1 cells expressing PAmCherry-KFERQ CMA reporter. Cells were treated with the TGFβ inhibitor (Tranilast) or AC220+Spautin1 to activate CMA for 16 h. (Left panel) Immunofluorescence confocal images of merged red (PAmCherry-KFERQ) and blue (nuclei, DAPI) highlighted with (circular insets) magnification. The scale: 50 µm. (Right panel) Quantification of number of puncta per cell (n cell > 50) represented by mean ± sd. P values refer to P *** < 0.001 ( P Ctrl vs Tranilast < 0.0001, P Ctrl vs CMA < 0.0001; ANOVA). ( B ) Immunoblot detection of indicated TGFβ signaling and CMA substrate protein level in ES2 cells treated with Tranilast or CMA activation (AC220+Spautin1) for 16 h. β -actin: loading control. ( C ) Immunoblot detection of TGFβR2 protein level in FUOV1 and ES2 cell lines following treatment with chloroquine (CQ) alone, Tranilast, Tranilast+CQ, CMA (AC220+Spautin1), or CMA + CQ for 16 h. β -actin: loading control. LE: long exposure, SE: short exposure. ( D ) Fold TMT intensity enrichment of TGFβR2 level in isolated lysosomes from control and siRNA-mediated LAMP-2A KD ES2 cells analyzed by mass spectrometry. Data presented (n exp = 4) as indicated by the data points P values refer to ** P < 0.01 ( P = 0.0025; Student’s t -test). The boxes extend from the 25th to 75th percentile, the middle line shows the median, whiskers extend to the most extreme data. ( E ) Immunoblot detection of EMT-TF proteins in control and CMA activated (AC220+Spautin1) FUOV1 and ES2 cells. β -actin: loading control. ( F ) Immunoblot detection of Snail protein in OVPA8 cells treated with the TGFβ ligand for 48 h and/or with CMA activation (AC220+Spautin1). β -actin: loading control. ( G ) Immunoblot detection of TGFβR2 following siRNA-mediated LAMP-2A KD in OVPA8 cells. β -actin: loading control. Immunoblot detection of TGFβ signaling proteins in WT or LAMP-2A KO HT1080 and A549 ( H ) cell lines, and ( I ) tumor lysates. β -actin or vinculin: loading control. ( J ) Immunoblot detection of LAMP-2A and TGFβR2 following re-expression (RE) of LAMP-2A in the LAMP-2A KO HT1080 cells. β -actin: loading control. All immunoblot data are presented with their quantification (n exp = 3) indicated by the average signal intensity for each protein band. The statistical significance is presented as Appendix Table . .

Article Snippet: AC220 , Selleckchem , S1526.

Techniques: Activity Assay, Expressing, Immunofluorescence, Western Blot, Activation Assay, Control, Isolation, Mass Spectrometry

Journal: EMBO Molecular Medicine

Article Title: Chaperone-mediated autophagy regulates the metastatic state of mesenchymal tumors

doi: 10.1038/s44321-025-00210-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: AC220 , Selleckchem , S1526.

Techniques: Recombinant, Sequencing, Isolation, Plasmid Preparation, In Vivo, In Vitro, Protease Inhibitor, Bicinchoninic Acid Protein Assay, cDNA Synthesis, SYBR Green Assay, H2O2 Assay, Software, Microscopy, Inverted Microscopy, Real-time Polymerase Chain Reaction