Journal: Cell reports
Article Title: High-glucose-associated YTHDC1 lactylation reduces the sensitivity of bladder cancer to enfortumab vedotin therapy.
doi: 10.1016/j.celrep.2025.115545
Figure Lengend Snippet: Figure 3. AARS1-mediated lactylation of YTHDC1 promotes ubiquitination degradation of YTHDC1 induced by RNF183 (A) HT1376 cells were treated with 1.0 nM EV for 48 h. Cells were collected for RNA-seq analysis. (B and C) KEGG enrichment analysis (B) and GSEA enrichment analysis (C) of RNA-seq in HT1376 cells. (D) HT1376 cells were transfected with the specified plasmid for 72 h; after puromycin screening, the cells were treated with the specified chemical MG132 for 8 h. The harvested cells were coIP assays with anti-hemagglutinin (HA) antibodies. (E and G) HT1376 and RT112 cells were treated with the specified chemical for 24 h, then the cells were treated with MG132 for 8 h, and the cells were harvested for western blot analysis. (F and H) HT1376 and RT112 cells were transfected with the specified shRNA and plasmid for 72 h; after puromycin screening, the cells were treated with the specified chemical for 24 h, and the cells were harvested for western blot analysis. (I) HT1376 and RT112 cells were transfected with the specified plasmid for 72 h; after puromycin screening, the cells were treated with the specified chemical MG132 for 8 h, and the cells were harvested for western blot analysis. (J) HT1376 and RT112 cells were transfected with the specified shRNA and plasmids for 72 h after puromycin screening and the cells were harvested for western blot analysis. (K) HT1376 and RT112 cells were transfected with the specified shRNA for 72 h; after puromycin screening, the harvested cells were coIP assayed with anti- YTHDC1 or RNF183 antibodies. (L) HT1376 and RT112 cells were transfected with the specified shRNA and plasmid for 72 h; after puromycin screening, the cells were treated with MG132 for 8 h, and IP and western blot analysis were performed. (M) HT1376 and RT112 cells were transfected with the specified shRNA for 72 h; after puromycin screening, the cells were treated with cycloheximide (CHX), and the cells were collected at various time points for western blot analysis. The relative quantitative analysis of proteins was performed using ImageJ software. (N) HT1376 cells were transfected with the specified shRNA and plasmids for 72 h; after puromycin screening, the cells were treated with the specified chemical MG132 for 8 h, and the cells were harvested for western blot analysis. (O) HT1376 cells were transfected with the specified plasmids for 24 h. After puromycin screening, the cells were treated with lactate (10 nM) for 24 h, and the harvested cells were coIP assayed with anti-HA antibodies or RNF183 antibodies. (P) HT1376 cells were transfected with the specified shRNA and plasmids for 72 h; after puromycin screening, the harvested cells were coIP assayed with anti-HA antibodies or RNF183 antibodies.
Article Snippet: The following antibodies were used for western blot analysis: Pan-Kla (#PTM-1401RM, Pioneer in Proteomics, 1:1000 dilution), YTHDC1 (#29441-1-AP, Proteintech, 1:500 dilution), NECTIN4 (#67721-1-lg, Proteintech, 1:2000 dilution), AARS1 (#17394-1-AP, Proteintech, 1:2000 dilution), RNF183 (#ab197321, Abcam, 1:1000 dilution), JUND (#AF6200, Affinity Biosciences, 1:2000 dilution), JNK (#HY-P80728, MedChemExpress, 1:2000 dilution), p-JNK (#ab124956, Abcam, 1:2000 dilution), cleaved caspase-3 (#19677-1- AP, Proteintech, 1:1000 dilution), b-actin (#81115-1-RR, Proteintech, 1:100000 dilution), HA-tag (#CL594-51064, Proteintech, 1:2000 dilution), FLAG tag (#P55001, ProMab Biotechnologies Inc., 1:50000 dilution), and MYC-tag (#16286-1-AP, Proteintech, 1:5000 dilution).
Techniques: Ubiquitin Proteomics, RNA Sequencing, Transfection, Plasmid Preparation, Western Blot, shRNA, Software
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