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Figure 2. AARS1-mediated lactylation of YTHDC1 promotes downregulation of YTHDC1 protein levels (A) <t>HT1376</t> and RT112 cells were treated with the glucose (40 mM) or lactate (10 mM) for 48 h, then the cells were collected for western blotting and ﬂow cytometry analysis. (B) RNA-seq analyzed after knockdown YTHDC1 in 5637 cells. (C) Protein levels of YTHDC1 and NECTIN4 in BC samples from patients with normal blood glucose and diabetes were analyzed by western blotting; the relative quantitative analysis of proteins was performed using ImageJ software. N indicates normal glucose. D indicates diabetes. (D and E) HT1376 and RT112 cells were transfected with the speciﬁed shRNA or plasmid for 72 h, and, after puromycin screening, the harvested cells were used for protein imprinting, RT-qPCR analysis, and ﬂow cytometry analysis. (F and G) Tissue samples from patients with or without diabetic BC were analyzed by IHC staining using anti-YTHDC1 antibodies and anti-NECTIN4 antibodies (F); paired tests were performed on patients with IHC (G). (H and I) IHC staining of BC tissue microarrays using YTHDC1 and NECTIN4 antibodies. Typical images are shown in (H). The correlation between YTHDC1 and NECTIN4 is shown in (I). (J and K) HT1376 cells were treated with the speciﬁed chemical for 48 or 72 h. Then the cells were harvested for western blot analysis and RT-qPCR analysis. (L and M) Endogenous YTHDC1 lactylation was analyzed in HT1376 and RT112 cells treated with the speciﬁed chemical for 48 h by immunoprecipitation western blotting with pan-Kla antibodies; the relative quantitative analysis of proteins was performed using ImageJ software. (N) HT1376 and RT112 cells were transfected with the speciﬁed shRNA for 72 h, after puromycin screening, and the cells were harvested. Endogenous YTHDC1 lactylation was analyzed by immunoprecipitation western blotting with pan-Kla antibodies and the YTHDC1 levels were analyzed by RT-qPCR. (O) Diagram of YTHDC1 K82 site.

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1) Product Images from "High-glucose-associated YTHDC1 lactylation reduces the sensitivity of bladder cancer to enfortumab vedotin therapy."

Article Title: High-glucose-associated YTHDC1 lactylation reduces the sensitivity of bladder cancer to enfortumab vedotin therapy.

Journal: Cell reports

doi: 10.1016/j.celrep.2025.115545

Figure 2. AARS1-mediated lactylation of YTHDC1 promotes downregulation of YTHDC1 protein levels (A) HT1376 and RT112 cells were treated with the glucose (40 mM) or lactate (10 mM) for 48 h, then the cells were collected for western blotting and ﬂow cytometry analysis. (B) RNA-seq analyzed after knockdown YTHDC1 in 5637 cells. (C) Protein levels of YTHDC1 and NECTIN4 in BC samples from patients with normal blood glucose and diabetes were analyzed by western blotting; the relative quantitative analysis of proteins was performed using ImageJ software. N indicates normal glucose. D indicates diabetes. (D and E) HT1376 and RT112 cells were transfected with the speciﬁed shRNA or plasmid for 72 h, and, after puromycin screening, the harvested cells were used for protein imprinting, RT-qPCR analysis, and ﬂow cytometry analysis. (F and G) Tissue samples from patients with or without diabetic BC were analyzed by IHC staining using anti-YTHDC1 antibodies and anti-NECTIN4 antibodies (F); paired tests were performed on patients with IHC (G). (H and I) IHC staining of BC tissue microarrays using YTHDC1 and NECTIN4 antibodies. Typical images are shown in (H). The correlation between YTHDC1 and NECTIN4 is shown in (I). (J and K) HT1376 cells were treated with the speciﬁed chemical for 48 or 72 h. Then the cells were harvested for western blot analysis and RT-qPCR analysis. (L and M) Endogenous YTHDC1 lactylation was analyzed in HT1376 and RT112 cells treated with the speciﬁed chemical for 48 h by immunoprecipitation western blotting with pan-Kla antibodies; the relative quantitative analysis of proteins was performed using ImageJ software. (N) HT1376 and RT112 cells were transfected with the speciﬁed shRNA for 72 h, after puromycin screening, and the cells were harvested. Endogenous YTHDC1 lactylation was analyzed by immunoprecipitation western blotting with pan-Kla antibodies and the YTHDC1 levels were analyzed by RT-qPCR. (O) Diagram of YTHDC1 K82 site.

Figure Legend Snippet: Figure 2. AARS1-mediated lactylation of YTHDC1 promotes downregulation of YTHDC1 protein levels (A) HT1376 and RT112 cells were treated with the glucose (40 mM) or lactate (10 mM) for 48 h, then the cells were collected for western blotting and ﬂow cytometry analysis. (B) RNA-seq analyzed after knockdown YTHDC1 in 5637 cells. (C) Protein levels of YTHDC1 and NECTIN4 in BC samples from patients with normal blood glucose and diabetes were analyzed by western blotting; the relative quantitative analysis of proteins was performed using ImageJ software. N indicates normal glucose. D indicates diabetes. (D and E) HT1376 and RT112 cells were transfected with the speciﬁed shRNA or plasmid for 72 h, and, after puromycin screening, the harvested cells were used for protein imprinting, RT-qPCR analysis, and ﬂow cytometry analysis. (F and G) Tissue samples from patients with or without diabetic BC were analyzed by IHC staining using anti-YTHDC1 antibodies and anti-NECTIN4 antibodies (F); paired tests were performed on patients with IHC (G). (H and I) IHC staining of BC tissue microarrays using YTHDC1 and NECTIN4 antibodies. Typical images are shown in (H). The correlation between YTHDC1 and NECTIN4 is shown in (I). (J and K) HT1376 cells were treated with the speciﬁed chemical for 48 or 72 h. Then the cells were harvested for western blot analysis and RT-qPCR analysis. (L and M) Endogenous YTHDC1 lactylation was analyzed in HT1376 and RT112 cells treated with the speciﬁed chemical for 48 h by immunoprecipitation western blotting with pan-Kla antibodies; the relative quantitative analysis of proteins was performed using ImageJ software. (N) HT1376 and RT112 cells were transfected with the speciﬁed shRNA for 72 h, after puromycin screening, and the cells were harvested. Endogenous YTHDC1 lactylation was analyzed by immunoprecipitation western blotting with pan-Kla antibodies and the YTHDC1 levels were analyzed by RT-qPCR. (O) Diagram of YTHDC1 K82 site.

Techniques Used: Western Blot, Cytometry, RNA Sequencing, Knockdown, Software, Transfection, shRNA, Plasmid Preparation, Quantitative RT-PCR, Immunohistochemistry, Immunoprecipitation

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.

Figure Legend 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.

Techniques Used: Ubiquitin Proteomics, RNA Sequencing, Transfection, Plasmid Preparation, Western Blot, shRNA, Software

Figure 4. YTHDC1 plays a key role in regulating BC sensitivity to EV in a m6A-dependent manner (A and B) HT1376 and RT112 cells were transfected with the speciﬁed shRNA for 72 h; after puromycin screening, harvested cells were analyzed for protein imprints (A), treated with continuous doses of EV for 24 h, and harvested for CCK-8 assays to measure the IC50 values of EV (B). (C–G) HT1376 and RT112 cells were transfected with the speciﬁed shRNA for 72 h. After puromycin selection, cells were treated with or without EV (1.0 nM) and subjected to CCK-8 assay (C), colony-formation assay (D), western blot analysis (E), caspase-3 activity assay (F), and annexin V-FITC/PI assay (G). (H–J) HT1376 cells were transfected with the designated shRNA for 72 h. After puromycin screening, these cells were injected subcutaneously into nude mice. These mice received or did not receive EV (intraperitoneal injection, 1 mg/kg, once every 3 days ﬁve times in a row). Tumor image (H); tumor volume (I); and tumor growth curve (J). Data were expressed as the mean ± SD and repeated ﬁve times. *p < 0.05; ***p < 0.001. (K–N) HT1376 and RT112 cells were transfected with the speciﬁed shRNA for 72 h. After puromycin screening, the cells were treated with glucose (40 mM) or lactate (10 mM) for another 48 h. Then the cells were harvested for protein imprints (K and M) or treated with continuous doses of EV for 24 h and harvested for CCK-8 assays to measure the IC50 values of EV (L and N). (O–R) HT1376 and RT112 cells were transfected with the speciﬁed shRNA for 72 h. After puromycin selection, the cells were treated with glucose (40 mM) or lactate (10 mM) or EV (1.0 nM) and subjected to CCK-8 assay (O and P) or caspase-3 activity assay (Q and R). Data are representative of three independent experiments (C, D, F, G, and O–R). All data were expressed as the mean ± SD and repeated three times. ns, not signiﬁcant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure Legend Snippet: Figure 4. YTHDC1 plays a key role in regulating BC sensitivity to EV in a m6A-dependent manner (A and B) HT1376 and RT112 cells were transfected with the speciﬁed shRNA for 72 h; after puromycin screening, harvested cells were analyzed for protein imprints (A), treated with continuous doses of EV for 24 h, and harvested for CCK-8 assays to measure the IC50 values of EV (B). (C–G) HT1376 and RT112 cells were transfected with the speciﬁed shRNA for 72 h. After puromycin selection, cells were treated with or without EV (1.0 nM) and subjected to CCK-8 assay (C), colony-formation assay (D), western blot analysis (E), caspase-3 activity assay (F), and annexin V-FITC/PI assay (G). (H–J) HT1376 cells were transfected with the designated shRNA for 72 h. After puromycin screening, these cells were injected subcutaneously into nude mice. These mice received or did not receive EV (intraperitoneal injection, 1 mg/kg, once every 3 days ﬁve times in a row). Tumor image (H); tumor volume (I); and tumor growth curve (J). Data were expressed as the mean ± SD and repeated ﬁve times. *p < 0.05; ***p < 0.001. (K–N) HT1376 and RT112 cells were transfected with the speciﬁed shRNA for 72 h. After puromycin screening, the cells were treated with glucose (40 mM) or lactate (10 mM) for another 48 h. Then the cells were harvested for protein imprints (K and M) or treated with continuous doses of EV for 24 h and harvested for CCK-8 assays to measure the IC50 values of EV (L and N). (O–R) HT1376 and RT112 cells were transfected with the speciﬁed shRNA for 72 h. After puromycin selection, the cells were treated with glucose (40 mM) or lactate (10 mM) or EV (1.0 nM) and subjected to CCK-8 assay (O and P) or caspase-3 activity assay (Q and R). Data are representative of three independent experiments (C, D, F, G, and O–R). All data were expressed as the mean ± SD and repeated three times. ns, not signiﬁcant; *p < 0.05; **p < 0.01; ***p < 0.001.

Techniques Used: Transfection, shRNA, CCK-8 Assay, Selection, Colony Assay, Western Blot, Caspase-3 Activity Assay, Injection

Figure 5. YTHDC1 enhances the mRNA stability of NECTIN4 transcription factor JUND in a m6A-dependent manner (A) The Ominer network tool predicts potential transcription factors for NECTIN4. (B) ChIP-seq from JUND on the NECTIN4 promoter region of the ChIP-Atlas. (C and D) HT1376 and RT112 cells were transfected with the speciﬁed shRNA or plasmid for 72 h, and, after puromycin screening, the cells were collected for ChIP-qPCR analysis using immunoglobulin (Ig)G or JUND antibodies.

Figure Legend Snippet: Figure 5. YTHDC1 enhances the mRNA stability of NECTIN4 transcription factor JUND in a m6A-dependent manner (A) The Ominer network tool predicts potential transcription factors for NECTIN4. (B) ChIP-seq from JUND on the NECTIN4 promoter region of the ChIP-Atlas. (C and D) HT1376 and RT112 cells were transfected with the speciﬁed shRNA or plasmid for 72 h, and, after puromycin screening, the cells were collected for ChIP-qPCR analysis using immunoglobulin (Ig)G or JUND antibodies.

Techniques Used: ChIP-sequencing, Transfection, shRNA, Plasmid Preparation, ChIP-qPCR

Figure 6. YTHDC1 regulates the sensitivity of BC to EV through the JUND-NECTIN4 axis (A and B) HT1376 and RT112 cells were transfected with the speciﬁed shRNA and plasmids for 72 h after puromycin screening. Then the cells were harvested for western blot analysis (A) and RT-qPCR analysis (B). (C) HT1376 and RT112 cells were transfected with the speciﬁed shRNA and plasmids for 72 h after puromycin screening. The cells were treated with continuous doses of EV for 24 h and harvested for CCK-8 assays to measure the IC50 values of EV. (D–I) HT1376 cells were transfected with the speciﬁed shRNA and plasmids for 72 h. After puromycin selection, the cells were collected for colony-formation assay (D), CCK-8 assay (E), western blot analysis (F), caspase-3 activity assay (G), and annexin V-FITC/PI assay (H and I). (J–L) HT1376 cells were transfected with designated shRNA for 72 h. After puromycin screening, these cells were injected subcutaneously into nude mice. These mice received or did not receive EV (intraperitoneal injection, 1 mg/kg once every 3 days ﬁve times in a row). Tumor image (J); tumor volume (K); tumor growth curve (L). Data were expressed as the mean ± SD and repeated ﬁve times. ns, not signiﬁcant; *p < 0.05; **p < 0.01; ***p < 0.001. Data are representative of three independent experiments (B, D, E, G, and I). All data were expressed as the mean ± SD and repeated three times. ns, not signiﬁcant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure Legend Snippet: Figure 6. YTHDC1 regulates the sensitivity of BC to EV through the JUND-NECTIN4 axis (A and B) HT1376 and RT112 cells were transfected with the speciﬁed shRNA and plasmids for 72 h after puromycin screening. Then the cells were harvested for western blot analysis (A) and RT-qPCR analysis (B). (C) HT1376 and RT112 cells were transfected with the speciﬁed shRNA and plasmids for 72 h after puromycin screening. The cells were treated with continuous doses of EV for 24 h and harvested for CCK-8 assays to measure the IC50 values of EV. (D–I) HT1376 cells were transfected with the speciﬁed shRNA and plasmids for 72 h. After puromycin selection, the cells were collected for colony-formation assay (D), CCK-8 assay (E), western blot analysis (F), caspase-3 activity assay (G), and annexin V-FITC/PI assay (H and I). (J–L) HT1376 cells were transfected with designated shRNA for 72 h. After puromycin screening, these cells were injected subcutaneously into nude mice. These mice received or did not receive EV (intraperitoneal injection, 1 mg/kg once every 3 days ﬁve times in a row). Tumor image (J); tumor volume (K); tumor growth curve (L). Data were expressed as the mean ± SD and repeated ﬁve times. ns, not signiﬁcant; *p < 0.05; **p < 0.01; ***p < 0.001. Data are representative of three independent experiments (B, D, E, G, and I). All data were expressed as the mean ± SD and repeated three times. ns, not signiﬁcant; *p < 0.05; **p < 0.01; ***p < 0.001.

Techniques Used: Transfection, shRNA, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Selection, Colony Assay, Caspase-3 Activity Assay, Injection

Figure 7. JNK activators enhance the sensitivity of BC to EV (A) HT1376 and RT112 cells were treated with or without anisomycin (10 mM) for 24 h. The cells were treated with a series of doses of EV, harvested for CCK-8 assays, and the IC50 value of EV was measured. (B–E) HT1376 and RT112 cells were treated with the speciﬁed chemical for 24 h, then the cells were collected for colony formation (B), western blot analysis (C), caspase-3 activity assay (D), and annexin V-FITC/PI assay (E). (F and G) HT1376 and RT112 cells were treated with the speciﬁed chemical for 48 h. Then the cells were treated with a series of doses of EV, harvested for CCK-8 assays, and the IC50 value of EV was measured. (H–M) HT1376 and RT112 cells were treated with the speciﬁed chemical for 48 h. Then the cells were collected for the CCK-8 assay (H and I), western blot analysis (J and K), and caspase-3 activity assay (L and M). (N–P) HT1376 cells were treated with or without lactate (10 mM) for 48 h; these cells were injected subcutaneously into nude mice pretreated with WFI or lactate (intraperitoneal injection, 1 g/kg once daily for 7 days). These mice received WFI/lactate (intraperitoneal injection, 1 g/kg, once every 3 days ﬁve times in a row)/EV (intraperitoneal injection, 1 mg/kg once every 3 days ﬁve times in a row) or anisomycin (intraperitoneal injection, 15 mg/kg once every 3 days ﬁve times in a row) treatment. Tumor image (N); tumor volume (O); tumor growth curve (P). Data were expressed as the mean ± SD and repeated ﬁve times. *p < 0.05; **p < 0.01; ***p < 0.001. (Q) HT1376 and RT112 cells were treated with or without b-alanine (50 mM) for 24 h. The cells were treated with a series of doses of EV, harvested for CCK-8 assays, and the IC50 value of EV was measured. (R–U) HT1376 and RT112 cells were treated with b-alanine (50 mM) for 24 h. Then the cells were treated with or without EV (1.0 nM) for 24 h and subjected to colony (R), western blot analysis (S), caspase-3 activity assay (T), and annexin V-FITC/PI assay (U). Data are representative of three independent experiments (B, D, E, H–K, and R–U). All data were expressed as the mean ± SD and repeated three times. ns, not signiﬁcant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure Legend Snippet: Figure 7. JNK activators enhance the sensitivity of BC to EV (A) HT1376 and RT112 cells were treated with or without anisomycin (10 mM) for 24 h. The cells were treated with a series of doses of EV, harvested for CCK-8 assays, and the IC50 value of EV was measured. (B–E) HT1376 and RT112 cells were treated with the speciﬁed chemical for 24 h, then the cells were collected for colony formation (B), western blot analysis (C), caspase-3 activity assay (D), and annexin V-FITC/PI assay (E). (F and G) HT1376 and RT112 cells were treated with the speciﬁed chemical for 48 h. Then the cells were treated with a series of doses of EV, harvested for CCK-8 assays, and the IC50 value of EV was measured. (H–M) HT1376 and RT112 cells were treated with the speciﬁed chemical for 48 h. Then the cells were collected for the CCK-8 assay (H and I), western blot analysis (J and K), and caspase-3 activity assay (L and M). (N–P) HT1376 cells were treated with or without lactate (10 mM) for 48 h; these cells were injected subcutaneously into nude mice pretreated with WFI or lactate (intraperitoneal injection, 1 g/kg once daily for 7 days). These mice received WFI/lactate (intraperitoneal injection, 1 g/kg, once every 3 days ﬁve times in a row)/EV (intraperitoneal injection, 1 mg/kg once every 3 days ﬁve times in a row) or anisomycin (intraperitoneal injection, 15 mg/kg once every 3 days ﬁve times in a row) treatment. Tumor image (N); tumor volume (O); tumor growth curve (P). Data were expressed as the mean ± SD and repeated ﬁve times. *p < 0.05; **p < 0.01; ***p < 0.001. (Q) HT1376 and RT112 cells were treated with or without b-alanine (50 mM) for 24 h. The cells were treated with a series of doses of EV, harvested for CCK-8 assays, and the IC50 value of EV was measured. (R–U) HT1376 and RT112 cells were treated with b-alanine (50 mM) for 24 h. Then the cells were treated with or without EV (1.0 nM) for 24 h and subjected to colony (R), western blot analysis (S), caspase-3 activity assay (T), and annexin V-FITC/PI assay (U). Data are representative of three independent experiments (B, D, E, H–K, and R–U). All data were expressed as the mean ± SD and repeated three times. ns, not signiﬁcant; *p < 0.05; **p < 0.01; ***p < 0.001.

Techniques Used: CCK-8 Assay, Western Blot, Caspase-3 Activity Assay, Injection

CCK-8 Assay:

Article Title: High-glucose-associated YTHDC1 lactylation reduces the sensitivity of bladder cancer to enfortumab vedotin therapy.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal Anti-L-Lactyl Lysine Pioneer in Proteomics Cat# PTM-1401RM; RRID: AB_2942013 Rabbit polyclonal anti-YTHDC1 Proteintech Cat# 29441-1-AP; RRID: AB_2918307 Mouse monoclonal Anti-NECTIN4 Proteintech Cat# 67721-1-Ig; RRID: AB_2882908 Rabbit polyclonal anti-AARS1 Proteintech Cat# 17394-1-AP; RRID: AB_2219748 Rabbit polyclonal anti- Cleaved Caspase 3 Proteintech Cat# 25128-1-AP; RRID: AB_3073913 Rabbit polyclonal anti- Beta Actin Proteintech Cat# 81115-1-RR; RRID: AB_2923704 Rabbit polyclonal anti- JNK Proteintech Cat# 17337-1-AP; RRID: AB_2878388 Mouse monoclonal anti- Phospho-JNK Proteintech Cat# 60666-1-Ig; RRID: - Rat polyclonal anti- HA tag Proteintech Cat# 7c9; RRID: AB_2631399 Rabbit polyclonal anti- FLAG tag Proteintech Cat# 80801-2-RR; RRID: - Rabbit polyclonal anti- MYC tag Proteintech Cat# 16286-1-AP; RRID: AB_11182162 Rabbit polyclonal anti-RNF183 Abcam Cat# ab197321; RRID: - Rabbit polyclonal antibody-JUND Affinity Biosciences Cat # AF6200; RRID: AB_2835081 Rabbit polyclonal antibody-NECTIN4 ABclonal Cat # A25200; RRID: AB_3675333 Rabbit IgG isotype control ABclonal Cat # A22070; RRID: AB_367533 488-conjugated Goat anti-Rabbit IgG (H + L) ABclonal Cat # AS073; RRID: AB_2768317 Bacterial and virus strains E.coli HB101 DL-Stbl3 Tsingke Cat# DLC106 Biological samples Clinical specimens Bladder cancer tissue from Henan Cancer Hospital N/A Tissue microarray slides Bioaitech Cat# U100BI01T Patient-derived xenografts (PDX) Nectin4-positive bladder cancer tissue from Henan Cancer Hospital N/A Chemicals, peptides, and recombinant proteins Water for injection (WFI) ThermoFisher Cat# A1287301 L-lactate MedChemExpress Cat# 867-56-1 Lactate sodium (NaLac) MedChemExpress Cat# 72-17-3 Oxamate MedChemExpress Cat# 565-73-1 Glucose Selleck Cat# S3131 (Continued on next page) 18 Cell Reports 44, 115545, April 22, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Mannitol Selleck Cat# S2381 Streptozocin (STZ) Beyotime Cat# 18883-66-4 MG132 Selleck Cat# S2619 Anisomycin Beyotime Cat# SC0132 b-Alanine MedChemExpress Cat# 107-95-9 Enfortumab Vedotin MedChemExpress Cat# 346452-25-2 Lipofectamine 2000 reagent Thermo Fisher Cat# 11668019 Trizol Accurate Biotechnology Cat# AG21102 Critical commercial assays L-Lactate Assay Kit Solarbio Cat# BC2235 Cell Counting Kit-8 Beyotime Cat# C0037 Caspase-3 assay kit Abcam Cat# ab39401 Annexin V-FITC/PI assay Vazyme Cat# A211-02 EndoFree Plasmid Midi Kit CWBIO Cat# CW2105S ClonExpress II One Step Cloning Kit Vazyme Cat# C112-01 BeyoChIPTM Enzymatic ChIP Assay Kit Beyotime Cat# P2083S Evo M-MLV reverse transcription premix kit Accurate Cat# AG11728 SYBR Green Pro Taq HS premixed qPCR kit Accurate Cat# AG11701 Deposited data Raw and analyzed sequencing data This paper GEO: GSE291591/GSE291592 Experimental models: Cell lines Human: HT1376 ATCC Cat# SC0215 Human: RT112 ATCC Cat# SC0651 Human: 5637 ATCC Cat# SC0214 Human: T24 ATCC Cat# SC0113 Experimental models: Organisms/strains Mouse: BALB/c-nu/nu Hunan SJA Experimental Animal Company N/A Mouse: NOD-PrkdcscidIl2rgem1/Smoc (NSG) Shanghai model organisms Cat# NM-NSG-001 Oligonucleotides See Tables S3–S5 for primer sequences Recombinant DNA CMV-HA-YTHDC1 WT Miaoling Biology Cat# P44598 CMV-HA-YTHDC1 Mut (W377A, W428A) This paper Xiao, Wen et al., 201644 CMV-HA-YTHDC1 K82R This paper N/A CMV-HA-YTHDC1 K127R This paper N/A CMV-HA-YTHDC1 2KR (K82R, K127R) This paper N/A CMV- Myc-UB Miaoling Biology Cat# P59265 CMV- Flag-AARS1 Miaoling Biology Cat# P68100 CMV- Flag-JUND Miaoling Biology Cat# P20773 PCDH-33Myc This paper Deng, Zebin et al., 202377 PCDH-33Flag This paper N/A PCDH-33HA This paper N/A Software and algorithms ImageJ NIH N/A FlowJo FlowJo LLC N/A GraphPad Prism 8 Graphpad, lnc N/A R 4.2.2 R-Project N/A Cell Reports 44, 115545, April 22, 2025 19 ..