Journal: Microorganisms

Article Title: Coxsackievirus B3-Induced m 6 A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

doi: 10.3390/microorganisms12112152

Figure Lengend Snippet: CVB3 infection induces subcellular redistribution of METTL3, ALKBH5 and YTHDF2. ( A ) METTL3 and ( B ) ALKBH5 translocate from the nucleus to the cytoplasm. HeLa cells were infected with CVB3 (10 MOI) or sham-infected with PBS for 5 h. Confocal images show immunofluorescent staining results. The nucleus (blue) was stained with DAPI. Viral protein VP1 (pink) and METTL3 or ALKBH5 (green) were stained using specific antibodies. ( C ) m 6 A reader protein YTHDF2 translocates from the cytoplasm to the nucleus. Cells were treated as described above but stained with an anti-YTHDF2 antibody. Note that at 5 h post infection (hpi), some VP1 also translocated to the nucleus.

Article Snippet: Polyclonal rabbit anti-human YTHDF1 (66745-1AP), YTHDF2 (24744-1AP), YTHDF3 (25537-1-AP), METTL3 (15073-AP) and METTL14 (23158-1-AP) (Proteintech, Rosemont, IL, USA); polyclonal rabbit anti-human ALKBH5 (Abcam, Cambridge, United Kingdom); monoclonal mouse anti-Flag tag (Sigma) and monoclonal mouse anti-3D antibody (GeneTex, Irvine, CA, USA).

Techniques: Infection, Staining

Journal: Microorganisms

Article Title: Coxsackievirus B3-Induced m 6 A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

doi: 10.3390/microorganisms12112152

Figure Lengend Snippet: CVB3 infection induces cleavage of reader proteins YTHDF1-3 and increases CVB3 VP1 production and 2A transcription. HeLa cells were infected with CVB3 or sham-infected with PBS. Cell lysates were collected at 3 and 5 hpi for Western blot analysis to detect YTHDF2 ( A ), YTHDF1 ( B ), and YTHDF3 ( C ), as well as viral VP1. ( D ) HeLa cells were transfected with YTHDF2 siRNA (siY2) or control siRNA (siCtrl) and then infected with CVB3. ( G ) HeLa cells were transfected with a YTHDF2 plasmid (pY2) or an empty vector and then infected with CVB3. Western blot was conducted using the indicated antibodies ( D , G ), and protein levels were quantified using the ImageJ program ( E , H ). RT-qPCR was conducted using samples from ( D , G ) to measure the transcripts of the viral gene 2A ( F , I ). Data are presented as means ± SEM, n = 3, * p < 0.05, ** p < 0.01.

Article Snippet: Polyclonal rabbit anti-human YTHDF1 (66745-1AP), YTHDF2 (24744-1AP), YTHDF3 (25537-1-AP), METTL3 (15073-AP) and METTL14 (23158-1-AP) (Proteintech, Rosemont, IL, USA); polyclonal rabbit anti-human ALKBH5 (Abcam, Cambridge, United Kingdom); monoclonal mouse anti-Flag tag (Sigma) and monoclonal mouse anti-3D antibody (GeneTex, Irvine, CA, USA).

Techniques: Infection, Western Blot, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR

Journal: Microorganisms

Article Title: Coxsackievirus B3-Induced m 6 A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

doi: 10.3390/microorganisms12112152

Figure Lengend Snippet: Colocalization and interaction of YTHDF2 with HuR in SGs. ( A ) HeLa cells were either infected with CVB3 (MOI 10) or sham-infected with PBS. The cells were fixed at 3 and 5 hpi and then subjected to immunofluorescent staining for YTHDF2, HuR and DAPI. Images were captured by confocal microscopy. Red indicates YTHDF2, green indicates HuR and blue indicates DAPI. Scale bar: 10 µm. ( B ) The number of SGs per cell was quantified using a total of 25 cells from 5 random microscopic views (n = 25). Data were analyzed by Student’s t -test with Welch’s correction and are presented as means ± SEM, **** p < 0.0001. ( C ) HeLa cells were infected with CVB3 (MOI 10) for 3 h, and the collected cell lysates were used for co-immunoprecipitation (IP) with HuR antibody. The pulled HuR-protein complexes were detected by Western blot using a YTHDF2 antibody. Conversely, cell lysates were also incubated with the YTHDF2 antibody to pull down the complexes and then detected using the HuR antibody. IgG was used as a control.

Article Snippet: Polyclonal rabbit anti-human YTHDF1 (66745-1AP), YTHDF2 (24744-1AP), YTHDF3 (25537-1-AP), METTL3 (15073-AP) and METTL14 (23158-1-AP) (Proteintech, Rosemont, IL, USA); polyclonal rabbit anti-human ALKBH5 (Abcam, Cambridge, United Kingdom); monoclonal mouse anti-Flag tag (Sigma) and monoclonal mouse anti-3D antibody (GeneTex, Irvine, CA, USA).

Techniques: Infection, Staining, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation, Control

Journal: Microorganisms

Article Title: Coxsackievirus B3-Induced m 6 A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

doi: 10.3390/microorganisms12112152

Figure Lengend Snippet: Silencing m 6 A machinery suppresses SG formation. HeLa cells were transfected with specific siRNAs to knock down either METTL3 ( A ) or YTHDF2 ( C ) and then infected with CVB3 at an MOI of 10. Cells were fixed at 3 hpi and then subjected to immunofluorescent staining for HuR and DAPI. Images were captured by confocal microscopy. Green indicates HuR and blue indicates DAPI. Scale bar: 10 µm. ( B , D ) The number of SGs per cell was quantified using a total of 25 cells from 5 random microscopic views (n = 25). Data were analyzed by Student’s t -test with Welch’s correction and are presented as means ± SEM, **** p < 0.0001.

Article Snippet: Polyclonal rabbit anti-human YTHDF1 (66745-1AP), YTHDF2 (24744-1AP), YTHDF3 (25537-1-AP), METTL3 (15073-AP) and METTL14 (23158-1-AP) (Proteintech, Rosemont, IL, USA); polyclonal rabbit anti-human ALKBH5 (Abcam, Cambridge, United Kingdom); monoclonal mouse anti-Flag tag (Sigma) and monoclonal mouse anti-3D antibody (GeneTex, Irvine, CA, USA).

Techniques: Transfection, Knockdown, Infection, Staining, Confocal Microscopy

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: Analysis of the expression of YTHDF2 and its impact on the progression of NPC. (A) Comparison of the transcription levels of YTHDF2 in the combined GSE12452 , GSE180272 , GSE53819 , GSE61218 , and GSE64634 datasets. (B) Comparison of the expression level of YTHDF2 between malignant and nonmalignant cells in single-cell NPC dataset GSE150430 . (C) ROC curve analysis of YTHDF2 in diagnosing NPC. (D) Kaplan–Meier survival curve for YTHDF2 expression of NPC in GSE102349 .

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: Expressing, Comparison

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: The expression of FOXO1 in the transcription and translation of NPC cells and tissues. (A) Comparison of the transcription level of YTHDF2 in NPC cell lines with NP69. (B) Comparison of the transcription level of YTHDF2 in NPC tissues with Rhinitis.(A: n = 3 biological replicates, one-way ANOVA, B: t-test). (C-D) Comparison of the protein level of YTHDF2 in NPC cell lines with NP69. (E-F) Comparison of the protein level of YTHDF2 in NPC tissues with Rhinitis. (D: n = 3 biological replicates, one-way ANOVA, F: t-test). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns: not significant.

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: Expressing, Comparison

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: In vitro functional experiments of YTHDF2 knockdown in NPC cells ( n = 3 biological replicates, student's t-test). (A-C) YTHDF2 knockdown inhibited the proliferation in 5-8F, CNE1 and HONE1cells. (D-G) YTHDF2 knockdown reduced the migration in 5-8F, CNE1 and HONE1 cells. (H-I) YTHDF2 knockdown reduced the invasive capabilities of 5-8F, CNE1 and HONE1 cells. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: In Vitro, Functional Assay, Knockdown, Migration

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: In vitro functional experiments of YTHDF2 overexpression in NPC cells ( n = 3 biological replicates, student's t-test). (A-C) YTHDF2-transfected 5-8F, CNE1, and HONE1 cells exhibited accelerated proliferation. (D-G) Enhanced migratory capacity was observed in YTHDF2-overexpressing cells. (H-I) Matrigel invasion assays revealed significantly increased invasiveness following YTHDF2 overexpression. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: In Vitro, Functional Assay, Over Expression, Transfection

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: Correlation between YTHDF2 and FOXO1 expression ( n = 3 biological replicates, C-D: t-test, E-F: one-way ANOVA). (A) GSEA in GSE102349 revealed enrichment pathways in the YTHDF2-activated group. (B) Correlation analysis between YTHDF2 and FOXO1 expression in GSE102349 . Analysis of FOXO1 expression in 5-8F and CNE1 cells following YTHDF2 knockdown (C) or overexpression (D). Comparison of FOXO1 expression in NPC cell lines 5-8F (E) and CNE1 (F) which were treated with the m6A demethylase inhibitor 3-DAA for 48 h. * P < 0.05; ** P < 0.01; *** P < 0.001; ns: not significant.

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: Expressing, Knockdown, Over Expression, Comparison

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: YTHDF2 participates in m6A-mediated regulation of FOXO1. (A) Distribution of m6A modification sites of FOXO1 mRNA in 5-8F. (B) Volcano plot of MeRIP-seq analysis for YTHDF2. (C) RIP-qPCR exhibited the relative expression of FOXO1. (D) YFHDF2 binding interval in FOXO1. (E) M6A modification site on the 3'UTR of FOXO1.

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: Modification, Expressing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation

doi: 10.1016/j.jbc.2024.107152

Figure Lengend Snippet: OTUB1 regulates YTHDF2 protein stability. A , 4 μg Myc-OTUB1 and FLAG-YTHDF2 plasmids were cotransfected into HEK-293T cells in a 6-cm dish. Forty eight hours later, cells were collected for Co-IP–WB analysis as indicated. "Input" denotes 1% input material for each IP. B , PC-3 cells were analyzed with Co-IP–WB as indicated. "Input" denotes 1% input material for each IP. C , OTUB1 was knocked down in PC-3 cells with shRNA. Whole-cell extracts (WCE) were then analyzed with WB. D , shRNA-resistant Myc-OTUB1 was introduced into OTUB1-KD PC-3 cells. WCE were then analyzed with WB. E , HA-OTUB1 was expressed in PC-3 cells. WCE were then analyzed with WB. F , control or OTUB1-KD PC-3 cells were treated with 25 μg/ml cycloheximide (Chx) for the indicated time. WCE were analyzed with WB. For YTHDF2, separate exposures for control and OTUB1-KD cells were presented to make the signals of "Chx 0 h" comparable and facilitate the comparison of protein stability. G , shRNA-resistant Myc-OTUB1 was rescue-expressed into OTUB1-KD cells. Cells were then treated with 25 μg/ml Chx for the indicated time. WCE were analyzed with WB. For YTHDF2, separate exposures for control, "OTUB1-KD" and "OTUB1-KD + Myc-OTUB1" cells were presented to make the signals of "Chx 0 h" comparable and facilitate comparison of protein stability. H , Myc-OTUB1 was expressed in PC-3 cells. Cells were then treated with 25 μg/ml Chx for different time. WCE were analyzed with WB. For YTHDF2, separate exposures for control and "Myc-OTUB1" cells were presented to make the signals of "Chx 0 h" comparable and facilitate comparison of protein stability. Co-immunoprecipitation; HA, hemagglutinin; IP, immunoprecipitation; KD, knock down; m6A, methylation of adenine N6; OTUB1, OUT domain–containing ubiquitin aldehyde-binding protein 1; PRSS8, protease serine S1 family member 8; WB, Western blot; WCE, whole-cell extract; YTHDF2, YTH domain–containing family protein 2.

Article Snippet: Primary antibodies used are as follows: OTUB1 (Cell Signaling Technology #3783), YTHDF2 (Cell Signaling Technology #80014 for C and A , Proteintech #24744-1-AP for all the others), PRSS8 (Abcam #185236), GAPDH (Abclonal #AC033), β-actin (Abclonal #AC026), E-cadherin (BD Transduction Laboratories#610182), β-catenin (Cell Signaling Technology #9562), histone H3 (Abcam #ab1791), ubiquitin (Santa Cruz #sc-8017), YTHDF1 (Cell Signaling Technology #86463), YTHDF3 (Santa Cruz #sc-377119), METTL14 (Cell Signaling Technology #51104), ALKBH5 (Abcam #195377), FTO (Cell Signaling Technology #31687), hemagglutinin (HA)-tag (Cell Signaling Technology # 3724), FLAG-tag (Sigma #A8592), Myc-Tag (Santa Cruz #sc-40 for H , B , and E , Proteintech #16286-1-AP for all others).

Techniques: Co-Immunoprecipitation Assay, shRNA, Comparison, Immunoprecipitation, Methylation, Binding Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation

doi: 10.1016/j.jbc.2024.107152

Figure Lengend Snippet: OTUB1 decreases YTHDF2 ubiquitination independent of the DUB activity. A , Ctrl, OTUB1-KD, or YTHDF2-KD PC-3 cells were analyzed with IP-WB as indicated. "Ub" denotes ubiquitin. B , 2 μg FLAG-YTHDF2, HA-ubiquitin, and Myc-OTUB1 plasmids were cotransfected into HEK-293T cells. Cells were analyzed with IP-WB as indicated. C , the result of the in vitro ubiquitination reaction as analyzed with WB. FLAG-YTHDF2 immunopurified from transfected HEK-293T cells was used as the substrate. It was incubated in vitro with recombinant His-ubiquitin and His-OTUB1. Then HEK-293T cell extract was added as a source of E2 and E3. "Ub" denotes ubiquitin. D , HA-OTUB1 WT or D88A mutant was expressed in PC-3 cells. WCE were analyzed with WB. E , HA-OTUB1 WT or D88A mutant was expressed in PC-3 cells. Cells were treated with 25 μg/ml Chx for different time. WCE were analyzed with WB. For YTHDF2, separate exposures for control and HA-OTUB1 cells were presented to make the signals of "Chx 0 h" comparable and facilitate the comparison of protein stability. Chx, cycloheximide; HA, hemagglutinin; IP, immunoprecipitation; KD, knock down; OTUB1, OUT domain–containing ubiquitin aldehyde-binding protein 1; PRSS8, protease serine S1 family member 8; WB, Western blot; WCE, whole-cell extract; YTHDF2, YTH domain–containing family protein 2.

Article Snippet: Primary antibodies used are as follows: OTUB1 (Cell Signaling Technology #3783), YTHDF2 (Cell Signaling Technology #80014 for C and A , Proteintech #24744-1-AP for all the others), PRSS8 (Abcam #185236), GAPDH (Abclonal #AC033), β-actin (Abclonal #AC026), E-cadherin (BD Transduction Laboratories#610182), β-catenin (Cell Signaling Technology #9562), histone H3 (Abcam #ab1791), ubiquitin (Santa Cruz #sc-8017), YTHDF1 (Cell Signaling Technology #86463), YTHDF3 (Santa Cruz #sc-377119), METTL14 (Cell Signaling Technology #51104), ALKBH5 (Abcam #195377), FTO (Cell Signaling Technology #31687), hemagglutinin (HA)-tag (Cell Signaling Technology # 3724), FLAG-tag (Sigma #A8592), Myc-Tag (Santa Cruz #sc-40 for H , B , and E , Proteintech #16286-1-AP for all others).

Techniques: Activity Assay, In Vitro, Transfection, Incubation, Recombinant, Mutagenesis, Comparison, Immunoprecipitation, Binding Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation

doi: 10.1016/j.jbc.2024.107152

Figure Lengend Snippet: OTUB1 promotes prostate cancer cell proliferation through YTHDF2. A , left : relative proliferation during 3 days. The proliferation of control or OTUB1-KD PC-3 cells was analyzed with cell counting. Error bars denote the SD of four biological replicates. p values were from one-way ANOVA. The right shows WB results for WCE. B , proliferation of control or OTUB1-KD cells was analyzed with CCK-8. Shown are cell growth curves during 4 days. Error bars denote the SD of six biological replicates. p values were from two-way ANOVA. C , 1000 control or OTUB1-KD cells were seeded into 3.5 cm dishes. Fourteen days later, cells were fixed and stained with crystal violet. In the bar graph , error bars denote the SD of three biological replicates, and all data was normalized to the first group. p values were from one-way ANOVA. Below the bar graph shows the result of a representative experiment. D , left : relative proliferation during 4 days. Proliferation of control or YTHDF2-KD cells was analyzed with cell counting. Error bars denote the SD of four biological replicates. p values were from one-way ANOVA. The right shows WB results for WCE. E , proliferation of Control or YTHDF2-KD cells was analyzed with CCK-8. Shown are cell growth curves during 4 days. Error bars denote the SD of six biological replicates. p values were from two-way ANOVA. F , 1000 control or YTHDF2-KD cells were seeded into 3.5 cm dishes. Eighteen days later, cells were fixed and stained with crystal violet. In the bar graph , error bars denote the SD of three biological replicates, and all data was normalized to the first group. p values were from one-way ANOVA. Below the bar graph shows the result of a representative experiment. G , YTHDF2 was overexpressed in OTUB1-KD cells. On the left , the proliferation of Control, OTUB1-KD and "OTUB1-KD + YTHDF2" cells was analyzed with CCK-8. Shown are proliferation curves during 4 days. Error bars denote the SD of six biological replicates. p values were from two-way ANOVA. On the right is the WB result for WCE. H , YTHDF2 was overexpressed in OTUB1-KD cells. Five hundred cells were seeded into 3.5 cm dishes. Twelve days later, cells were fixed and stained with crystal violet. In the bar graph , error bars denote the SD of three biological replicates, and all data was normalized to the first group. p values were from one-way ANOVA. Below the bar graph shows the result of a representative experiment. KD, knock down; OTUB1, OUT domain–containing ubiquitin aldehyde-binding protein 1; WB, Western blot; WCE, whole-cell extract; YTHDF2, YTH domain–containing family protein 2.

Article Snippet: Primary antibodies used are as follows: OTUB1 (Cell Signaling Technology #3783), YTHDF2 (Cell Signaling Technology #80014 for C and A , Proteintech #24744-1-AP for all the others), PRSS8 (Abcam #185236), GAPDH (Abclonal #AC033), β-actin (Abclonal #AC026), E-cadherin (BD Transduction Laboratories#610182), β-catenin (Cell Signaling Technology #9562), histone H3 (Abcam #ab1791), ubiquitin (Santa Cruz #sc-8017), YTHDF1 (Cell Signaling Technology #86463), YTHDF3 (Santa Cruz #sc-377119), METTL14 (Cell Signaling Technology #51104), ALKBH5 (Abcam #195377), FTO (Cell Signaling Technology #31687), hemagglutinin (HA)-tag (Cell Signaling Technology # 3724), FLAG-tag (Sigma #A8592), Myc-Tag (Santa Cruz #sc-40 for H , B , and E , Proteintech #16286-1-AP for all others).

Techniques: Cell Counting, CCK-8 Assay, Staining, Binding Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation

doi: 10.1016/j.jbc.2024.107152

Figure Lengend Snippet: OTUB1-YTHDF2 regulates PRSS8 mRNA level. A , control or YTHDF2-KD cells were analyzed with real-time RT-PCR. Shown are relative PRSS8 mRNA levels normalized to β-actin. Error bars denote the SD of three technical replicates. p values were from one-way ANOVA. The result from a second independent experiment is shown in the Fig. S4 A . B , control or OTUB1-KD cells were analyzed with real-time RT-PCR. Shown are relative PRSS8 mRNA levels normalized to β-actin. Error bars denote the SD of three technical replicates. p values were from one-way ANOVA. The result from a second independent experiment is shown in the Fig. S4 B . C , WCE of Control or YTHDF2-KD PC-3 cells were analyzed with WB. D , WCE of control or OTUB1-KD PC-3 cells were analyzed with WB. E , Myc-YTHDF2 was expressed in OTUB1-KD cells. WCE of control, OTUB1-KD, and "OTUB1-KD + YTHDF2" cells were analyzed with WB. F , Myc-YTHDF2 was overexpressed in OTUB1-KD cells. Control, OTUB1-KD, and "OTUB1-KD + YTHDF2" cells were analyzed with real-time RT-PCR. Shown are relative PRSS8 mRNA levels normalized to β-ACTIN. Error bars denote the SD of three technical replicates. p values were from one-way ANOVA. The result from a second independent experiment is shown in the Fig. S4 I . KD, knock down; OTUB1, OUT domain–containing ubiquitin aldehyde-binding protein 1; PRSS8, protease serine S1 family member 8; WB, Western blot; WCE, whole-cell extract; YTHDF2, YTH domain–containing family protein 2.

Article Snippet: Primary antibodies used are as follows: OTUB1 (Cell Signaling Technology #3783), YTHDF2 (Cell Signaling Technology #80014 for C and A , Proteintech #24744-1-AP for all the others), PRSS8 (Abcam #185236), GAPDH (Abclonal #AC033), β-actin (Abclonal #AC026), E-cadherin (BD Transduction Laboratories#610182), β-catenin (Cell Signaling Technology #9562), histone H3 (Abcam #ab1791), ubiquitin (Santa Cruz #sc-8017), YTHDF1 (Cell Signaling Technology #86463), YTHDF3 (Santa Cruz #sc-377119), METTL14 (Cell Signaling Technology #51104), ALKBH5 (Abcam #195377), FTO (Cell Signaling Technology #31687), hemagglutinin (HA)-tag (Cell Signaling Technology # 3724), FLAG-tag (Sigma #A8592), Myc-Tag (Santa Cruz #sc-40 for H , B , and E , Proteintech #16286-1-AP for all others).

Techniques: Quantitative RT-PCR, Binding Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation

doi: 10.1016/j.jbc.2024.107152

Figure Lengend Snippet: OTUB1-YTHDF2 regulates PRSS8 in an m6A-dependent manner. A , cell lysates were subject to RNA immunoprecipitation (RIP) with YTHDF2 antibody or nonspecific IgG. Immunoprecipitated RNA was then analyzed with real-time RT-PCR. Shown are levels of PRSS8 mRNA immunoprecipitated as normalized to 1% input. The companion WB results show the immunoprecipitation efficiency. Error bars denote the SD of three technical replicates. p values were from one-way ANOVA. "Input" denotes 1% input. The result from a second independent experiment is shown in the Fig. S5 A . B , control or METTL14-KD cell lysates were subject to RNA immunoprecipitation (RIP) with YTHDF2 antibody or nonspecific IgG. Immunoprecipitated RNA was then analyzed with real-time RT-PCR. Shown are the relative PRSS8 mRNA levels as first normalized to 1% "input" in each group and then normalized to the IgG group. The companion WB results show the immunoprecipitation efficiency. Error bars denote the SD of three technical replicates. p values were from unpaired t test. "Input" denotes 1% input. The result from a second independent experiment is shown in the Fig. S5 C . C , control, YTHDF2 WT, or W432A/W486A mutant cells were subject to RIP with YTHDF2 antibody or nonspecific IgG. Shown are relative PRSS8 mRNA levels as first normalized to 1% “input” in each group and then normalized to the IgG group. The companion WB results show the immunoprecipitation efficiency. Error bars denote the SD of three technical replicates. p values were from unpaired t test. "Input" denotes 1% input. The result from a second independent experiment is shown in the Fig. S5 E . D , control or YTHDF2-KD cells were treated with 5 μg/ml actinomycin for different time. Cells were analyzed with real-time RT-PCR. Shown are the relative PRSS8 mRNA levels normalized to β-ACTIN. Error bars denote the SD of three technical replicates. p values were from two-way ANOVA. The result from a second independent experiment is shown in the Fig. S5 G . E , control and YTHDF2-KD or OTUB1-KD cells were subject to RIP with m6A antibody. Immunoprecipitated RNA was analyzed with real-time RT-PCR. Shown are relative PRSS8 mRNA levels as first normalized to "input" in each group and then normalized to control cells. Error bars denote the SD of three technical replicates. p values were from unpaired t test. The result from a second independent experiment is shown in the Fig. S5 I . F , YTHDF2 WT or W432A/W486A mutant was expressed in PC-3 cells. Cells were then analyzed with real-time RT-PCR ( left ) or WB ( right ). Error bars denote the SD of three technical replicates. p values were from one-way ANOVA. The result from a second independent experiment is shown in the Fig. S5 J . G , YTHDF2 WT or W432A/W486A mutant was expressed in PC-3 cells. Cells were subject to RIP with m6A antibody. Immunoprecipitated RNA was analyzed with real-time RT-PCR. Shown is relative PRSS8 mRNA as first normalized to "input" in each group and then normalized to the control group. Error bars denote the SD of three technical replicates. p values were from one-way ANOVA. The result from a second independent experiment is shown in the Fig. S5 L . H , METTL14 was knocked down with shRNA. WCE were analyzed with WB. I , fat mass and obesity-associated protein was knocked down with shRNA. WCE were analyzed with WB. J , ALKBH5 was knocked down with shRNA. WCE were analyzed with WB. KD, knock down; m6A, methylation of adenine N6; OTUB1, OUT domain–containing ubiquitin aldehyde-binding protein 1; PRSS8, protease serine S1 family member 8; WB, Western blot; WCE, whole-cell extract; YTHDF2, YTH domain–containing family protein 2.

Article Snippet: Primary antibodies used are as follows: OTUB1 (Cell Signaling Technology #3783), YTHDF2 (Cell Signaling Technology #80014 for C and A , Proteintech #24744-1-AP for all the others), PRSS8 (Abcam #185236), GAPDH (Abclonal #AC033), β-actin (Abclonal #AC026), E-cadherin (BD Transduction Laboratories#610182), β-catenin (Cell Signaling Technology #9562), histone H3 (Abcam #ab1791), ubiquitin (Santa Cruz #sc-8017), YTHDF1 (Cell Signaling Technology #86463), YTHDF3 (Santa Cruz #sc-377119), METTL14 (Cell Signaling Technology #51104), ALKBH5 (Abcam #195377), FTO (Cell Signaling Technology #31687), hemagglutinin (HA)-tag (Cell Signaling Technology # 3724), FLAG-tag (Sigma #A8592), Myc-Tag (Santa Cruz #sc-40 for H , B , and E , Proteintech #16286-1-AP for all others).

Techniques: Immunoprecipitation, Quantitative RT-PCR, Mutagenesis, shRNA, Methylation, Binding Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation

doi: 10.1016/j.jbc.2024.107152

Figure Lengend Snippet: YTHDF2 and OTUB1 promote prostate cancer cell proliferation through PRSS8. A , PRSS8 was knocked down in control or YTHDF2-KD PC-3 cells. Shown on the left are proliferation curves during 4 days as determined with CCK-8. Error bars denote the SD of six biological replicates. p values were from two-way ANOVA. Shown on the right are WB results to validate gene expression and/or knockdown. B , PRSS8 was knocked down in control or YTHDF2-KD PC-3 cells. One thousand cells were seeded into 3.5 cm dishes. Fourteen days later, cells were fixed and stained with crystal violet. In the bar graph , error bars denote the SD of three biological replicates and all data was normalized to the first group. p values were from one-way ANOVA. Below the bar graph shows the result of a representative experiment. C , PRSS8 was knocked down in OTUB1-KD PC-3 cells. Shown on the left is relative cell proliferation during 4 days as analyzed with cell counting. Error bars denote the SD of four biological replicates. p values were from one-way ANOVA. Shown on the right are WB results for WCE to validate gene expression and/or knockdown. D , PRSS8 was knocked down in control or OTUB1-KD PC-3 cells. Shown on the left are proliferation curves during 4 days. Cell proliferation was analyzed with CCK-8. E , PRSS8 was knocked down in OTUB1-KD PC-3 cells. One thousand cells were seeded into 3.5 cm dishes. Twenty days later, cells were fixed and stained with crystal violet. In the bar graph , error bars denote the SD of three biological replicates and all data was normalized to the first group. p values were from one-way ANOVA. Below the bar graph shows the result of a representative experiment. F , working model of this study, where denotes ubiquitin and denotes m6A. OTUB1 inhibits YTHDF2 ubiquitination independent of its DUB activity, which protects YTHDF2 from degradation. YTHDF2 in turn can bind PRSS8 mRNA and promotes its degradation, which increases cell proliferation. m6A, methylation of adenine N6; OTUB1, OUT domain–containing ubiquitin aldehyde-binding protein 1; PRSS8, protease serine S1 family member 8; WB, Western blot; KD, knock down; WCE, whole-cell extract; YTHDF2, YTH domain–containing family protein 2.

Article Snippet: Primary antibodies used are as follows: OTUB1 (Cell Signaling Technology #3783), YTHDF2 (Cell Signaling Technology #80014 for C and A , Proteintech #24744-1-AP for all the others), PRSS8 (Abcam #185236), GAPDH (Abclonal #AC033), β-actin (Abclonal #AC026), E-cadherin (BD Transduction Laboratories#610182), β-catenin (Cell Signaling Technology #9562), histone H3 (Abcam #ab1791), ubiquitin (Santa Cruz #sc-8017), YTHDF1 (Cell Signaling Technology #86463), YTHDF3 (Santa Cruz #sc-377119), METTL14 (Cell Signaling Technology #51104), ALKBH5 (Abcam #195377), FTO (Cell Signaling Technology #31687), hemagglutinin (HA)-tag (Cell Signaling Technology # 3724), FLAG-tag (Sigma #A8592), Myc-Tag (Santa Cruz #sc-40 for H , B , and E , Proteintech #16286-1-AP for all others).

Techniques: CCK-8 Assay, Expressing, Staining, Cell Counting, Activity Assay, Methylation, Binding Assay, Western Blot

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Mettl3 is essential for female mice fertility. ( A ). Phylogenetic tree of the protein sequences of Ythdf1, Ythdf2 and Ythdf3, based on UCSC database. The three readers appeared together in vertebrates, possibly after whole genome duplication. ( B ) Immunostaining of Ythdf1, Ythdf2, and Ythdf3 in ICR wild-type (WT) oocytes. (BF) Bright field. ( C ) H&E staining of ovaries, showing normal follicle structure in Mettl3 f/f VasaCre − control, and a severe abnormality in Mettl3 f/f VasaCre + females. ( D ) Gross morphology of Mettl3 f/f VasaCre + and control female ovaries. Cre + females show a smooth shape that lacks the typical follicular morphology. ( E ) Number of pups per plug produced by mating Mettl3 f/f Vasa Cre + females, compared with Mettl3 f/f Vasa Cre − control females. The fathers in both cases were WT. A significant difference between Cre + and Cre − female fertility is observed ( P < 0.0001, Mann–Whitney test). ( F ) H&E staining of ovaries, showing normal morphology both in Mettl3 f/f Zp3 Cre − and Mettl3 f/f Zp3 Cre + ovaries. ( G ) Number of pups per plug produced by mating a Mettl3 f/f Zp3 Cre + female, compared with a Mettl3 f/+ Zp3 Cre + control female. The fathers in both cases were WT. A significant difference between f/f and f/+ female fertility is observed ( P < 0.0001, Mann-Whitney test). ( H ) Number of oocytes per mouse flushed from Mettl3 f/f Zp3 Cre + females, compared with Mettl3 f/f Zp3 Cre − control females. A significant difference between the number of oocytes of Mettl3 f/f ZP3 Cre + and Mettl3 f/f ZP3 Cre − is observed ( P < 0.0002, Mann–Whitney test). ( I , top ) Experimental design – Mett3 f/f Zp3 Cre + and Cre − as control underwent hormone priming, oocyte flushing, fixation, and staining for tubulin. ( Bottom ) Number of oocytes observed in the different stages of meiosis. In the control, most of the oocytes were in MI stage, in KO (Cre + ) all of the observed oocytes were in the GV state. ( J ) Staining examples of oocytes in the different stages of meiosis as observed in KO (Cre + ) and control (Cre − ). ( K ) Differentially expressed genes between Mettl3 f/f Zp3 Cre − (control) and Mettl3 f/f Zp3 Cre + (KO) oocytes, along with selected enriched categories. m 6 A methylated genes appear in bold. Ninety-six genes are up-regulated in the KO, and 117 are down-regulated in the KO.

Article Snippet: The following primary antibodies were used: Ythdf2 (Aviva Systems Biology ARP67917_P050), Ythdf3 (Santa Cruz Biotechnology SC-87503), Cnot1 (Proteintech 14276-1-A) and Hsp90 (Epitomics 1492-1).

Techniques: Immunostaining, Staining, Control, Produced, MANN-WHITNEY, Methylation

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Characterization of compound Ythdf1/2/3 KO mice. ( A , left ) Statistics of KO offspring received from crossing of Heterozygous mice from each of the indicated strains (Ythdf1 +/− , Ythdf2 +/− , and Ythdf3 +/− ). ( Right ) Distribution of Ythdf2 WT, HET, and KO offspring at days E13.5, 2 d postnatal (DPN), and 30 DPN (compared with expected ratios). ( B ) Crossing strategy for generating triple-heterozygous mice that were further crossed, and their offspring statistics are presented in C . ( C ) Percentage of genotypes received by crossing triple-heterozygous mice, out of 200 pups tested 30 DPN. (Red) Observed percentage; (gray) expected under null assumption. No pups with Ythdf2-KO genotype survived 30 DPN. In Ythdf2 +/− genotype, pups with KO in either Ythdf1 or Ythdf3 were born in a sub-Mendelian ratio. χ 2 test P -values are indicated. ( D ) Using tetraploid complementation assay, triple-KO embryos were generated and examined on E7.5. ( Bottom ) H&E staining showing aberrant morphology of triple-KO E7.5 embryos, compared with WT control.

Article Snippet: The following primary antibodies were used: Ythdf2 (Aviva Systems Biology ARP67917_P050), Ythdf3 (Santa Cruz Biotechnology SC-87503), Cnot1 (Proteintech 14276-1-A) and Hsp90 (Epitomics 1492-1).

Techniques: Generated, Staining, Control

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf2 is essential for male mice fertility. ( A ) H&E staining showing mild degenerative changes, including scattered vacuoles marked by arrows in the seminiferous tubules in Ythdf2-KO males, compared with WT control. ( B ) Schematic representation of spermatogenesis inside seminiferous tubules. Differentiation is progressing from spermatogonia at the periphery, via spermatocytes and round spermatids, and ends with elongated mature sperm in the center of the tubule. ( C , left ) H&E staining of the cauda epididymis, showing severe loss of sperm in Ythdf2-KO compared with control. ( Right ) Bright field of sperm extracted from the cauda epididymis of KO and control, showing a severe reduction in normal sperm quantity in the KO sample, compared with control. ( D ) Number of pups per plug produced by mating Ythdf2 −/− males, compared with Ythdf2 +/− control males. The mothers in both cases were WT. A significant difference between the fertility of KO and heterozygous males is observed ( P < 0.0001, Mann–Whitney test). ( E ) Immunostaining of Ythdf1, Ythdf2, Ythdf3, and Mettl3 in seminiferous tubules, showing that each of the Ythdf proteins is expressed at different stages of spermatogenesis. Costaining of Gata4 typically marks Sertoli cells, costaining of γ- H2AX marks different cells during early spermatogenesis. ( F ) Dimension reduction representation of single-cell RNA-seq (UMAP) measured in adult mouse testis, showing mild expression of Ythdf1 and Ythdf3 in spermatogonia and in Sertoli cells, and more substantial expression of Ythdf2 in spermatogonia and in spermatocytes.

Article Snippet: The following primary antibodies were used: Ythdf2 (Aviva Systems Biology ARP67917_P050), Ythdf3 (Santa Cruz Biotechnology SC-87503), Cnot1 (Proteintech 14276-1-A) and Hsp90 (Epitomics 1492-1).

Techniques: Staining, Control, Produced, MANN-WHITNEY, Immunostaining, RNA Sequencing, Expressing

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf1, Ythdf2, and Ythdf3 are redundant in mouse naïve ESC maintenance. ( A ) Immunostaining of Ythdf1, Ythdf2, and Ythdf3 in WT mESCs, showing a protein expression in the cytosolic compartment of the cell. ( B ) Cell growth curve of all KO lines and WT control. Cells were grown on mouse feeders, in serum/LIF conditions. ( C ) Bright field and immunostaining of Nanog (green), Esrrb (red), and DAPI (blue) in KO cells (single, triple, and Mettl3) and WT control, showing that all cell lines express Nanog and Esrrb.

Article Snippet: The following primary antibodies were used: Ythdf2 (Aviva Systems Biology ARP67917_P050), Ythdf3 (Santa Cruz Biotechnology SC-87503), Cnot1 (Proteintech 14276-1-A) and Hsp90 (Epitomics 1492-1).

Techniques: Immunostaining, Expressing, Control

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf1, Ythdf2, and Ythdf3 are functionally redundant in ESC differentiation. ( A ) Teratomas generated from KO cell line and from WT control. Single-KO teratomas show all germ layers, while triple-KO (TKO) teratomas are poorly differentiated. Selected differentiated structures are marked by arrows. ( B ) Immunostaining of triple-KO and WT control to Oct4 (red), Foxa2 (green), Tuj1(purple) and DAPI (blue). Triple-KO teratomas contain patches of Oct4 staining, unlike WT teratomas. ( C ) Alkaline phosphatase (AP) staining of disassociated teratomas from triple-KO and WT control samples, showing a greater AP staining in the Ythdf triple-KO. ( D ) RT-PCR of pluripotency genes ( left ) and differentiation genes ( right ), measured in WT mESCs, and mEBs from the following cell lines: WT control, Ythdf single-KO, Ythdf triple-KO, and Ythdf triple-KO + overexpression (OE) of Ythdf1/2/3 (rescued cell lines). Triple-KO EBs express pluripotent markers and repress differentiation markers similarly to mESCs. Rescued EBs express differentiation markers, similarly to single-KO EBs. ( E ) EBs were induced for 7 d from the indicated cell lines, and dissociated into single cells at day 7 and comparable amounts of cells were replated in ES medium. Undifferentiated AP + colonies number per 1000 plated cells was evaluated 5 d later.

Article Snippet: The following primary antibodies were used: Ythdf2 (Aviva Systems Biology ARP67917_P050), Ythdf3 (Santa Cruz Biotechnology SC-87503), Cnot1 (Proteintech 14276-1-A) and Hsp90 (Epitomics 1492-1).

Techniques: Generated, Control, Immunostaining, Staining, Reverse Transcription Polymerase Chain Reaction, Over Expression

Journal: Genes & Development

Article Title: Context-dependent functional compensation between Ythdf m 6 A reader proteins

doi: 10.1101/gad.340695.120

Figure Lengend Snippet: Ythdf triple-KO has a dramatic effect on gene expression. ( A ) Hierarchical clustering of samples based on Pearson correlation, showing that single-KO samples are highly similar to WT. ( B ) Number of differentially expressed genes in each of the KO cell lines, compared with WT. (Black) Down-regulated genes, (gray) up-regulated genes. ( C ) RNA-seq and m 6 A methylation landscape of selected genes. Normalized coverage is presented. Only Nanog and Dnmt3l are m 6 A methylated. Dnmt3l, Zscan4a, Zscan4b, Zscan4d, and Dppa3 are overexpressed in triple-KO. ( D ) Enrichment of up-regulated genes in each category, to early embryo genes . Genes that are up-regulated in KO of Ythdf1 and Ythdf3 are specifically enriched for two-cell embryo genes. ( E ) Normalized expression of Ythdf1, Ythdf2, and Ythdf3, as measured in early mouse embryo . ( F ) CLIP coverage over m 6 A peaks of Ythdf readers as measured in humans ( n = 41,885) , and in mice ( n = 9861), showing high correlation between coverage by different Ythdf readers.

Article Snippet: The following primary antibodies were used: Ythdf2 (Aviva Systems Biology ARP67917_P050), Ythdf3 (Santa Cruz Biotechnology SC-87503), Cnot1 (Proteintech 14276-1-A) and Hsp90 (Epitomics 1492-1).

Techniques: Gene Expression, RNA Sequencing, Methylation, Expressing