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Addgene inc pgex 4t 1 construct
Pgex 4t 1 Construct, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pgex+4t+1+ythdf1/pGEx-4T-1-YTHDF1+(Plasmid+%2370087)/pmc11791202-376-6-8
Average 93 stars, based on 6 article reviews

pgex 4t 1 construct - by Bioz Stars, 2026-10

93/100 stars

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Article Title: Poly(GR) interacts with key stress granule factors promoting its assembly into cytoplasmic inclusions
Article Snippet: pGEX-4T-1 YTHDF1 , Wang, X. et al. , Addgene Cat#70087.

Construct:

Article Title: Endoribonucleolytic Cleavage of m 6 A-Containing RNAs by RNase P/MRP Complex.
Article Snippet: Where indicated, the cells were treated with 100 nM Dex (Sigma-Aldrich) for 1 h before cell harvesting. .. The following constructs were described previously: pMS2-HA, pMS2-HA-SMG5, pcb-6bs, and pMS2-HA-PNRC2 (Cho et al., 2013); pcDNA3-FLAG-HRSP12-WT, pcDNA3-FLAG-HRSP12-WT(P105A/R107E), and pCMV-MYC-HRSP12 (Park et al., 2016). pBG-SONWT, pBG-SON-Mut, pBG-PLAC2-WT, and pBG-PLAC2-Mut were provided by Dr. Ligang Wu (Du et al., 2016). pCMV-hPOP13xFLAG (#53968), pGEx-4T-1-YTHDF1 (#70087), pcDNA3-FLAG-YTHDF2 (#52300), and pGEx-4T-1-YTHDF3 (#70088) were purchased from Addgene. .. To construct pCMV-MYC-YTHDF1 and pCMV-MYC-YTHDF3, a XhoI/Klenow-filled EcoRI fragment of pGEx-4T-1-YTHDF1 containing YTHDF1 cDNA and XhoI/Klenow-filled BamHI fragment of pGEx-4T-1-YTHDF3 containing YTHDF3 cDNA were ligated to an XhoXI/Klenow-filled SalI fragment of pCMV-MYC (Clontech), respectively.

Amplification:

Article Title: TRACE-seq: A transgenic system for unbiased and non-invasive transcriptome profiling of living cells
Article Snippet: This fragment was cloned in the pCMV-mCherry-CD9-10 plasmid which was a gift from Michael Davidson (Addgene, #55013) to generated, the pCMV-GBP1-CD9 plasmid. .. The C terminal part of the YTHDF1 gene was amplified (a flexible linker SGGGGGGGGGG on the N-terminal part of the sequence was added) from pGEx-4T-1-YTHDF1 which was a gift from Chuan He (Wang et al., 2014) (Addgene, #70087) and cloned into the pT7-eGFP-HseIF4E gifted from Elisa Izaurralde (Peter et al., 2015) (Addgene, #79437) and gave the pCMV-eGFP-C-YTHDF1 . ..

Sequencing:

Clone Assay:

Enzyme-linked Immunosorbent Assay:

Image Search Results

a Immunoblot (IB) analysis of whole-cell lysates (WCL) and anti-Flag immunoprecipitates (IP) from HEK293T cells transfected with GFP-Ub and Flag-YTHDF1. b YTHDF1 polyubiquitination could largely be detected in cells transfected with indicated constructs. c IB analysis of WCL and His immunoprecipitate from HEK293T cells transfected with indicated constructs. d PLC/PRF/5 cells were immunoprecipitated with either anti-USP5 or anti-YTHDF1 antibody and then analyzed by IB. e IB and IP products analysis of USP5-YTHDF1 interaction in HEK293T cells expressing HA-USP5 WT or the indicated truncated YTHDF1 mutants. f , g IB analysis of YTHDF1 levels in HEK293T cells expressing HA-USP5 (DNA content of 250 ng or 500 ng) or indicated plasmids. h IB and QRT-PCR analysis of YTHDF1 from Hepa1-6 cells with Usp5 knockout. n = 3. i , j IB analysis of WCL from Hepa1-6 cells with the depletion of Usp5 or HEK293T cells transfected with indicated constructs for 36 h. Cells were treated with 100 μg/ml CHX at indicated time points. The YTHDF1 levels was quantified by the ImageJ software. k IB analysis of WCL and IP products from HEK293T cells transfected with indicated constructs. Cells were treated with 20 μM MG132 for 8 h. l Effects of Usp5 knockout in Hepa1-6 cells on Ythdf1 K11-linked polyubiquitination were evaluated by IB. m Effects of WP1130 on USP5-mediated YTHDF1 K11-linked polyubiquitination. Cells expressing indicated plasmids were treated with different doses of WP1130. n IB analysis of proteins labeled with puromycin using anti-puromycin antibody upon Usp5 depletion with or without expressing Ythdf1. o , p Assessment of subcutaneous tumor formation from PLC/PRF/5 cells after depletion of USP5, or YTHDF1, or stably expressing YTHDF1 with endogenous USP5 knockdown. Tumor weight was measured at the endpoint of the study. Tumor growth was measured at the indicated time points. n = 5. * p < 0.05, t -test. q Kaplan-Meier analysis revealed a relationship between YTHDF1 and USP5 expression and overall survival in HCC patients. All data are presented as mean ± SEM. All IB data are representative of three independent experiments. Source data are provided as a file.

Journal: Nature Communications

Article Title: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation

doi: 10.1038/s41467-025-56564-9

Figure Lengend Snippet: a Immunoblot (IB) analysis of whole-cell lysates (WCL) and anti-Flag immunoprecipitates (IP) from HEK293T cells transfected with GFP-Ub and Flag-YTHDF1. b YTHDF1 polyubiquitination could largely be detected in cells transfected with indicated constructs. c IB analysis of WCL and His immunoprecipitate from HEK293T cells transfected with indicated constructs. d PLC/PRF/5 cells were immunoprecipitated with either anti-USP5 or anti-YTHDF1 antibody and then analyzed by IB. e IB and IP products analysis of USP5-YTHDF1 interaction in HEK293T cells expressing HA-USP5 WT or the indicated truncated YTHDF1 mutants. f , g IB analysis of YTHDF1 levels in HEK293T cells expressing HA-USP5 (DNA content of 250 ng or 500 ng) or indicated plasmids. h IB and QRT-PCR analysis of YTHDF1 from Hepa1-6 cells with Usp5 knockout. n = 3. i , j IB analysis of WCL from Hepa1-6 cells with the depletion of Usp5 or HEK293T cells transfected with indicated constructs for 36 h. Cells were treated with 100 μg/ml CHX at indicated time points. The YTHDF1 levels was quantified by the ImageJ software. k IB analysis of WCL and IP products from HEK293T cells transfected with indicated constructs. Cells were treated with 20 μM MG132 for 8 h. l Effects of Usp5 knockout in Hepa1-6 cells on Ythdf1 K11-linked polyubiquitination were evaluated by IB. m Effects of WP1130 on USP5-mediated YTHDF1 K11-linked polyubiquitination. Cells expressing indicated plasmids were treated with different doses of WP1130. n IB analysis of proteins labeled with puromycin using anti-puromycin antibody upon Usp5 depletion with or without expressing Ythdf1. o , p Assessment of subcutaneous tumor formation from PLC/PRF/5 cells after depletion of USP5, or YTHDF1, or stably expressing YTHDF1 with endogenous USP5 knockdown. Tumor weight was measured at the endpoint of the study. Tumor growth was measured at the indicated time points. n = 5. * p < 0.05, t -test. q Kaplan-Meier analysis revealed a relationship between YTHDF1 and USP5 expression and overall survival in HCC patients. All data are presented as mean ± SEM. All IB data are representative of three independent experiments. Source data are provided as a file.

Article Snippet: Human YTHDF1 was subcloned into the pGEX-4T-1 construct (Addgene 70087).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Expressing, Quantitative RT-PCR, Knock-Out, Software, Labeling, Stable Transfection, Knockdown

a IB analysis of WCL and anti-Flag IPs from HEK293T cells transfected with indicated constructs and treated with the 20 μM MG132 for 8 h. b In vivo ubiquitination assay of YTHDF1 in HEK293T cells expressing Flag-YTHDF1 WT and indicated truncated YTHDF1 mutants in the presence or absence of ectopic USP5 expression. Cells were treated with 20 μM MG132 for 8 h. c IB analysis of the protein levels of the indicated truncated YTHDF1 mutants in HEK293T cells expressing increasing amounts of HA-USP5. d IB analysis of WCL and IP derived from HEK293T cells transfected with indicated constructs. Cells were treated with 20 μM MG132 for 8 h. e IB analysis of WCL derived from HEK293T cells transfected with indicated constructs. 36 h post-transfection, cells were treated with 100 μg/ml CHX at indicated time points. The YTHDF1 protein abundance was quantified by the ImageJ software. f In vivo ubiquitination assays of WCL and anti-Flag IPs derived from HEK293T cells transfected with plasmids expressing the indicated proteins. Cells were treated with 20 μM MG132 for 8 h. g In vivo ubiquitination assay of USP5 knockout in PLC/PRF/5 cells transfected with WT Flag-YTHDF1 or indicated mutant constructs. YTHDF1 polyubiquitination was evaluated by IB analysis. Cells were treated with 20 μM MG132 for 8 h. h Colony formation assays of PLC/PRF/5 cells stably expressing YTHDF1-WT or -4KR mutant with endogenous YTHDF1 knockout. n = 3 per group. ** p < 0.01. t -test. i , j Assessment of subcutaneous tumor formation from PLC/PRF/5 cells stably expressing YTHDF1-WT or -4KR mutant with endogenous YTHDF1 knockout. Tumor weight was measured at the endpoint of the study. In vivo tumor growth was measured at the indicated time points and tumors were dissected at the endpoint. n = 6 per group. ** p < 0.01, t -test. All data are presented as mean ± SEM. All IB data are representative of two independent experiments. Source data are provided as a file.

Journal: Nature Communications

Article Title: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation

doi: 10.1038/s41467-025-56564-9

Figure Lengend Snippet: a IB analysis of WCL and anti-Flag IPs from HEK293T cells transfected with indicated constructs and treated with the 20 μM MG132 for 8 h. b In vivo ubiquitination assay of YTHDF1 in HEK293T cells expressing Flag-YTHDF1 WT and indicated truncated YTHDF1 mutants in the presence or absence of ectopic USP5 expression. Cells were treated with 20 μM MG132 for 8 h. c IB analysis of the protein levels of the indicated truncated YTHDF1 mutants in HEK293T cells expressing increasing amounts of HA-USP5. d IB analysis of WCL and IP derived from HEK293T cells transfected with indicated constructs. Cells were treated with 20 μM MG132 for 8 h. e IB analysis of WCL derived from HEK293T cells transfected with indicated constructs. 36 h post-transfection, cells were treated with 100 μg/ml CHX at indicated time points. The YTHDF1 protein abundance was quantified by the ImageJ software. f In vivo ubiquitination assays of WCL and anti-Flag IPs derived from HEK293T cells transfected with plasmids expressing the indicated proteins. Cells were treated with 20 μM MG132 for 8 h. g In vivo ubiquitination assay of USP5 knockout in PLC/PRF/5 cells transfected with WT Flag-YTHDF1 or indicated mutant constructs. YTHDF1 polyubiquitination was evaluated by IB analysis. Cells were treated with 20 μM MG132 for 8 h. h Colony formation assays of PLC/PRF/5 cells stably expressing YTHDF1-WT or -4KR mutant with endogenous YTHDF1 knockout. n = 3 per group. ** p < 0.01. t -test. i , j Assessment of subcutaneous tumor formation from PLC/PRF/5 cells stably expressing YTHDF1-WT or -4KR mutant with endogenous YTHDF1 knockout. Tumor weight was measured at the endpoint of the study. In vivo tumor growth was measured at the indicated time points and tumors were dissected at the endpoint. n = 6 per group. ** p < 0.01, t -test. All data are presented as mean ± SEM. All IB data are representative of two independent experiments. Source data are provided as a file.

Article Snippet: Human YTHDF1 was subcloned into the pGEX-4T-1 construct (Addgene 70087).

Techniques: Transfection, Construct, In Vivo, Ubiquitin Proteomics, Expressing, Derivative Assay, Quantitative Proteomics, Software, Knock-Out, Mutagenesis, Stable Transfection

a IB analysis using the indicated antibodies in PLC/PRF/5 and Hepa1-6 cells treated with increasing concentrations of insulin for 30 min. b Reduced YTHDF1 polyubiquitination upon growth factor stimulation. HEK293T cells transfected with ubiquitin constructs were lysed for anti-YTHDF1 IP and IB. c The WT and Usp5-KO cells were treated with insulin (100 ng/ml) or Torin1 (1.0 μM). After 30 min or 24 h, cells were collected and analyzed by IB. d IB analysis of YTHDF1 protein levels in PLC/PRF/5 cells expressing the indicated constructs. e Knockdown of USP5 inhibits mTOR or RPTOR-induced accumulation of YTHDF1. The WT and USP5-KD PLC/PRF/5 cells were transfected with Flag-mTOR or HA-RPTOR constructs for 36 h. f IB analysis of YTHDF1 immunoprecipitate or WCL from PLC/PRF/5 cells transfected with Flag-YTHDF1. Cells were then serum starved for 16 h and stimulated with insulin (100 ng/ml) for 30 min. g IB analysis of WCL and IP derived from HEK293T cells transfected with various HA-USP5 constructs as well as Flag-USP5. Cells were pretreated with 1.0 μM Torin1 for 24 h. h IB analysis of WCL and IP derived from HEK293T cells transfected with Flag-mTOR together with the HA-USP5 constructs for 36 h. i In vivo ubiquitination analysis of YTHDF1 in PLC/PRF/5 cells expressing the indicated HA-USP5 constructs. Cells were treated with 20 μM MG132 for 8 h. j In vivo phosphorylation assay of HA-USP5 WT and S149A mutant in HEK293T cells with or without ectopic Flag-mTOR expression. k The S149 mutant disrupted USP5 dimerization process in cells. IB analysis of WCL and IP derived from HEK293T cells transfected with constructs indicated. l Co-IP analysis of USP5/YTHDF1 interaction in HEK293T cells with expressing the indicated constructs. m A simplified model depicting the regulatory mechanism of USP5 by insulin signaling pathways. The active mTORC1 phosphorylates USP5 at S149, which promotes its dimerization and stabilizes YTHDF1, and then feedback positively regulates mTORC1 activity by increasing RPTOR mRNA translation. All data are representative of two independent experiments. Source data are provided as a file.

Journal: Nature Communications

Article Title: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation

doi: 10.1038/s41467-025-56564-9

Figure Lengend Snippet: a IB analysis using the indicated antibodies in PLC/PRF/5 and Hepa1-6 cells treated with increasing concentrations of insulin for 30 min. b Reduced YTHDF1 polyubiquitination upon growth factor stimulation. HEK293T cells transfected with ubiquitin constructs were lysed for anti-YTHDF1 IP and IB. c The WT and Usp5-KO cells were treated with insulin (100 ng/ml) or Torin1 (1.0 μM). After 30 min or 24 h, cells were collected and analyzed by IB. d IB analysis of YTHDF1 protein levels in PLC/PRF/5 cells expressing the indicated constructs. e Knockdown of USP5 inhibits mTOR or RPTOR-induced accumulation of YTHDF1. The WT and USP5-KD PLC/PRF/5 cells were transfected with Flag-mTOR or HA-RPTOR constructs for 36 h. f IB analysis of YTHDF1 immunoprecipitate or WCL from PLC/PRF/5 cells transfected with Flag-YTHDF1. Cells were then serum starved for 16 h and stimulated with insulin (100 ng/ml) for 30 min. g IB analysis of WCL and IP derived from HEK293T cells transfected with various HA-USP5 constructs as well as Flag-USP5. Cells were pretreated with 1.0 μM Torin1 for 24 h. h IB analysis of WCL and IP derived from HEK293T cells transfected with Flag-mTOR together with the HA-USP5 constructs for 36 h. i In vivo ubiquitination analysis of YTHDF1 in PLC/PRF/5 cells expressing the indicated HA-USP5 constructs. Cells were treated with 20 μM MG132 for 8 h. j In vivo phosphorylation assay of HA-USP5 WT and S149A mutant in HEK293T cells with or without ectopic Flag-mTOR expression. k The S149 mutant disrupted USP5 dimerization process in cells. IB analysis of WCL and IP derived from HEK293T cells transfected with constructs indicated. l Co-IP analysis of USP5/YTHDF1 interaction in HEK293T cells with expressing the indicated constructs. m A simplified model depicting the regulatory mechanism of USP5 by insulin signaling pathways. The active mTORC1 phosphorylates USP5 at S149, which promotes its dimerization and stabilizes YTHDF1, and then feedback positively regulates mTORC1 activity by increasing RPTOR mRNA translation. All data are representative of two independent experiments. Source data are provided as a file.

Article Snippet: Human YTHDF1 was subcloned into the pGEX-4T-1 construct (Addgene 70087).

Techniques: Transfection, Ubiquitin Proteomics, Construct, Expressing, Knockdown, Derivative Assay, In Vivo, Phospho-proteomics, Mutagenesis, Co-Immunoprecipitation Assay, Protein-Protein interactions, Activity Assay

a IB analysis of YTHDF1 protein levels in Huh7 and PLC/PRF/5 cells after treatment of 20 μM MG132, 10 μM Bortezomib, or 5 μM MLN4924 for 8 h. b IB analysis of YTHDF1 protein levels in PLC/PRF/5 cells transfected with an empty vector (EV) or vectors encoding various Myc-tagged Cullin proteins. c IB analysis of YTHDF1 protein levels in PLC/PRF/5 cells expressing increasing amount of T7-FBXW8. Cells were treated with 20 μM MG132 for 8 h. d FBXW8 and USP5 control K11-linked ubiquitination of YTHDF1. IB analysis of IP and WCL derived from HEK293T cells transfected with indicated constructs. e IB and IP products analysis of FBXW8-YTHDF1 interaction in HEK293T cells expressing T7-FBXW8 WT or the indicated truncated YTHDF1 mutants. Cells were treated with 20 μM MG132 for 8 h before harvesting. f IB and IP products analysis of FBXW8-YTHDF1 interaction in HEK293T cells expressing Flag-YTHDF1 or the indicated truncated FBXW8 mutants. Cells were treated with 20 μM MG132 for 8 h before harvesting. g In vivo ubiquitination assay of YTHDF1 in HEK293T cells expressing Flag-YTHDF1 or the indicated truncated YTHDF1 mutants in the presence or absence of ectopic FBXW8 expression. Cells were treated with 20 μM MG132 for 8 h before harvesting. h In vivo ubiquitination assay of YTHDF1 in HEK293T cells expressing Flag-YTHDF1 in the presence ectopic FBXW8 expression or the indicated truncated FBXW8 mutants. Cells were treated with 20 μM MG132 for 8 h before harvesting. i IB analysis of YTHDF1 protein levels in PLC/PRF/5 cells expressing the indicated mTOR components. j FBXW8 antagonism USP5-YTHDF1 interaction. IB and IP products analysis of USP5/YTHDF1 or FBXW8/YTHDF1 interaction in HEK293T cells expressing Flag-YTHDF1 or HA-USP5, plus with or without expressing increasing amounts of T7-FBXW8. k IB and IP products analysis of USP5/YTHDF1 or FBXW8/YTHDF1 interaction in HEK293T cells with or without ectopic mTOR expression. l A model illustrating the roles of mTORC1 pathway in governing USP5/FBXW8-controlled YTHDF1 K11-linked poly-ubiquitination. All data are representative of two independent experiments. Source data are provided as a file.

Journal: Nature Communications

Article Title: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation

doi: 10.1038/s41467-025-56564-9

Figure Lengend Snippet: a IB analysis of YTHDF1 protein levels in Huh7 and PLC/PRF/5 cells after treatment of 20 μM MG132, 10 μM Bortezomib, or 5 μM MLN4924 for 8 h. b IB analysis of YTHDF1 protein levels in PLC/PRF/5 cells transfected with an empty vector (EV) or vectors encoding various Myc-tagged Cullin proteins. c IB analysis of YTHDF1 protein levels in PLC/PRF/5 cells expressing increasing amount of T7-FBXW8. Cells were treated with 20 μM MG132 for 8 h. d FBXW8 and USP5 control K11-linked ubiquitination of YTHDF1. IB analysis of IP and WCL derived from HEK293T cells transfected with indicated constructs. e IB and IP products analysis of FBXW8-YTHDF1 interaction in HEK293T cells expressing T7-FBXW8 WT or the indicated truncated YTHDF1 mutants. Cells were treated with 20 μM MG132 for 8 h before harvesting. f IB and IP products analysis of FBXW8-YTHDF1 interaction in HEK293T cells expressing Flag-YTHDF1 or the indicated truncated FBXW8 mutants. Cells were treated with 20 μM MG132 for 8 h before harvesting. g In vivo ubiquitination assay of YTHDF1 in HEK293T cells expressing Flag-YTHDF1 or the indicated truncated YTHDF1 mutants in the presence or absence of ectopic FBXW8 expression. Cells were treated with 20 μM MG132 for 8 h before harvesting. h In vivo ubiquitination assay of YTHDF1 in HEK293T cells expressing Flag-YTHDF1 in the presence ectopic FBXW8 expression or the indicated truncated FBXW8 mutants. Cells were treated with 20 μM MG132 for 8 h before harvesting. i IB analysis of YTHDF1 protein levels in PLC/PRF/5 cells expressing the indicated mTOR components. j FBXW8 antagonism USP5-YTHDF1 interaction. IB and IP products analysis of USP5/YTHDF1 or FBXW8/YTHDF1 interaction in HEK293T cells expressing Flag-YTHDF1 or HA-USP5, plus with or without expressing increasing amounts of T7-FBXW8. k IB and IP products analysis of USP5/YTHDF1 or FBXW8/YTHDF1 interaction in HEK293T cells with or without ectopic mTOR expression. l A model illustrating the roles of mTORC1 pathway in governing USP5/FBXW8-controlled YTHDF1 K11-linked poly-ubiquitination. All data are representative of two independent experiments. Source data are provided as a file.

Article Snippet: Human YTHDF1 was subcloned into the pGEX-4T-1 construct (Addgene 70087).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Ubiquitin Proteomics, Derivative Assay, Construct, In Vivo

a Kaplan-Meier analysis of USP5 and CD4 or CD8 expression and overall survival of 90 HCC patients. b A total of 5.0 × 10 6 WT or Usp5-KO Hepa1-6 cells were subcutaneously implanted in C57BL/6 mice pretreated with anti-IgG, anti-NK1.1, anti-CD4, and anti-CD8 antibodies (n = 6). Tumor growth was monitored at the indicated time points. *** p < 0.001 by two-way ANOVA. c IB analysis of lysates derived from dissected xenografts formed by Usp5-depleted H22 or Hepa1-6 cells. d , e IB analysis of Pd-l1 in the WT and Usp5-KO Hepa1-6 cells transfected with Flag-Ythdf1 constructs for 36 h or treated with 2.5 μM WP1130 for 24 h. f Activated T cells and tumor cells were co-cultured in 24-well plates for 4 days and surviving tumor cells were visualized by crystal violet staining. Relative fold ratios of surviving cell intensities are shown. n = 3. ** p < 0.01. t- test. g Correlation between the fold changes of differentially expressed genes in Ythdf1 and Usp5 knockout Hepa1-6 cells. h The top 5 terms in GO analysis of the immune-related genes suppressed by both Usp5 and Ythdf1 knockout Hepa1-6 cells. n = 3. i GSEA and heatmap showing the differential expression of genes in Fig. 5g. n = 3. j mRNA levels of indicated genes from sgUsp5 or sgCtrl Hepa1-6 cells were analyzed using QRT-PCR. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. t- test. k The mRNA and protein levels in Usp5 and Ythdf1 knockout Hepa1-6 cells are measured by RNA-Seq and proteomics. l , m Usp5 or Ythdf1 depletion affects tumor growth in mouse xenograft in NOD/SCID and C57BL/6 mice ( n = 8). Tumor growth was monitored at the indicated time points, and tumor volume were measured at the endpoint ( l ). Tumor image and tumor weight are presented ( m ). *** p < 0.001 by two-way ANOVA. All data are presented as mean ± SEM. All IB data are representative of two independent experiments. Source data are provided as a file.

Journal: Nature Communications

Article Title: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation

doi: 10.1038/s41467-025-56564-9

Figure Lengend Snippet: a Kaplan-Meier analysis of USP5 and CD4 or CD8 expression and overall survival of 90 HCC patients. b A total of 5.0 × 10 6 WT or Usp5-KO Hepa1-6 cells were subcutaneously implanted in C57BL/6 mice pretreated with anti-IgG, anti-NK1.1, anti-CD4, and anti-CD8 antibodies (n = 6). Tumor growth was monitored at the indicated time points. *** p < 0.001 by two-way ANOVA. c IB analysis of lysates derived from dissected xenografts formed by Usp5-depleted H22 or Hepa1-6 cells. d , e IB analysis of Pd-l1 in the WT and Usp5-KO Hepa1-6 cells transfected with Flag-Ythdf1 constructs for 36 h or treated with 2.5 μM WP1130 for 24 h. f Activated T cells and tumor cells were co-cultured in 24-well plates for 4 days and surviving tumor cells were visualized by crystal violet staining. Relative fold ratios of surviving cell intensities are shown. n = 3. ** p < 0.01. t- test. g Correlation between the fold changes of differentially expressed genes in Ythdf1 and Usp5 knockout Hepa1-6 cells. h The top 5 terms in GO analysis of the immune-related genes suppressed by both Usp5 and Ythdf1 knockout Hepa1-6 cells. n = 3. i GSEA and heatmap showing the differential expression of genes in Fig. 5g. n = 3. j mRNA levels of indicated genes from sgUsp5 or sgCtrl Hepa1-6 cells were analyzed using QRT-PCR. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. t- test. k The mRNA and protein levels in Usp5 and Ythdf1 knockout Hepa1-6 cells are measured by RNA-Seq and proteomics. l , m Usp5 or Ythdf1 depletion affects tumor growth in mouse xenograft in NOD/SCID and C57BL/6 mice ( n = 8). Tumor growth was monitored at the indicated time points, and tumor volume were measured at the endpoint ( l ). Tumor image and tumor weight are presented ( m ). *** p < 0.001 by two-way ANOVA. All data are presented as mean ± SEM. All IB data are representative of two independent experiments. Source data are provided as a file.

Article Snippet: Human YTHDF1 was subcloned into the pGEX-4T-1 construct (Addgene 70087).

Techniques: Expressing, Derivative Assay, Transfection, Construct, Cell Culture, Staining, Knock-Out, Quantitative Proteomics, Quantitative RT-PCR, RNA Sequencing

a Tumor growth of sgCtrl and sgUsp5 Hepa1-6 cells in C57BL/6 mice with anti-IgG mAb or anti-PD-L1 mAb treatments. n = 10. b Kaplan-Meier survival curves for four treatment groups demonstrate the improved efficacy of PD-L1 mAb after knockout Usp5. ** P < 0.01. c A schematic model illustrating the treatment plan for mice bearing subcutaneous Hepa1-6 or H22 tumors. Male C57BL/6 mice were implanted with Hepa1-6 or H22 cells subcutaneously and treated with four arms: control antibody (anti-IgG mAb) treatment, anti-PD-L1 mAb treatment, USP5 inhibitor WP1130 treatment, and anti-PD-L1 mAb plus USP5 inhibitor combination treatment. d Hepa1-6 and H22 implanted tumor-bearing mice were enrolled in different treatment groups as indicated. Tumor volumes of mice treated with control antibody, anti-PD-L1 mAb, the USP5 inhibitor WP1130, or combined therapy were measured every 3 days and plotted individually. n = 10 mice per group. e Kaplan-Meier survival curves for each treatment group demonstrate the improved efficacy of combining PD-L1 mAb with the USP5 inhibitor WP1130. *** P < 0.001. f Immunohistochemical (IHC) analysis of Cd8, Granzyme B (GzmB), Tim-3, Ythdf1 and Pd-l1 expression in Hepa1-6 tumors after indicated treatments. g Representative multiplex immunohistochemistry (mIHC) images of Cd8 (Gray), GzmB (Green), Tim-3 (Red), and DAPI nuclear staining (blue) in Hepa1-6 tumors after indicated treatments. All data are presented as mean ± SEM. Source data are provided as a file.

Journal: Nature Communications

Article Title: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation

doi: 10.1038/s41467-025-56564-9

Figure Lengend Snippet: a Tumor growth of sgCtrl and sgUsp5 Hepa1-6 cells in C57BL/6 mice with anti-IgG mAb or anti-PD-L1 mAb treatments. n = 10. b Kaplan-Meier survival curves for four treatment groups demonstrate the improved efficacy of PD-L1 mAb after knockout Usp5. ** P < 0.01. c A schematic model illustrating the treatment plan for mice bearing subcutaneous Hepa1-6 or H22 tumors. Male C57BL/6 mice were implanted with Hepa1-6 or H22 cells subcutaneously and treated with four arms: control antibody (anti-IgG mAb) treatment, anti-PD-L1 mAb treatment, USP5 inhibitor WP1130 treatment, and anti-PD-L1 mAb plus USP5 inhibitor combination treatment. d Hepa1-6 and H22 implanted tumor-bearing mice were enrolled in different treatment groups as indicated. Tumor volumes of mice treated with control antibody, anti-PD-L1 mAb, the USP5 inhibitor WP1130, or combined therapy were measured every 3 days and plotted individually. n = 10 mice per group. e Kaplan-Meier survival curves for each treatment group demonstrate the improved efficacy of combining PD-L1 mAb with the USP5 inhibitor WP1130. *** P < 0.001. f Immunohistochemical (IHC) analysis of Cd8, Granzyme B (GzmB), Tim-3, Ythdf1 and Pd-l1 expression in Hepa1-6 tumors after indicated treatments. g Representative multiplex immunohistochemistry (mIHC) images of Cd8 (Gray), GzmB (Green), Tim-3 (Red), and DAPI nuclear staining (blue) in Hepa1-6 tumors after indicated treatments. All data are presented as mean ± SEM. Source data are provided as a file.

Article Snippet: Human YTHDF1 was subcloned into the pGEX-4T-1 construct (Addgene 70087).

Techniques: Knock-Out, Control, Immunohistochemical staining, Expressing, Multiplex Assay, Immunohistochemistry, Staining

(A) Representative images of immunohistochemical analysis of YTHDF1 in the cortex of 12-month-old (G 4 C 2 ) 2 or (G 4 C 2 ) 149 mice (n = 6 per group). Black arrows indicate inclusions. Scale bars, 20 μm. (B) Double-immunofluorescence staining for poly(GR) and YTHDF1 in the cortex of 12-month-old (G 4 C 2 ) 2 or (G 4 C 2 ) 149 mice (n = 6 per group). Scale bars, 2 μm. (C) Double-immunofluorescence staining for poly(GR) and m6A-modified RNAs in the cortex of 12-month-old (G 4 C 2 ) 2 or (G 4 C 2 ) 149 mice. Scale bars, 2 μm. (D) Double-immunofluorescence staining for poly(GR) and YTHDF1 in the cortex of 2-week-old GFP-(GR) 200 mice. Scale bars, 2 μm. NT, non-transduced cells; Diffusion, cells with diffuse poly(GR); Inclusion, cells with poly(GR) inclusions. (E) Quantification of the percentage of NT cells and transduced cells with diffuse poly(GR) or poly(GR) inclusions with YTHDF1 inclusions (n = 6). (F) Double-immunofluorescence staining for poly(GR) and m6A-modified RNAs in the cortex of 2-week-old GFP-(GR) 200 mice. Scale bars, 2 μm. (G) Quantification of the percentage of NT cells or transduced cells with diffuse poly(GR) or poly(GR) inclusion with m6A-modified RNA-containing inclusions (n = 6). (H) Double-immunofluorescence staining for poly(GA) and YTHDF1 in the cortex of 3.5-month-old (GA) 100 -V5 mice (n = 3). Scale bars, 2 μm. (I) Double-immunofluorescence staining for poly(GA) and m6A-modified RNAs in the cortex of 3.5-month-old (GA) 100 -V5 mice (n = 3). Scale bars, 2 μm. (J) Double-immunofluorescence staining for poly(GR) and YTHDF1 in the mid-frontal cortex of patients with c9FTD. The intracellular localization of YTHDF1 is shown for cells without or with poly(GR) inclusions (n = 6). Scale bars, 2 μm. (K) Double-immunofluorescence staining for poly(GR) and m6A-modified RNAs in the mid-frontal cortex of patients with c9FTD. The intracellular localization of m6A-modified RNA is shown for cells without and with poly(GR) inclusions (n = 6). Scale bars, 2 μm. Data are shown as the mean ± SEM. In (E), ****p < 0.0001, one-way ANOVA, Tukey’s post hoc analysis. In (G), ****p < 0.0001, one-way ANOVA, Tukey’s post hoc analysis.

Journal: Cell reports

Article Title: Poly(GR) interacts with key stress granule factors promoting its assembly into cytoplasmic inclusions

doi: 10.1016/j.celrep.2023.112822

Figure Lengend Snippet: (A) Representative images of immunohistochemical analysis of YTHDF1 in the cortex of 12-month-old (G 4 C 2 ) 2 or (G 4 C 2 ) 149 mice (n = 6 per group). Black arrows indicate inclusions. Scale bars, 20 μm. (B) Double-immunofluorescence staining for poly(GR) and YTHDF1 in the cortex of 12-month-old (G 4 C 2 ) 2 or (G 4 C 2 ) 149 mice (n = 6 per group). Scale bars, 2 μm. (C) Double-immunofluorescence staining for poly(GR) and m6A-modified RNAs in the cortex of 12-month-old (G 4 C 2 ) 2 or (G 4 C 2 ) 149 mice. Scale bars, 2 μm. (D) Double-immunofluorescence staining for poly(GR) and YTHDF1 in the cortex of 2-week-old GFP-(GR) 200 mice. Scale bars, 2 μm. NT, non-transduced cells; Diffusion, cells with diffuse poly(GR); Inclusion, cells with poly(GR) inclusions. (E) Quantification of the percentage of NT cells and transduced cells with diffuse poly(GR) or poly(GR) inclusions with YTHDF1 inclusions (n = 6). (F) Double-immunofluorescence staining for poly(GR) and m6A-modified RNAs in the cortex of 2-week-old GFP-(GR) 200 mice. Scale bars, 2 μm. (G) Quantification of the percentage of NT cells or transduced cells with diffuse poly(GR) or poly(GR) inclusion with m6A-modified RNA-containing inclusions (n = 6). (H) Double-immunofluorescence staining for poly(GA) and YTHDF1 in the cortex of 3.5-month-old (GA) 100 -V5 mice (n = 3). Scale bars, 2 μm. (I) Double-immunofluorescence staining for poly(GA) and m6A-modified RNAs in the cortex of 3.5-month-old (GA) 100 -V5 mice (n = 3). Scale bars, 2 μm. (J) Double-immunofluorescence staining for poly(GR) and YTHDF1 in the mid-frontal cortex of patients with c9FTD. The intracellular localization of YTHDF1 is shown for cells without or with poly(GR) inclusions (n = 6). Scale bars, 2 μm. (K) Double-immunofluorescence staining for poly(GR) and m6A-modified RNAs in the mid-frontal cortex of patients with c9FTD. The intracellular localization of m6A-modified RNA is shown for cells without and with poly(GR) inclusions (n = 6). Scale bars, 2 μm. Data are shown as the mean ± SEM. In (E), ****p < 0.0001, one-way ANOVA, Tukey’s post hoc analysis. In (G), ****p < 0.0001, one-way ANOVA, Tukey’s post hoc analysis.

Article Snippet: pGEX-4T-1 YTHDF1 , Wang, X. et al. , Addgene Cat#70087.

Techniques: Immunohistochemical staining, Double Immunofluorescence Staining, Modification, Diffusion-based Assay

(A) Double-immunofluorescence staining for YTHDF1 and G3BP1 in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 48 h post transfection. Scale bars, 10 μm. (B) Quantification of the percentage of GFP-positive cells containing poly(GR) inclusions in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 (n = 3 independent experiments). (C) Quantification of the size of poly(GR) inclusions in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 (n = 3 independent experiments). (D) Double-immunofluorescence staining for V5 and G3BP1 in HEK293T cells co-expressing GFP-(GR) 100 and either tagBFP-V5 or tagBFP/V5 tagged wild-type YTHDF1 (tagBFP-DF1-V5). Scale bars, 10 μm. (E) Quantification of the percentage of GFP-positive cells containing poly(GR) inclusions in HEK293T cells co-expressing GFP-(GR) 100 and either tagBFP-V5 or tagBFP-DF1-V5 (n = 3 independent experiments). (F) Quantification of the size of poly(GR) inclusions in GFP-(GR) 100 overexpressing HEK293T cells co-expressing either tagBFP-V5 or tagBFP-DF1-V5 (n = 3 independent experiments). (G) Double-immunofluorescence staining for YTHDF1 and G3BP1 in ALKBH5-depleted HEK293T cells expressing GFP-(GR) 100 . Staining was performed 24 h after GFP-(GR) 100 transfection. Scale bars, 20 μm. (H) Quantification of the percentage of GFP-positive cells containing poly(GR) inclusions in ALKBH5-depleted HEK293T cells expressing GFP-(GR) 100 (n = 4 independent experiments). (I) Quantification of the size of poly(GR) inclusions in ALKBH5-depleted HEK293T cells expressing GFP-(GR) 100 (n = 4 independent experiments). (J) Double-immunofluorescence staining for Flag and G3BP1 in Flag-ALKBH5 overexpressing HEK293T cells co-expressing GFP-(GR) 100 . Scale bars, 20 μm. (K) Quantification of the percentage of GFP-positive cells with poly(GR) inclusions in Flag-ALKBH5 overexpressing HEK293T cells co-expressing GFP-(GR) 100 (n = 3 independent experiments). (L) Quantification of the size of poly(GR) inclusions in Flag-ALKBH5 and GFP-(GR) 100 co-expressing HEK293T cells (n = 3 independent experiments). (M) Double-immunofluorescence staining for YTHDF1 and G3BP1 in HEK293T cells expressing GFP-(GR) 100 in which only ALKBH5 was depleted or in which ALKBH5 and YTHDF1/3 were depleted. Scale bars, 20 μm. (N) Quantification of the percentage of GFP-positive cells containing poly(GR) inclusions in HEK293T cells expressing GFP-(GR) 100 in which only ALKBH5 was depleted or in which ALKBH5 and YTHDF1/3 were depleted (n = 3 independent experiments). (O) Quantification of the size of poly(GR) inclusions in ALKBH5-depleted or ALKBH5 and YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 (n = 3 independent experiments). Data are shown as mean ± SEM. In (B), **p = 0.0095, unpaired two-tailed t test. In (C), ****p < 0.0001, unpaired two-tailed t test. In (E), **p = 0.0011, unpaired two-tailed t test. In (F), ****p < 0.0001, unpaired two-tailed t test. In (H), ****p < 0.0001, unpaired two-tailed t test. In (I), *p = 0.0101, unpaired two-tailed t test. In (K), **p = 0.0033, unpaired two-tailed t test. In (L), ***p = 0.0007, unpaired two-tailed t test. In (N), ***p = 0.0006 and *p = 0.0193, one-way ANOVA, Tukey’s post hoc analysis. In (O), *** (left to right) p = 0.0001 and p = 0.0001, one-way ANOVA, Tukey’s post hoc analysis.

Journal: Cell reports

Article Title: Poly(GR) interacts with key stress granule factors promoting its assembly into cytoplasmic inclusions

doi: 10.1016/j.celrep.2023.112822

Figure Lengend Snippet: (A) Double-immunofluorescence staining for YTHDF1 and G3BP1 in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 48 h post transfection. Scale bars, 10 μm. (B) Quantification of the percentage of GFP-positive cells containing poly(GR) inclusions in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 (n = 3 independent experiments). (C) Quantification of the size of poly(GR) inclusions in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 (n = 3 independent experiments). (D) Double-immunofluorescence staining for V5 and G3BP1 in HEK293T cells co-expressing GFP-(GR) 100 and either tagBFP-V5 or tagBFP/V5 tagged wild-type YTHDF1 (tagBFP-DF1-V5). Scale bars, 10 μm. (E) Quantification of the percentage of GFP-positive cells containing poly(GR) inclusions in HEK293T cells co-expressing GFP-(GR) 100 and either tagBFP-V5 or tagBFP-DF1-V5 (n = 3 independent experiments). (F) Quantification of the size of poly(GR) inclusions in GFP-(GR) 100 overexpressing HEK293T cells co-expressing either tagBFP-V5 or tagBFP-DF1-V5 (n = 3 independent experiments). (G) Double-immunofluorescence staining for YTHDF1 and G3BP1 in ALKBH5-depleted HEK293T cells expressing GFP-(GR) 100 . Staining was performed 24 h after GFP-(GR) 100 transfection. Scale bars, 20 μm. (H) Quantification of the percentage of GFP-positive cells containing poly(GR) inclusions in ALKBH5-depleted HEK293T cells expressing GFP-(GR) 100 (n = 4 independent experiments). (I) Quantification of the size of poly(GR) inclusions in ALKBH5-depleted HEK293T cells expressing GFP-(GR) 100 (n = 4 independent experiments). (J) Double-immunofluorescence staining for Flag and G3BP1 in Flag-ALKBH5 overexpressing HEK293T cells co-expressing GFP-(GR) 100 . Scale bars, 20 μm. (K) Quantification of the percentage of GFP-positive cells with poly(GR) inclusions in Flag-ALKBH5 overexpressing HEK293T cells co-expressing GFP-(GR) 100 (n = 3 independent experiments). (L) Quantification of the size of poly(GR) inclusions in Flag-ALKBH5 and GFP-(GR) 100 co-expressing HEK293T cells (n = 3 independent experiments). (M) Double-immunofluorescence staining for YTHDF1 and G3BP1 in HEK293T cells expressing GFP-(GR) 100 in which only ALKBH5 was depleted or in which ALKBH5 and YTHDF1/3 were depleted. Scale bars, 20 μm. (N) Quantification of the percentage of GFP-positive cells containing poly(GR) inclusions in HEK293T cells expressing GFP-(GR) 100 in which only ALKBH5 was depleted or in which ALKBH5 and YTHDF1/3 were depleted (n = 3 independent experiments). (O) Quantification of the size of poly(GR) inclusions in ALKBH5-depleted or ALKBH5 and YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 (n = 3 independent experiments). Data are shown as mean ± SEM. In (B), **p = 0.0095, unpaired two-tailed t test. In (C), ****p < 0.0001, unpaired two-tailed t test. In (E), **p = 0.0011, unpaired two-tailed t test. In (F), ****p < 0.0001, unpaired two-tailed t test. In (H), ****p < 0.0001, unpaired two-tailed t test. In (I), *p = 0.0101, unpaired two-tailed t test. In (K), **p = 0.0033, unpaired two-tailed t test. In (L), ***p = 0.0007, unpaired two-tailed t test. In (N), ***p = 0.0006 and *p = 0.0193, one-way ANOVA, Tukey’s post hoc analysis. In (O), *** (left to right) p = 0.0001 and p = 0.0001, one-way ANOVA, Tukey’s post hoc analysis.

Article Snippet: pGEX-4T-1 YTHDF1 , Wang, X. et al. , Addgene Cat#70087.

Techniques: Double Immunofluorescence Staining, Expressing, Transfection, Staining, Two Tailed Test

(A) Schematic of the tagBFP/V5 tagged YTHDF1 wild-type (tagBFP-DF1-WT-V5) and mutant (tagBFP-DF1-mut-V5) constructs, the latter having mutations in the YTH domain that impair the ability of YTHDF1 to bind m6A-modified RNA (top). (B) Representative images of proximity ligation assay (PLA) for GFP-(GR) 100 and tagBFP or tagBFP-YTHDF1 species in HEK293T cells co-expressing GFP-(GR) 100 and tagBFP or tagBFP-YTHDF1 species. Scale bars, 2 μm. (C) Quantification of the intensity for PLA signal in HEK293T cells co-expressing GFP-(GR) 100 and tagBFP or tagBFP-YTHDF1 species (n = 152–176 cells). (D) Immunofluorescence staining for V5 in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5 or tagBFP-DF1-mut-V5. Scale bars, 10 μm. (E) Quantification of the percentage of cells with poly(GR) inclusions in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5 (n = 3 independent experiments). (F) Quantification of the size of poly(GR) inclusions in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5 (n = 3 independent experiments). (G) Triple-immunofluorescence staining for GFP, V5, and m6A in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5. Scale bars, 5 μm. (H) Quantification of the relative ratio of m6A intensity (poly(GR) inclusions/total) from YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5 (n = 3 independent experiments). (I) Immunofluorescence staining for V5 followed by RNA-FISH for oligo(dT) in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5. Scale bars, 5 μm. (J) Quantification of the relative ratio of oligo(dT) intensity (poly(GR) inclusions/total) from YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5 (n = 3 independent experiments). Data are shown as mean ± SEM. In (C), ****p < 0.0001 and ns p = 0.0547, one-way ANOVA, Tukey’s post hoc analysis. In (E), ***p = 0.0007, **p = 0.0022, and ns (not significant) p = 0.3670, one-way ANOVA, Tukey’s post hoc analysis. In (F), ****p < 0.0001 and ns p = 0.0576, one-way ANOVA, Tukey’s post hoc analysis. In (H), ****p < 0.0001 and ns p = 0.4376, one-way ANOVA, Tukey’s post hoc analysis. In (J), ****p < 0.0001 and ns p = 0.9690, one-way ANOVA, Tukey’s post hoc analysis.

Journal: Cell reports

Article Title: Poly(GR) interacts with key stress granule factors promoting its assembly into cytoplasmic inclusions

doi: 10.1016/j.celrep.2023.112822

Figure Lengend Snippet: (A) Schematic of the tagBFP/V5 tagged YTHDF1 wild-type (tagBFP-DF1-WT-V5) and mutant (tagBFP-DF1-mut-V5) constructs, the latter having mutations in the YTH domain that impair the ability of YTHDF1 to bind m6A-modified RNA (top). (B) Representative images of proximity ligation assay (PLA) for GFP-(GR) 100 and tagBFP or tagBFP-YTHDF1 species in HEK293T cells co-expressing GFP-(GR) 100 and tagBFP or tagBFP-YTHDF1 species. Scale bars, 2 μm. (C) Quantification of the intensity for PLA signal in HEK293T cells co-expressing GFP-(GR) 100 and tagBFP or tagBFP-YTHDF1 species (n = 152–176 cells). (D) Immunofluorescence staining for V5 in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5 or tagBFP-DF1-mut-V5. Scale bars, 10 μm. (E) Quantification of the percentage of cells with poly(GR) inclusions in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5 (n = 3 independent experiments). (F) Quantification of the size of poly(GR) inclusions in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5 (n = 3 independent experiments). (G) Triple-immunofluorescence staining for GFP, V5, and m6A in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5. Scale bars, 5 μm. (H) Quantification of the relative ratio of m6A intensity (poly(GR) inclusions/total) from YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5 (n = 3 independent experiments). (I) Immunofluorescence staining for V5 followed by RNA-FISH for oligo(dT) in YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5. Scale bars, 5 μm. (J) Quantification of the relative ratio of oligo(dT) intensity (poly(GR) inclusions/total) from YTHDF1/3-depleted HEK293T cells expressing GFP-(GR) 100 and either tagBFP-V5, tagBFP-DF1-WT-V5, or tagBFP-DF1-mut-V5 (n = 3 independent experiments). Data are shown as mean ± SEM. In (C), ****p < 0.0001 and ns p = 0.0547, one-way ANOVA, Tukey’s post hoc analysis. In (E), ***p = 0.0007, **p = 0.0022, and ns (not significant) p = 0.3670, one-way ANOVA, Tukey’s post hoc analysis. In (F), ****p < 0.0001 and ns p = 0.0576, one-way ANOVA, Tukey’s post hoc analysis. In (H), ****p < 0.0001 and ns p = 0.4376, one-way ANOVA, Tukey’s post hoc analysis. In (J), ****p < 0.0001 and ns p = 0.9690, one-way ANOVA, Tukey’s post hoc analysis.

Article Snippet: pGEX-4T-1 YTHDF1 , Wang, X. et al. , Addgene Cat#70087.

Techniques: Mutagenesis, Construct, Modification, Proximity Ligation Assay, Expressing, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Poly(GR) interacts with key stress granule factors promoting its assembly into cytoplasmic inclusions

doi: 10.1016/j.celrep.2023.112822

Figure Lengend Snippet:

Article Snippet: pGEX-4T-1 YTHDF1 , Wang, X. et al. , Addgene Cat#70087.

Techniques: Purification, Virus, Recombinant, Protease Inhibitor, Imaging, Saline, Staining, Hybridization, Blocking Assay, In Situ, Proximity Ligation Assay, Fluorescence, Methylation, Bradford Assay, Bicinchoninic Acid Protein Assay, Mutagenesis, Software, Microscopy

The 10 most abundant m 6 A-centered 5-mers and 11-mers enriched upon in vitro binding selections with YTH readers. 5-mers and 11-mers shared between selections are highlighted in yellow and purple, respectively.

Journal: Biochemistry

Article Title: In vitro selection with a site-specifically modified RNA library reveals the binding preferences of N 6 -methyladenosine (m 6 A) reader proteins

doi: 10.1021/acs.biochem.9b00485

Figure Lengend Snippet: The 10 most abundant m 6 A-centered 5-mers and 11-mers enriched upon in vitro binding selections with YTH readers. 5-mers and 11-mers shared between selections are highlighted in yellow and purple, respectively.

Article Snippet: Plasmids encoding cDNA for YTH proteins were obtained from Addgene: YTHDC1 ( {"type":"entrez-protein","attrs":{"text":"NP_001026902.1","term_id":"72534750","term_text":"NP_001026902.1"}} NP_001026902.1 ) (#85167) 46 , YTHDF1 ( {"type":"entrez-protein","attrs":{"text":"NP_060268.2","term_id":"31377750","term_text":"NP_060268.2"}} NP_060268.2 ) (# 70087) 5 , and YTHDF2 ( {"type":"entrez-protein","attrs":{"text":"NP_057342.2","term_id":"116812575","term_text":"NP_057342.2"}} NP_057342.2 ) (# 52300) 5 .

Techniques: In Vitro, Binding Assay

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pgex 4t 1 construct (Addgene inc)

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