Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: N6-methyladenosine (m 6 A) depletion regulates pluripotency exit by activating signaling pathways in embryonic stem cells

doi: 10.1073/pnas.2105192118

Figure Lengend Snippet: Immediate m 6 A depletion promotes Nanog-state heterogeneity in mESCs. ( A – J ) mESCs were transduced with control shRNA (Scr) or shRNAs targeting Mettl3 (shM3-1 and shM3-2). ( A ) Mettl3, Mettl14, and Wtap immunoblots. GAPDH is a loading control. ( B ) Reduction in m6A levels on mRNA upon Mettl3 KD by m6A dot blot ( Left ) and quantification ( Right ). Methylene blue is a loading control. ( C ) Mettl3 KD resulted in flatter colony morphology in NanogVENUS mESCs. (Scale bars, 100 μm.) ( D ) Flow cytometric analysis of Nanog VENUS -negative and -positive cell percentages upon Mettl3 KD in Nanog VENUS mESCs. “Relative counts” indicates normalized cell counts as cumulative percentages up to 100%. ( E ) Proportions of Nanog VENUS -negative cells upon Mettl3 KD, as quantified by flow cytometry using n = 9 independent repeats. ( F ) Representative immunostaining for Oct4 (cyan), Sox2 (magenta), Nanog (yellow), and DAPI (blue, cell nuclei) for Scr and shM3-1 cells. (Scale bars, 50 μm.) ( G and H ) Multispectral quantitative analysis of immunostaining in F . Scatter plot showing fluorescence intensity per cell in arbitrary units (a.u.) for Oct4 versus Nanog ( G ) and Sox2 versus Nanog ( H ). Dashed gray lines mark electronic gates ( Methods ) delineating positive and negative cells. Contour lines indicate probability distribution. Percentages of cells in each compartment are annotated. Analysis of n = 6,239 (Scr), n = 5,169 (shM3-1), and n = 5,257 (shM3-2) cells. ( I and J ) Relative mRNA levels ( Left ) and mRNA half-lives ( Right ) for Nanog ( I ) and Esrrb ( J ) in control and m6A-depleted mESCs. mRNA lifetime was determined by monitoring transcript abundance after transcription inhibition (TI), detailed in Methods . All statistics include error bars indicating mean ± SD and were calculated using two-tailed independent t test and unequal variance, * P < 0.1, ** P < 0.05, and *** P < 0.01. ( A ) n = 3 ( B ), n = 9 ( D ), n = 3 ( F – H ), and n = 3 ( I and J ) independent repeats. Unless stated otherwise, Nanog VENUS cells were used for experiments and analyzed 10 d after transductions.

Article Snippet: Separated proteins were transferred onto nitrocellulose membranes then detected with primary antibodies against Mettl3 (Abcam ab195352), Mettl14 (Sigma-Aldrich, HPA038002), Wtap (Proteintech, 60188–1-Ig), Oct4 (Santa Cruz, sc-8628), Nanog (Abcam ab80892), Oct6 (Merck Millipore, MABN738), Otx2 (R&D systems, AF1979), GSK-3β (Cell Signaling, 9315S), pGSK3β (Ser9) (Cell Signaling, 9323S), β-catenin (Santa Cruz, sc-7199), pβ-catenin(Ser33/37/Thr41) (Cell Signaling, 9561S), Phlpp2 (Abcam, ab71973), Smad1/5/9 (Abcam, ab66737), pSmad1(Ser463/465)/Smad5(Ser463/465)/Smad9 (Ser465/467) (Cell Signaling, 13820S), Ras (Abcam, ab52939), Smad2/3 (Cell Signaling, 8685S), pSmad2(Ser465/467)/Smad3(Ser423/425) (Cell Signaling, 8828S), mTOR (Cell Signaling, 2983S), pmTOR(S2481) (Abcam, ab137133), Eras (Abcam, ab192868), Pten (Cell Signaling, 9559S), PP2A C subunit (Cell Signaling, 2038S), Akt (Cell Signaling, 4691S), pAkt(Ser473) (Cell Signaling, 4060S), pAkt(Thr308) (Cell Signaling, 13038S), c-Raf (Cell Signaling, 9422S), pc-Raf(Ser338) (Cell Signaling, 9427S), Erk1/2 (Cell Signaling, 4348S), pErk1/2 (Thr202/Tyr204) (Cell Signaling, 9101S), Mek1(Abcam, ab32091), Mek2 (Abcam, ab32517), pMek1/2 (Ser217/221) (Cell Signaling, 9154S), Fgfr1 (Abcam, ab10646), pFgfr1(Tyr653/Tyr654) (Merck Millipore, 06–1433), Histone H3 (Abcam, ab85869), and GAPDH (Abcam, ab125247).

Techniques: Transduction, shRNA, Western Blot, Dot Blot, Flow Cytometry, Immunostaining, Fluorescence, Inhibition, Two Tailed Test

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