rabbit anti ythdf3  (Proteintech)

Journal: Nature Communications

Article Title: INCENP and CDCA8 predict neoadjuvant chemotherapy response and outcomes in esophageal squamous cell carcinoma

doi: 10.1038/s41467-026-68371-x

Figure Lengend Snippet: a , b Representative images and analysis of INCENP and CDCA8 protein expression levels after YTHDF3 knockdown in PDX-derived organoids (PDXO) by immunofluorescence. Scale bar = 10 μm. Mean protein fluorescence intensity was determined using ImageJ software. c , d Gene plots illustrating YTHDF3 binding to INCENP and CDCA8 mRNAs, as measured by RIP-seq. The normalized read distribution: input (yellow) and YTHDF3 (purple) along the mRNAs. e RNA immunoprecipitation-qPCR (RIP-qPCR) highlighting the indicated mRNA transcripts bound by YTHDF3 in ESCC cells. f , g Representative immunofluorescence staining of newly synthesized proteins and statistical analysis in KYSE30 and KYSE150 cells after YTHDF3 overexpression. Relative fluorescence intensity was determined using ImageJ software. Puromycin staining (green) indicates newly synthesized proteins; nuclei are stained with DAPI (blue). Scale bar = 50 μm. h , i Mock or YTHDF3 knockdown KYSE30 and KYSE150 cells were transfected with pmirGLO-INCENP or pmirGLO-CDCA8 reporters for 48 h. Translation efficiency of INCENP or CDCA8 is defined as the quotient of reporter protein production (F-luc/R-luc). j , k Co-immunoprecipitation of endogenous YTHDF3 and eIF3A in ESCC cells. Representative immunoblots shown in figures were repeated three times independently with similar results. l , m In situ detection and quantification of YTHDF3–eIF3A interactions in the indicated ESCC cell lines were measured using PLA assays, and puncta per cell were determined using ImageJ software. Scale bar = 10 μm, n = 5 biological replicates. n RIP-qPCR illustrating the association of eIF3A with the indicated transcripts in YTHDF3 knockdown KYSE30 cells. o RIP-qPCR illustrating the association of eIF3A with the indicated transcripts in Ebselen-treated KYSE30 cells. RIP-qPCR illustrating the association of YTHDF3 ( p ) and eIF3A ( q ) with the indicated transcripts in METTL3 knockdown KYSE30 cells. Data were analyzed by two-tailed unpaired t-test ( b , e , g , h , i , m ) or one-way ANOVA ( n , o , p , q ). n = 3 biological replicates ( b , e , g , h , i , n , o , p , q ). Data were presented as means ± SD. Source data are provided as a file.

Article Snippet: Ten percent (10%) of the lysate was stored as “input”, 80% was used in immunoprecipitation reactions with anti-YTHDF3 antibody (Abcam, ab220161), and the remaining 10% was incubated with rabbit IgG (Cell Signaling Technology) as a negative control and named “IgG”, respectively.

Techniques: Expressing, Knockdown, Derivative Assay, Immunofluorescence, Fluorescence, Software, Binding Assay, RNA Immunoprecipitation, Staining, Synthesized, Over Expression, Transfection, Immunoprecipitation, Western Blot, In Situ, Two Tailed Test

Journal: Nature Communications

Article Title: INCENP and CDCA8 predict neoadjuvant chemotherapy response and outcomes in esophageal squamous cell carcinoma

doi: 10.1038/s41467-026-68371-x

Figure Lengend Snippet: a An experimental design of the present study. Responders (tumor regression grade [TRG] scores of 0–1 or patients who achieved clinical complete or partial response; n = 6) and non-responders (TRG scores of 2–3 or patients who achieved clinical minimal response; n = 8). Created in BioRender. GU, T. (2025) https://BioRender.com/ppcp3bu . b Transcriptomic data from clinical samples (Cohort 1) were subjected to GO enrichment analysis to determine alterations in biological processes, molecular functions, and cellular components. Data are visualized in the provided bubble diagram. c Bubble diagram visualizing the alteration of m 6 A regulators in clinical ESCC sample Cohort 1. d GO enrichment analysis of differentially expressed proteins between shYTHDF3 and Mock cells. The Rich Factor represents the ratio of enriched genes; bubble size denotes gene count, and color indicates -log₁₀P value. Statistical significance was calculated using a two-sided Fisher’s exact test with Benjamini–Hochberg FDR correction. e Rank-based gene set enrichment analysis (GSEA) of signaling pathway alterations and representative protein changes in shYTHDF3 cells. Terms in red represent the primary targets. f Representative immunohistochemical staining images of INCENP, CDCA8, and YTHDF3 in ESCC Cohort 1 tissue samples from the indicated responders and non-responders. Scale bar = 250 μm. g INCENP, CDCA8, and YTHDF3 score levels were measured in tissue derived from ESCC Cohort 1 samples. Responders (TRG1, n = 4) and non-responders (TRG 2-3, n = 8). h Correlation between YTHDF3 protein scores and INCENP or CDCA8 protein scores in Cohort 1. Correlation coefficients (R) and P values were determined using a two-sided Pearson correlation test. Shaded areas represent the 95% confidence interval of the regression line. i Representative radiological images and INCENP, CDCA8 IHC-stained tissues (scale bar = 250 μm) in Cohort 2, ESCC patients with favorable and unfavorable responses to NACT [responders (TRG 0–1, n = 31) and non-responders (TRG 2–3, n = 37)]. Red arrows represent the lesion sites. j Immunohistochemical analysis of INCENP and CDCA8 scores in ESCC tissues from Cohort 2, including responders (TRG 1, n = 10) and non-responders (TRG 2-3, n = 37). k Stacked bar plots showing distributions of clinical outcomes (death, n = 10, progression, n = 10, remission, n = 10) in patients with low and high expression of INCENP and CDCA8. Statistical significance was assessed using a two-sided Chi-square test. Data were presented as means ± SD. P -values were determined by a two-tailed unpaired t-test ( g , j ). Source data are provided as a file.

Article Snippet: Ten percent (10%) of the lysate was stored as “input”, 80% was used in immunoprecipitation reactions with anti-YTHDF3 antibody (Abcam, ab220161), and the remaining 10% was incubated with rabbit IgG (Cell Signaling Technology) as a negative control and named “IgG”, respectively.

Techniques: Immunohistochemical staining, Staining, Derivative Assay, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: INCENP and CDCA8 predict neoadjuvant chemotherapy response and outcomes in esophageal squamous cell carcinoma

doi: 10.1038/s41467-026-68371-x

Figure Lengend Snippet: a Cell cycle kinetics were measured by flow cytometry in YTHDF3 knockdown ESCC cells. b Representative confocal images and statistical analysis results of multipolar spindle formation in ESCC cells upon YTHDF3 knockdown. scale bar = 5 μm. IC50 values of ( c ) paclitaxel and d cisplatin after YTHDF3 knockdown in KYSE30 and KYSE150 cells. IC50 values of ( e ) paclitaxel and f cisplatin in KYSE30 and KYSE150 cells with Ebselen treatment. g IC50 values of paclitaxel and cisplatin after overexpression of YTHDF3 in KYSE450 cells. h Plate colony formation assays showing and analyzing overexpression of YTHDF3 in KYSE450 cells treated with paclitaxel and cisplatin. i–n Tumors generated from ESCC cells were subcutaneously xenografted into female NU/NU mice ( i , l ) and divided into the indicated four groups ( n = 6 per group). j , m Effects of each treatment group on tumor growth and k , n tumor weight. Tumor volume = (length × width²)/2 was measured twice per week.IC50 values of paclitaxel ( o ) and cisplatin ( p ) in the shYTHDF3-rescued INCENP or CDCA8 group. Data were analyzed by two-tailed unpaired t-test ( a , b ), one-way ANOVA ( k , n ), and two-way ANOVA ( h , j , m ). n = 3 biological replicates ( a , b , h ). n = 4 biological replicates ( c , d , e , f , g , o , p ). Data were presented as means ± SD. Source data are provided in the file.

Article Snippet: Ten percent (10%) of the lysate was stored as “input”, 80% was used in immunoprecipitation reactions with anti-YTHDF3 antibody (Abcam, ab220161), and the remaining 10% was incubated with rabbit IgG (Cell Signaling Technology) as a negative control and named “IgG”, respectively.

Techniques: Flow Cytometry, Knockdown, Over Expression, Generated, Two Tailed Test

Journal: Nature Communications

Article Title: INCENP and CDCA8 predict neoadjuvant chemotherapy response and outcomes in esophageal squamous cell carcinoma

doi: 10.1038/s41467-026-68371-x

Figure Lengend Snippet: INCENP and CDCA8 expression levels were measured in Flag-YTHDF3-WT or Flag-YTHDF3 Mut transfected shYTHDF3#1 (targeting the 3’ UTR of YTHDF3) KYSE30 ( a ) and KYSE150 ( b ) cells. c Cell cycle kinetics were measured in Flag-YTHDF3-WT or Flag-YTHDF3 Mut transfected KYSE150 shYTHDF3#1 cells, n = 3 biological replicates. d Expression of cell cycle proteins in Flag-YTHDF3-WT or Flag-YTHDF3 Mut plasmid-transfected shYTHDF3#1 KYSE150 cells was analyzed by Western blotting. Representative confocal images ( e ) and statistical analysis of metaphase alterations ( f ) in WT or mutant YTHDF3 cells, n = 3 biological replicates, Scale bar = 5 μm. g Western blot analysis showing INCENP and CDCA8 expression levels in shMETTL3 cells. Representative images ( h , j ) and statistical analysis of protein expression levels ( i , k ) after METTL3 knockdown in PDXO cells. n = 3 biological replicates, Scale bar = 10 μm. Relative fluorescence intensity was determined using ImageJ software. Representative confocal images ( l ) and statistical analysis of metaphase alterations ( m ) in shMETTL3 KYSE30 cells, n = 3 biological replicates, Scale bar = 5 μm. n Representative immunohistochemical staining images of METTL3 in Cohort 2 ESCC tissues with the indicated TRG. Scale bar = 250 μm. o Immunohistochemical analysis of METTL3 scores in ESCC tissues from cohort 2, including responders (TRG 1, n = 10) and non-responders (TRG 2-3, n = 37). The box plot shows the median (center line), upper and lower quartiles (box limits), and 1.5 × the interquartile range (whiskers). p Two-sided Pearson correlation was calculated between METTL3 protein scores and INCENP or CDCA8 protein scores in Cohort 2. Shaded areas represent the 95% confidence interval of the regression line. Data were analyzed by two-tailed unpaired t-test ( i , k , m , o ) or one-way ANOVA ( c , f ). Data were presented as means ± SD. Representative immunoblots shown in figures were repeated three times independently with similar results. Source data are provided as a a file.

Article Snippet: Ten percent (10%) of the lysate was stored as “input”, 80% was used in immunoprecipitation reactions with anti-YTHDF3 antibody (Abcam, ab220161), and the remaining 10% was incubated with rabbit IgG (Cell Signaling Technology) as a negative control and named “IgG”, respectively.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Knockdown, Fluorescence, Software, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: INCENP and CDCA8 predict neoadjuvant chemotherapy response and outcomes in esophageal squamous cell carcinoma

doi: 10.1038/s41467-026-68371-x

Figure Lengend Snippet: a , b Representative images and analysis of INCENP and CDCA8 protein expression levels after YTHDF3 knockdown in PDX-derived organoids (PDXO) by immunofluorescence. Scale bar = 10 μm. Mean protein fluorescence intensity was determined using ImageJ software. c , d Gene plots illustrating YTHDF3 binding to INCENP and CDCA8 mRNAs, as measured by RIP-seq. The normalized read distribution: input (yellow) and YTHDF3 (purple) along the mRNAs. e RNA immunoprecipitation-qPCR (RIP-qPCR) highlighting the indicated mRNA transcripts bound by YTHDF3 in ESCC cells. f , g Representative immunofluorescence staining of newly synthesized proteins and statistical analysis in KYSE30 and KYSE150 cells after YTHDF3 overexpression. Relative fluorescence intensity was determined using ImageJ software. Puromycin staining (green) indicates newly synthesized proteins; nuclei are stained with DAPI (blue). Scale bar = 50 μm. h , i Mock or YTHDF3 knockdown KYSE30 and KYSE150 cells were transfected with pmirGLO-INCENP or pmirGLO-CDCA8 reporters for 48 h. Translation efficiency of INCENP or CDCA8 is defined as the quotient of reporter protein production (F-luc/R-luc). j , k Co-immunoprecipitation of endogenous YTHDF3 and eIF3A in ESCC cells. Representative immunoblots shown in figures were repeated three times independently with similar results. l , m In situ detection and quantification of YTHDF3–eIF3A interactions in the indicated ESCC cell lines were measured using PLA assays, and puncta per cell were determined using ImageJ software. Scale bar = 10 μm, n = 5 biological replicates. n RIP-qPCR illustrating the association of eIF3A with the indicated transcripts in YTHDF3 knockdown KYSE30 cells. o RIP-qPCR illustrating the association of eIF3A with the indicated transcripts in Ebselen-treated KYSE30 cells. RIP-qPCR illustrating the association of YTHDF3 ( p ) and eIF3A ( q ) with the indicated transcripts in METTL3 knockdown KYSE30 cells. Data were analyzed by two-tailed unpaired t-test ( b , e , g , h , i , m ) or one-way ANOVA ( n , o , p , q ). n = 3 biological replicates ( b , e , g , h , i , n , o , p , q ). Data were presented as means ± SD. Source data are provided as a file.

Article Snippet: Ten percent (10%) of the lysate was stored as “input”, 80% was used in immunoprecipitation reactions with anti-YTHDF3 antibody (Abcam, ab220161), and the remaining 10% was incubated with rabbit IgG (Cell Signaling Technology) as a negative control and named “IgG”, respectively.

Techniques: Expressing, Knockdown, Derivative Assay, Immunofluorescence, Fluorescence, Software, Binding Assay, RNA Immunoprecipitation, Staining, Synthesized, Over Expression, Transfection, Immunoprecipitation, Western Blot, In Situ, Two Tailed Test

Journal: Nature Communications

Article Title: INCENP and CDCA8 predict neoadjuvant chemotherapy response and outcomes in esophageal squamous cell carcinoma

doi: 10.1038/s41467-026-68371-x

Figure Lengend Snippet: a Annotated clinical and molecular characteristics of ESCC patients from Cohort 3 ( n = 65, responder [TRG 0-1, n = 30]; non-responder [TRG 2-3, n = 35]). P -values on the right indicate statistically significant non-random distributions for each attribute. A two-sided Chi-square test was used for categorical variables, and a two-sided unpaired t-test was used for continuous variables. b Representative immunohistochemical staining images of indicated proteins from ESCC, lung cancer, and breast cancer patients of Cohort 3 with favorable and unfavorable NACT treatment responses. scale bar = 250 μm. Immunohistochemical analysis of the indicated proteins in NACT response-classified samples from ( c ) ESCC patients ( n = 65, responder n = 30; non-responder, n = 35]), d lung cancer patients ( n = 26, responder n = 12; non-responder n = 14]), and e breast cancer patients ( n = 22, responder n = 9; non-responder n = 13). f A two-sided Pearson correlation was calculated between the expression of YTHDF3 and INCENP or CDCA8 in biopsy tissues from ESCC patients. Shaded areas represent the 95% confidence interval of the regression line. Kaplan–Meier curves showing progression-free survival (PFS) for patients with ESCC according to high vs. low expression of INCENP ( g ), CDCA8 ( h ), and YTHDF3 ( i ). Statistical significance was assessed using a two-sided log-rank (Mantel–Cox) test; n and p values are shown in the plots. No multiple-comparison correction was applied. Receiver operating characteristic (ROC) curve analyses for predicting the NACT responses in ( j ) ESCC patients, k lung cancer patients, and l breast cancer patients based on the expression levels of YTHDF3, INCENP, and CDCA8. Data were presented as means ± SD. Statistical significance was determined by a two-tailed unpaired t-test ( c , d , e ). Source data are provided as a file.

Article Snippet: Ten percent (10%) of the lysate was stored as “input”, 80% was used in immunoprecipitation reactions with anti-YTHDF3 antibody (Abcam, ab220161), and the remaining 10% was incubated with rabbit IgG (Cell Signaling Technology) as a negative control and named “IgG”, respectively.

Techniques: Immunohistochemical staining, Staining, Expressing, Comparison, Two Tailed Test