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: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a)□ Multiple alignments of Ythdf1, Ythdf2 & Ythdf3 proteins, calculated using the Clustal Omega tool. The area of YTH-domain is highlighted in red. Ythdf1-Ythdf3 protein sequence similarity is 70.11%, Ythdf1-Ythdf2 is 67.15%, and Ythdf2-Ythdf3 is 67.78%. b)□ Phylogenetic tree of the protein sequences of Ythdf1, Ythdf2 and Ythdf3, based on the UCSC database. The three readers appear together in vertebrates, possibly due to whole genome duplication. c)□ CRISPR-Cas9 targeting strategy for knocking-out Ythdf readers in vivo. d)□ KO validation using PCR, showing successful primer integration in clones #12 (Ythdf1); #36, #46 (Ythdf2); #1, #3 & #4 (Ythdf3).

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

Techniques: Sequencing, CRISPR, In Vivo, Biomarker Discovery, Clone Assay

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a) Gross morphology of Cre+ and Cre−(control) female ovaries. Cre+ females show a smooth shape that lacks the typical follicular morphology. b) H&E staining of an ovary, showing a severe abnormality in Mettl3 f/f Vasa-Cre+ females. c) Number of pups per plug produced by mating Mettl3 f/f Vasa-Cre+ females, compared to Mettl3 f/f Vasa-Cre−control females. The fathers in both cases are WT. A significant difference between Cre+ and Cre−female fertility is observed (p<0.0001, Mann-Whitney test). d) H&E staining of ovaries, showing normal morphology in Mettl3 f/f Zp3-Cre+ ovaries. e) Number of pups per plug produced by mating a Mettl3 f/f Zp3-Cre+ female, compared to a Mettl3 f/+ Zp3-Cre+ control female. The fathers in both cases are WT. A significant difference between f/f and f/+ female fertility is observed (p<0.0001, Mann-Whitney test). f) Number of oocytes per mouse produced by mating Mettl3 f/f Zp3-Cre+ females, compared to Mettl3 f/f Zp3-Cre−control females. The fathers in both cases are WT. A significant difference between the number of oocytes of f/f Cre+ and f/f Cre−is observed (p<0.0002, Mann-Whitney test). g) Top: Experimental design - Mett3 f/f Zp3-Cre+ and Cre−as control underwent hormone priming, flush, 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. h) Staining examples of oocytes in the different stages of meiosis as observed in KO (Cre+) and control (Cre−). i) Immunostaining of Ythdf1, Ythdf2 and Ythdf3 in ICR WT oocytes after hormone priming (PMS & hCG).

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

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

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a) Gross morphology of testis and epididymis of Mettl3f/f Vasa-Cre+ and Mettl3f/f Vasa-Cre−males. Cre+ males show a massive decrease in testis and epididymis size compared to Cre−control. b) Number of pups per plug produced by Mettl3f/f Vasa-Cre+ males, compared to Mettl3f/f Vasa-Cre−control males. The mothers in both cases were WT. In this case there is a significant hypofertility of the KO (p<0.0001, Mann-Whitney test). c) Gross morphology of testis and epididymis of Mettl3f/f Stra8-Cre+ and Mettl3f/f Stra8-Cre−males. Cre+ males show a reduced-size testis and epididymis compared to Cre−control. d) Same as in (b), for Stra8-Cre, showing a significant hypofertility of the KO (p<0.0001, Mann-Whitney test). e) Vasa, Stra8 and Prm1 are expressed during spermatogenesis, in different stages, as indicated. f) Same as in (b), for Prm1-Cre, showing no significant difference between Cre+ and Cre−male fertility. g) Number of pups per plug produced by Ythdf2 −/− males, compared to 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). h) Immunostaining of Ythdf1, Ythdf2 and Ythdf3 in seminiferous tubules, showing that each of the proteins is expressed at different stages of spermatogenesis.

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

Techniques: Control, Produced, MANN-WHITNEY, Immunostaining

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a)□ H&E staining showing severe degenerative changes in the seminiferous tubules of Mettl3 f/f Vasa-Cre+ and lack of sperm in the cauda epididymis. b)□ H&E staining showing mild degenerative changes in the seminiferous tubules of Mettl3 f/f Stra8-Cre+ and ~75% reduction in sperm quantity in the cauda epididymis, compared to Mettl3 f/+ Stra8-Cre+ sibling control. c)□ H&E staining of seminiferous tubules showing a normal morphology in Mettl3 f/f Prm1-Cre+ males. d)□ H&E staining showing mild degenerative changes in the seminiferous tubules in Ythdf2-KO males, compared to WT control. e)□ Left: H&E staining in the cauda epididymis, showing severe loss of sperm in Ythdf2-KO compared to control. Right: Brightfield of sperm extracted from the cauda epididymis of KO and control, showing a severe reduction in normal sperm quantity in the KO sample, compared to control. f)□ Transcriptional profile of genes that are differentially expressed between Ythdf2-KO and WT round spermatids, along with selected enriched categories. m 6 A-methylated genes appear in bold; 145 downregulated in KO, and 156 upregulated in KO.

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

Techniques: Staining, Control, Methylation

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a) Left: statistics of KO offspring received from crossing heterozygous mice from each of the indicated strains (Ythdf1 +/− , Ythdf2 +/− , Ythdf3 +/− ). Right: distribution of Ythdf2 WT, HET and KO offspring in E13.5, two days postnatal (DPN), and 30 DPN (compared to expected ratios). b) Crossing strategy for generating triple-heterozygous mice, which were further crossed, and their offspring, statistics are presented in panel (c). c) Percentage of genotypes received by crossing triple-heterozygous mice, out of 200 pups tested 30 DPN. Red - observed percentage, grey - expected under null assumption. No pups with Ythdf2-KO genotype survived 30 DPN. In the Ythdf2 +/− genotype, pups with KO in either Ythdf1 or Ythdf3 were born at a sub-Mendelian ratio. Chi-square test p-values are indicated. d) H&E staining showing the impaired morphology of triple-KO E7.5 embryos, compared to WT control.

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

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a)□ The morphology of Ythdf2-KO flushed oocytes appears to be normal, similar to the WT flushed oocytes. b)□ Number of pups per plug produced by mating Ythdf2 −/− females, compared to Ythdf2 +/− control females. The fathers in both cases are WT. A significant difference between the fertility of KO and heterozygous females is observed (p<0.0001, Mann-Whitney test). c)□ Transcriptional profile of genes that are differentially expressed between Ythdf2-KO and WT oocytes, along with selected enriched categories; 311 downregulated in KO, and 339 upregulated in KO.

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

Techniques: Produced, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a)□ Immunostaining of Ythdf1, Ythdf2 and Ythdf3 in ICR WT oocytes after hormone priming (PMS & hCG). b)□ Immunostaining of Ythdf1, Ythdf2 and Ythdf3 in ICR WT oocytes after PMS & HCG - negative control (NC), without primary antibody. c)□ Immunostaining of Ythdf1, Ythdf2 and Ythdf3 in ICR WT oocytes without hormone priming.

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

Techniques: Immunostaining, Negative Control

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a) Immunostaining of Ythdf1, Ythdf2, and Ythdf3 in KH2 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) Brightfield 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. d) Teratomas generated by the KO cell line and by WT control. Single-KO cell lines show all germ layers, while triple-KO teratomas as poorly differentiated. e) Immunostaining of triple-KO and WT control with Oct4 (red), Foxa (green), Tuj1 (purple) and DAPI (blue). Triple-KO contains patches of Oct4 staining, unlike WT teratomas. f) Alkaline phosphatase (AP) staining of disassociated teratomas from Triple-KO and WT control, showing a greater AP staining in the triple-KO cells. g) RT-PCR of pluripotent genes (left) and differentiation genes (right), measured in WT and KO EBs, and in WT mESCs as a control. In the triple-KO EBs, pluripotent markers are higher than the control and differentiation markers are lower than the WT control.

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

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

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a)□ CRISPR-Cas9 targeting strategy for knocking out Ythdf readers in mESC cell lines. b)□ Sequencing validation of the single-KO lines. c)□ IGV browser view showing the missing fragments in the KO of Ythdf1, Ythdf2 & Ythdf3.

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

Techniques: CRISPR, Sequencing, Biomarker Discovery

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a)□ Targets of Ythdf1, Ythdf2 and Ythdf3 highly overlap targets that were published before in human cancer cell lines. b)□ Sequence logo of the GGACT containing motif which appears in 14% of YTHDF targets (enrichment fold-change 1.89, p<1e-24). c)□ Distribution of Ythdf peaks in various genomic entities, showing that the three readers have a tendency to bind 3' UTR, particularly Ythdf2. d)□ Significant overlap of Ythdf targets, with m 6 a-methylated genes. e)□ Significant overlap between Ythdf1, Ythdf2 and Ythdf3 targets f)□ Enrichment of Ythdf targets that were identified in mESCs, to early embryo genes (Gao et al. 2017), showing significant overlap with blastocyte genes.

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

Techniques: Sequencing, Methylation