Journal: BMC Cancer

Article Title: N6-methyladenosine RNA modification (m6A) is of prognostic value in HPV-dependent vulvar squamous cell carcinoma

doi: 10.1186/s12885-022-10010-x

Figure Lengend Snippet: Summary of the analyzed m6A proteins as indicated and their correlation with overall survival (indicated as %alive) for the entire study cohort, HPV-independent, and HPV-dependent VSCC. The HPV-status was not available for 24 patients. Samples were grouped according to high and low expression based on the staining intensities. p -values for the group comparisons are based on log-rank tests (significance threshold p < 0.5). q -values are based on multiple hypotheses testing using the method of Benjamini and Hochberg with a significance threshold of q < 0.1

Article Snippet: Immunostaining of METTL3, METTL4, METTL14, WTAP, KIAA1429, FTO, ALKBH5, HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1,YTHDF2, and YTHDF3 was performed on the TMAs using an automated staining system (BenchMark ULTRA; Ventana Medical Systems) which performed deparaffinization, pretreatment with cell conditioning buffer (CC1 buffer, pH8), and incubation with primary antibodies (FTO (1:50; Atlas Antibodies #HPA041086), ALKBH5 (1:200; Novus #NBP1-82,188), METTL3 (1:1000; Biorbyt #orb374082), METTL4 (1:40; Atlas Antibodies #HPA040061), METTL14 (1:100; Atlas Antibodies #HPA038002), WTAP (1:100; Atlas Antibodies #HPA010550), KIAA1429 (1:25; Atlas Antibodies #HPA031530), HNRNPC (1:25; Atlas Antibodies #HPA051075), HNRNPA2B1 (1:100; Atlas Antibodies #HPA001666), YTHDC1 (1:25; Atlas Antibodies #HPA036462), YTHDF1 (1:10; Biorbyt #orb179018), YTHDF2 (1:200; Biorbyt #orb39199), YTHDF3 (1:200; Biorbyt #orb374095) at 4 °C overnight.

Techniques: Expressing, Staining

Journal: Journal of Biological Chemistry

Article Title: Identification of Wilms' Tumor 1-associating Protein Complex and Its Role in Alternative Splicing and the Cell Cycle

doi: 10.1074/jbc.m113.500397

Figure Lengend Snippet: FIGURE 2. Proteomic profile of the WTAP complexes. A, the domains and unique amino acid repeats in the Hakai protein. Various Hakai deletion mutants are shown. The interaction of these mutants with WTAP was assessed by means of co-immunoprecipitation (IP) experiments. B, the cellular localization of Hakai-V5 and Hakai-delRING-V5. The nucleus was stained with TO-PRO-3 reagent. Bar, 10 m. C, immunoprecipitation of Hakai complexes from the tetracycline- inducibleHEK293stablecelllinesexpressingHakai-V5orHakai-delRING-V5.ImmunopurifiedHakaianditsinteractingproteinswereresolvedbySDS-PAGEand stained with SYPRO Ruby solution. The efficiency of immunoprecipitation was determined by Western blot. D, the proteomic profile of the WTAP complexes. This list shows the proteins isolated using the H1122, H1137, or Y6828 antibodies with a unique peptide number of 3 from HUVEC extracts or from PFA() HeLa cell extracts but not with the negative control antibody K7124 from HUVEC extracts or the anti-V5 antibody from the Hakai-delRING-V5 sample. The complete list of the identified proteins is presented in supplemental Table S1. The values represent the unweighted spectrum count (SPC) level divided by the molecular weight (MW) in kDa to determine the relative quantity of the immunopurified proteins. Hierarchical clustering was performed using JMP 7 software (SAS Institute, Cary, NC). E, immunopurification of the proteins cross-linked to WTAP. HeLa cells were cross-linked with paraformaldehyde, and the proteins cross-linked to WTAP were immunopurified with the H1122 antibody. Interacting proteins were resolved by SDS-PAGE and stained with SYPRO Ruby solution.

Article Snippet: Fluorescence Image Analysis—Cells were fixed with 4% (w/v) paraformaldehyde in PBS for 5 min, permeabilized with 0.5% (v/v) Triton X-100 in PBS for 5 min on ice, blocked with PBS containing 10% BlockAce (Yukijirushi, Japan), and incubated with the primary antibodies against WTAP (1:200; rabbit polyclonal antibody, 1:100; Y6828), SC-35 (1:2000; S4045, Sigma), BCLAF1 (1:100; A300-608A, Bethyl), THRAP3 (1:200; A300956A, Bethyl), Virilizer (1:250; A302-124A, Bethyl), KIAA0853 (1:100; ab70802, Abcam), Hakai (1:80; ARP39623_T100, Aviva Systems Biology), and RBM15 (1:80; ab70549, Abcam) for 30 min at room temperature.

Techniques: Immunoprecipitation, Staining, Western Blot, Isolation, Negative Control, Molecular Weight, Software, Immu-Puri, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Identification of Wilms' Tumor 1-associating Protein Complex and Its Role in Alternative Splicing and the Cell Cycle

doi: 10.1074/jbc.m113.500397

Figure Lengend Snippet: FIGURE 4. The intracellular localization of the WTAP complex. A, immunofluorescence analysis of HUVEC using anti-WTAP (rabbit polyclonal antibody), anti-BCLAF1, anti-THRAP3, anti-Virilizer, anti-KIAA0853, anti-Hakai, anti-RBM15, and anti-SC35 antibodies. WTAP, BCLAF1, THRAP3, Virilizer, KIAA0853, and RBM15 are all partially co-localized with SC35 in nuclear speckles and are also present in the nucleoplasm. B, the intracellular localization of WTAP (Y6828), BCLAF1, and THRAP3. WTAP is co-localized with BCLAF1 and THRAP3 in nuclear speckles and the nucleoplasm. C, the nuclear speckle localization of WTAP became dispersed upon knockdown of BCLAF1/THRAP3. HUVECs were treated with BCLAF1/THRAP3 siRNAs or control siRNA and, after 48 h, immunostained with an anti-WTAP antibody (Y6828) together with anti-BCLAF1 or anti-THRAP3 antibodies. The WTAP signal became evidently more dispersed in BCLAF1/ THRAP3 knockdown cells compared with the signal in control cells. D, confocal images of WTAP (rabbit polyclonal antibody) and SC35 in control or BCLAF1/ THRAP3siRNA-treatedHUVECs.TheDNAiscounterstainedwithDAPI(blue).E,thequantificationofthecolocalizationcoefficientbetweenWTAPandSC35.The values are the average of 20 independent single-cell images. *, p 0.05 (t test). Error bars, S.D. Bar, 10 m.

Article Snippet: Fluorescence Image Analysis—Cells were fixed with 4% (w/v) paraformaldehyde in PBS for 5 min, permeabilized with 0.5% (v/v) Triton X-100 in PBS for 5 min on ice, blocked with PBS containing 10% BlockAce (Yukijirushi, Japan), and incubated with the primary antibodies against WTAP (1:200; rabbit polyclonal antibody, 1:100; Y6828), SC-35 (1:2000; S4045, Sigma), BCLAF1 (1:100; A300-608A, Bethyl), THRAP3 (1:200; A300956A, Bethyl), Virilizer (1:250; A302-124A, Bethyl), KIAA0853 (1:100; ab70802, Abcam), Hakai (1:80; ARP39623_T100, Aviva Systems Biology), and RBM15 (1:80; ab70549, Abcam) for 30 min at room temperature.

Techniques: Immunofluorescence, Knockdown, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of Wilms' Tumor 1-associating Protein Complex and Its Role in Alternative Splicing and the Cell Cycle

doi: 10.1074/jbc.m113.500397

Figure Lengend Snippet: FIGURE 6. The WTAP complex autoregulates the alternative splicing of WTAP pre-mRNA. A, Western blot analysis of the WTAP protein. In the course ofthedepletionoftheWTAPcomplexproteins,theproteinlevelofWTAPwas increased compared with control cells. -Tublin was used as a loading con- trol. We did not detect the shorter isoform of the endogenous WTAP protein despite the application of several specific antibodies. B, the effect of the depletion of each protein in the WTAP complex on the WTAP transcript was determined by RNase protection assay. The region used for the probe is indi- cated. **, p 0.01; *, p 0.05 (t test), n 4. C, representative gel image of RT-PCR from the RIP samples using WTAP-specific primers (top). The interac- tion of WTAP with WTAP pre-mRNA was determined by RIP-quantitative PCR (bottom). GAPDH was used as a negative control (the same data as in Fig. 5A). The values are the average of five independent experiments; *, p 0.05 (t test). Error bars, S.D. IB, immunoblot.

Article Snippet: Fluorescence Image Analysis—Cells were fixed with 4% (w/v) paraformaldehyde in PBS for 5 min, permeabilized with 0.5% (v/v) Triton X-100 in PBS for 5 min on ice, blocked with PBS containing 10% BlockAce (Yukijirushi, Japan), and incubated with the primary antibodies against WTAP (1:200; rabbit polyclonal antibody, 1:100; Y6828), SC-35 (1:2000; S4045, Sigma), BCLAF1 (1:100; A300-608A, Bethyl), THRAP3 (1:200; A300956A, Bethyl), Virilizer (1:250; A302-124A, Bethyl), KIAA0853 (1:100; ab70802, Abcam), Hakai (1:80; ARP39623_T100, Aviva Systems Biology), and RBM15 (1:80; ab70549, Abcam) for 30 min at room temperature.

Techniques: Alternative Splicing, Western Blot, Control, Rnase Protection Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Identification of Wilms' Tumor 1-associating Protein Complex and Its Role in Alternative Splicing and the Cell Cycle

doi: 10.1074/jbc.m113.500397

Figure Lengend Snippet: FIGURE 5. The interaction of WTAP and the noncoding RNA MALAT1. A, a representative gel image of RT-PCR from the RIP samples using MALAT1- or GAPDH-specific primers (left). The interaction of WTAP with MALAT1 was determined by RIP-quantitative PCR (right). GAPDH was used as a negative control. The values are the average of five independent experiments; *, p 0.05 (t test). Error bars, S.D. B, RNA-FISH and immunostaining were performed using a probe against MALAT1 and an anti-SC35 antibody in the control or BCLAF1/THRAP3 siRNA-treated HUVECs. Bar, 10 m. C, the quantification of the colocalization coefficient between MALAT1 and SC35. The values are the average of 20 independent single-cell images. Error bars, S.D.

Article Snippet: Fluorescence Image Analysis—Cells were fixed with 4% (w/v) paraformaldehyde in PBS for 5 min, permeabilized with 0.5% (v/v) Triton X-100 in PBS for 5 min on ice, blocked with PBS containing 10% BlockAce (Yukijirushi, Japan), and incubated with the primary antibodies against WTAP (1:200; rabbit polyclonal antibody, 1:100; Y6828), SC-35 (1:2000; S4045, Sigma), BCLAF1 (1:100; A300-608A, Bethyl), THRAP3 (1:200; A300956A, Bethyl), Virilizer (1:250; A302-124A, Bethyl), KIAA0853 (1:100; ab70802, Abcam), Hakai (1:80; ARP39623_T100, Aviva Systems Biology), and RBM15 (1:80; ab70549, Abcam) for 30 min at room temperature.

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Negative Control, Immunostaining, Control

Journal: American Journal of Cancer Research

Article Title: Arsenic trioxide suppresses lung adenocarcinoma stem cell stemness by inhibiting m6A modification to promote ferroptosis

doi:

Figure Lengend Snippet: Inhibition of proliferation and m6A formation by ATO in LASCs from A549 cells. (A) Tumorsphere formation in A549 cells induced by treatment with the sphere formation medium. Tumor sphere formation after treatment for 1, 3, 5, and 7 days was evaluated using the sphere formation assay. (B) Increase in CD133-positive cells in A549 cells treated with sphere formation medium. The percentages of CD133+ cells were measured by flow cytometry. (C) Suppression of LASCs cell viability by ATO treatment for 24 or 48 h. LASCs from A549 cells were treated with 0, 0.625, 1.25, 2.5, 5, 10, 20, or 40 mM of ATO, followed by detection of cell viability by the CCK-8 method. (D and E) Effects of ATO treatment on the expression of m6A regulator genes in LASCs from A549 cells. The mRNA (D) and protein (E) levels of major m6A writers and erasers in LASCs from A549 cells were analyzed by quantitative RT-PCR and western blotting, respectively. (F) Decrease in total m6A content in LASCs from A549 cells induced by ATO treatment. The total m6A levels in LASCs from A549 cells were determined using the dot blot method. ATO: arsenic trioxide; LASCs: lung adenocarcinoma stem cells; NC: negative control; METTL14/16: methyltransferase-like protein 14/16; WTAP: Wilms’ tumor 1-associating protein; ZC3H13: zinc finger CCCH domain-containing protein 13; FTO: fat mass and obesity-associated gene; ALKBH5: alkylated DNA repair protein alkB homolog 5; *P < 0.05.

Article Snippet: The primary antibodies used in this study are as follows: anti-METTL14 (#A8530; ABCLONAL), anti-METTL16 (#A15894; ABCLONAL), anti-Wilms’ tumor 1-associating protein (WTAP) (#A14695; ABCLONAL), anti-ZC3H13 (#TD4623; ABMART), anti-FTO (#A1438; ABCLONAL), anti-ALKBH5 (#A11684; ABCLONAL), and anti-GAPDH (#60004-1-Ig; proteintech).

Techniques: Inhibition, Tube Formation Assay, Flow Cytometry, CCK-8 Assay, Expressing, Quantitative RT-PCR, Western Blot, Dot Blot, Negative Control, Wilms Tumor Assay