anti ythdc1 cst cat 77422  (Cell Signaling Technology Inc)

Journal: Advanced Science

Article Title: YTHDC1 Orchestrates Glucose and Glutamate Rewiring to Overcome Lethal Metabolic Stress in Triple‐Negative Breast Cancer

doi: 10.1002/advs.77492

Figure Lengend Snippet: YTHDC1 rewires glucose and glutamine metabolism in TNBC. (A,B) Survival analysis of TNBC patients from Kaplan–Meier plotter database reveals that high expression of YTHDC1 is significantly associated with poorer overall survival (OS) (A) and progression‐free survival (PFS) (B). (C,D) Immunohistochemistry of YTHDC1 in tissue microarray (TMA) reveals that expression of YTHDC1 in TNBC is significantly higher than in non‐TNBC and normal tissue. Scale bars, 20 µm. (E) Venn diagram analysis integrating RNA‐seq (MDA‐MB‐231and Hs578T), m6A‐seq (MDA‐MB‐231), and YTHDC1 RIP‐seq (MDA‐MB‐231) data identifies 347 protein‐coding genes as high‐confidence m6A‐dependent targets of YTHDC1. Hypergeometric testing confirms that the three‐way intersection is highly significant ( p < 0.05). (F) Gene Ontology (GO) analysis of the 347 candidate genes from (D) reveals significant enrichment in cellular metabolic process. (G) TNBC samples with high YTHDC1 expression exhibit higher ssGSEA scores of glutamine (Gln) metabolism and pentose phosphate pathways (PPP) signature compared to those with low YTHDC1 expression in METABRIC‐TNBC cohort (triple‐negative breast cancer cohort from the METABRIC database). (H) Metabolomic profiling in control and YTHDC1 KD MDA‐MB‐231 cells. Enrichment analysis illustrates that the citrate cycle (TCA cycle), glycolysis, and pentose phosphate pathways (PPP) are among the most significantly altered metabolic processes. (I) Heatmap depicting the relative abundance of metabolites from the pathways highlighted in (G) in YTHDC1 KD MDA‐MB‐231 cells. (J) A schematic diagram of the glucose and glutamine metabolism. The metabolites of these two processes were affected by YTHDC1 KD, as detected by metabolomic analysis. (K–M) Intracellular metabolite measurements show that YTHDC1 KD leads to decreased levels of glutamine (Gln) (K), glutamate (Glu) (L), and glucose‐6‐phosphate (G6P) (M). (N) YTHDC1 KD reduces glucose uptake of TNBC cells, as measured by the 2‐NBDG assay. Scale bars, 50 µm. Data are presented as mean ± SD of experimental triplicates (D,J–O). p values were assessed with Kaplan–Meier survival analysis (A,B), and two‐tailed Student's t‐test (C,J–N). ( * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: YTHDC1 antibody (ABclonal, A22992) (1:500) was incubated overnight at 4°C, and secondary antibodies was incubated for 1 h at 37°C.

Techniques: Expressing, Immunohistochemistry, Microarray, RNA Sequencing, Metabolomic, Control, Two Tailed Test

Journal: Advanced Science

Article Title: YTHDC1 Orchestrates Glucose and Glutamate Rewiring to Overcome Lethal Metabolic Stress in Triple‐Negative Breast Cancer

doi: 10.1002/advs.77492

Figure Lengend Snippet: YTHDC1 regulates ATF4 and GLUT3 mRNA in an m6A‐dependent manner to coordinate TNBC metabolism. (A,B) Overlap of YTHDC1 target genes with genes in the glucose and glutamine pathways. (C) Representative tracks of RNA‐seq, m6A RIP‐seq, and YTHDC1 RIP‐seq at the ATF4 and GLUT3 loci in control versus YTHDC1 KD MDA‐MB‐231 cells, demonstrating YTHDC1 binding, m6A modification, and transcript abundance changes. (D) m6A RIP followed by RT‐qPCR showing m6A enrichment on ATF4 and GLUT3 transcripts in TNBC cells. (E) YTHDC1 RIP followed by RT‐qPCR in TNBC cells validates the binding of ATF4 and GLUT3 mRNAs to YTHDC1. (F) Western blot analysis of ATF4 and GLUT3 protein levels in TNBC cells upon YTHDC1 KD. (G) YTHDC1 KD increases ATF4 mRNA stability. (H) YTHDC1 KD decreases the stability of GLUT3 mRNA. (I,J) RIP‐qPCR in TNBC cells overexpressing Flag‐tagged WT or mutant YTHDC1 shows that YTHDC1 W377A and W428A mutations abolish the binding to ATF4 (I) and GLUT3 (J) mRNAs. (K,L) WT YTHDC1, but not the W377A and W428A mutants rescue the mRNA expression of ATF4 (K) and GLUT3 (L) in YTHDC1‐KD cells. (M,N) Intracellular glucose (M) and glutamate (N) levels in YTHDC1‐KD TNBC cells rescued with WT, but not mutant YTHDC1. (O,P) RIP assay showing reduced binding of the ATF4 A670G mutant transcript (O) and GLUT3 A1723G mutant transcript (P) to YTHDC1 compared to WT transcript. (Q,R) mRNA stability analysis demonstrating significantly enhanced stability of the A670G mutant compared to WT ATF4 (Q) and decreased stability of the A1723G mutant compared to WT GLUT3. Data are presented as mean ± SD of experimental triplicates (D,E,G–R). p values were determined using two‐tailed Student's t‐test. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s means not significant).

Article Snippet: YTHDC1 antibody (ABclonal, A22992) (1:500) was incubated overnight at 4°C, and secondary antibodies was incubated for 1 h at 37°C.

Techniques: RNA Sequencing, Control, Binding Assay, Modification, Quantitative RT-PCR, Western Blot, Mutagenesis, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: YTHDC1 Orchestrates Glucose and Glutamate Rewiring to Overcome Lethal Metabolic Stress in Triple‐Negative Breast Cancer

doi: 10.1002/advs.77492

Figure Lengend Snippet: YTHDC1 knockdown induces disulfidptosis through ATF4‐mediated transcriptional upregulation of SLC7A11. (A) SLC7A11 is identified as a direct transcriptional target of ATF4 across multiple independent datasets in ChIPbase. (B,C) YTHDC1 knockdown in TNBC cells increases SLC7A11 expression at the (B) mRNA level and (C) protein level. (D,E) YTHDC1 knockdown decreases glucose uptake (D) and elevates intracellular cystine (E) in TNBC cells. (F) Schematic diagram of 1 3 C 5 ‐glutamine or 1 3 C 6 ‐glucose isotope tracing experiments followed by mass spectrometry in MDA‐MB‐231 cells. (G) YTHDC1 knockdown significantly reduced the abundance of 1 3 C‐labeled glucose‐6‐phosphate (G‐6‐P), ribose‐5‐phosphate (R‐5‐P), and pyruvate, compared with siNC cells. (H) YTHDC1 knockdown caused a marked reduction in intracellular 1 3 C‐labeled α‐ketoglutarate (α‐KG). Concomitantly, the abundance of 1 3 C‐labeled glutamate in the culture medium was significantly elevated. (I) A schematic diagram illustrating that upregulated SLC7A11 and downregulated GLUT3 together lead to disulfidptosis. (J) TNBC samples with low YTHDC1 expression exhibit higher disulfidptosis scores compared to those with high YTHDC1 expression in TCGA‐TNBC cohort. (K) Non‐reducing and reducing Western blotting show that YTHDC1 knockdown induces migration retardation of actin cytoskeleton proteins under non‐reducing conditions. (L) YTHDC1 knockdown induces disulfidptosis‐associated cell shrinkage and F‐actin contraction, which is rescued by 2‐mercaptoethanol (2‐ME) treatment. F‐actin is visualized by phalloidin staining, and F‐actin aggregation is quantified using the Fluorescence Aggregation Index (FAI). (M,N) F‐actin aggregation (M) and NADPH/NADP + ratio (N) in YTHDC1‐KD TNBC cells are rescued with siATF4. Scale bars, 20 µm. Data are presented as mean ± SD of experimental triplicates (B,D,E, L–N). p values were determined using two‐tailed Student's t‐test. ( * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: YTHDC1 antibody (ABclonal, A22992) (1:500) was incubated overnight at 4°C, and secondary antibodies was incubated for 1 h at 37°C.

Techniques: Knockdown, Expressing, Mass Spectrometry, Labeling, Western Blot, Migration, Staining, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: YTHDC1 Orchestrates Glucose and Glutamate Rewiring to Overcome Lethal Metabolic Stress in Triple‐Negative Breast Cancer

doi: 10.1002/advs.77492

Figure Lengend Snippet: YTHDC1 knockdown suppresses tumor growth and induces disulfidptosis in vivo. (A,B) YTHDC1 knockdown suppresses the growth of MDA‐MB‐231 xenografts in nude mice. 2 × 106 MDA‐MB‐231 cells stably expressing Tet‐on control or YTHDC1 shRNA were injected as xenografts in nude mice. Doxycycline (200 µg/ml) was supplied in drinking water to induce Tet‐on shRNA expression. (C) Validation of YTHDC1 expression in xenograft tumors by RT‐qPCR. (D–G) YTHDC1 knockdown upregulates ATF4 and SLC7A11, and downregulates GLUT3 at the mRNA (D–F) and protein level (G) in xenograft tumors. (H–J) YTHDC1 knockdown leads to decreased intracellular glucose (H), elevated cystine (I), and a reduced NADPH/NADP+ ratio (J). (K,L) Measurement of intracellular glutamate (K) and ATP (L) levels in tumor tissues, showing significant depletion upon YTHDC1 knockdown. (M) Representative phalloidin staining shows increased F‐actin contraction and aggregation upon YTHDC1 knockdow, as quantified by a significant increase in the Fluorescence Aggregation Index (FAI). Scale bars, 10 µm. (N,O) Immunohistochemistry of Ki67 in in YTHDC1 knockdown and control tumor tissues. Scale bars, 50 µm. (P–R) 30% Caloric restriction (CR) combined with YTHDC1 knockdown remarkably inhibits tumor growth of MDA‐MB‐231 xenografts in nude mice. Tumor image and tumor growth curves (P), tumor weights (Q) and intratumoral ATP levels (R). (S–U) Pharmacological glucose restriction using BAY‐876 combined with YTHDC1 knockdown dramatically suppresses tumor growth of MDA‐MB‐231 xenografts in nude mice. Tumor image and tumor growth curves (S), tumor weights (T) and intratumoral glucose levels (U). For G, K, N, O, n = 3 mice per group. For AF, HJ, L, M, n = 7 mice per group. For PU, n = 6 mice per group. Each dot represents one mouse (A–U). Data are presented as mean ± SD (A–O, Q–R, T–U). For P and S, mean ± SEM are shown, and p values were determined using two‐tailed Student's t‐test. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: YTHDC1 antibody (ABclonal, A22992) (1:500) was incubated overnight at 4°C, and secondary antibodies was incubated for 1 h at 37°C.

Techniques: Knockdown, In Vivo, Stable Transfection, Expressing, Control, shRNA, Injection, Biomarker Discovery, Quantitative RT-PCR, Staining, Fluorescence, Immunohistochemistry, Two Tailed Test

Journal: bioRxiv

Article Title: Regulation and Function of the HPV16 CircE7 RNA

doi: 10.64898/2026.03.09.710444

Figure Lengend Snippet: (A) Western blot from MDA-1483 cells transfected with WT or IRES mut circE7 plasmid. Representative blot (left), quantification (right). ***p<0.001 (B) RNA levels of the IRES mutant cricE7 RNA by RT-qPCR in MDA-1483 cells. Difference not statistically significant by t-test. Twice as much RNA was transfected in to get equivalent levels of RNA between IRES and WT. (C) Representative tracing of circE7-transfected cells after polysome enrichment assay with the monosome (M), light polysome (L), and heavy polysome (H) fractions indicated. Polysomes isolated from SCC-154 HPV+ (red) and MDA1483 HPV- (blue) cells. (D) Endpoint PCR of CircE7 or circHPK3, in light and heavy polysomes from SCC-154 (HPV+) cells, MDA1483 (HPV-) cells, and BJ cells transfected with hTERT circE7. All samples were RNAse R treated. (E) Western blot from HeLa cells co-transfected with a circE7-Flag expression vector and the indicated siRNA. HSP90, loading control. *indicates non-specific band. (F) RT-qPCR for circE7 RNA levels after YTHDC1 knockdown. (G) Quantification of western blot (n=3).

Article Snippet: Membranes were incubated overnight at 4°C with the following primary antibodies: YTHDC1 (Cell Signaling, 77422), YTHDF3 (Cell Signaling, 24206), YTHDC2 (Cell Signaling, 46324), FLAG (Sigma, A8592), GAPDH (Santa Cruz, sc-32233), HSP90 (Cell Signaling, 4877S).

Techniques: Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Isolation, Expressing, Control, Knockdown