Journal: Frontiers in immunology

Article Title: YTHDF1 Negatively Regulates Treponema pallidum -Induced Inflammation in THP-1 Macrophages by Promoting SOCS3 Translation in an m6A-Dependent Manner.

doi: 10.3389/fimmu.2022.857727

Figure Lengend Snippet: FIGURE 1 | m6A methylation: the m6A writer METTL3 and the reader YTHDF1 are upregulated in TP-infected macrophages. (A) Colorimetric quantification of m6A methylation in RNA from THP-1 cells with or without TP infection. (B, C) Western blot analysis of METTL3, METTL14, and YTHDF1 in THP-1 cells with or without TP infection. GAPDH was used as a control. *P < 0.05, **P < 0.01. (D, E) Immunofluorescence analysis of YTHDF1 or METTL3 in CD68+ macrophages in secondary syphilitic lesions and paired non-lesional skin tissue. Scale bar: 20 mm or 5 mm.

Article Snippet: For immunofluorescence analysis of the tissues, the sections were permeabilized with 0.25% Triton X-100 for 0.5 h and blocked with 5% goat serum for 1 h. Tissue sections were then incubated with primary antibodies against CD68 (Cat. No. 66231-2-Ig, Proteintech, Wuhan, Hubei, China) and YTHDF1 (Cat. No. 17479-1-AP, Proteintech) at 4°C overnight, followed by April 2022 | Volume 13 | Article 857727 incubation with the secondary antibody, Alexa Fluor® 488 donkey anti-rabbit IgG (H+L) (A21206, Life Technologies) or Alexa Fluor® 594 donkey anti-mouse IgG (H+L) (A21203, Life Technologies).

Techniques: Methylation, Infection, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: The m 6 A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

doi: 10.1093/nar/gkz157

Figure Lengend Snippet: Specific ablation of Ythdf1 from dorsal commissural neurons results in decrease of Robo3.1 protein level. ( A ) Schematic drawings are shown for the genetic deletion strategy for Ythdf1 . Exon 4 which contains YTH domain-coding sequence is deleted after Cre-mediated recombination. ( B ) Depletion of YTHDF1 protein in the dorsal spinal cord of Wnt1-Cre +/- ;Ythdf1 fl/fl cKO mouse embryos. Anti YTHDF1 immunostaining of E11.5 spinal cord sections confirmed cKO of YTHDF1 protein from dorsal spinal cord and dorsal root ganglia (DRG), illustrated by asterisks. ( C ) Specific ablation of YTHDF1 protein from Atoh1-Cre + commissural neurons. Anti YTHDF1 immunostaining of E11.5 spinal cord sections confirmed cKO of YTHDF1 protein in YFP + commissural neurons in Atoh1-Cre +/ - ;Rosa26-YFP +/ - ;Ythdf1 fl/fl cKO mouse embryos, while YTHDF1 expression was intact in Atoh1-Cre +/ - ;Rosa26-YFP +/ - control embryos. ( D ) Ythdf1 cKO with Atoh1-Cre led to dramatic reduction of Robo3.1 protein from dorsal commissural axons. E10.5 pre-crossing DSC explants was dissected and cultured in vitro . Anti-Robo3.1 IF showed significant decline of Robo3.1 protein level in TAG1 (TSA)-positive commissural axons. Representative images are shown from eight Ythdf1 fl/fl and nine Atoh1-Cre +/ - ;Ythdf1 fl/fl embryos, respectively. ( E ) Quantification of Robo3.1 IF in commissural axons of cultured DSC explants from Ythdf1 cKO mouse embryos and their littermate controls. All data are mean ± S.E.M. and represented as box and whisker plots: Ythdf1 fl/fl ( n = 30 confocal fields) versus Atoh1-Cre +/ - ;Ythdf1 fl/fl ( n = 47 confocal fields), ** P = 0.0014; by unpaired Student's t test. Scale bars, 100 μm (B and D) and 10 μm (C).

Article Snippet: The dilutions and sources of antibodies are as following: Robo3.1 (1:500), GFP (1:500, Abcam), YTHDF1 (1:1000, Proteintech), Isl1/2 (1:500, DSHB), Lhx2 (1:500, Santa Cruz Biotechnology), Lhx2 (1:500, Abcam), Lhx9 (1:200, Santa Cruz Biotechnology), TAG1 (1:1000, R&D Systems), TAG1 (1:200, DSHB).

Techniques: Sequencing, Immunostaining, Expressing, Control, Cell Culture, In Vitro, Whisker Assay

Journal: Nucleic Acids Research

Article Title: The m 6 A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

doi: 10.1093/nar/gkz157

Figure Lengend Snippet: Robo3.1 mRNA is modified by m 6 A and bound by the m 6 A reader YTHDF1. ( A ) Predicted m 6 A sites in Robo3.1 mRNA by SRAMP program. ( B ) Anti-m 6 A IP pulled down Robo3.1 mRNA from RNA of mouse embryonic spinal cord using two different m 6 A antibodies (one mAb and another pAb) with corresponding IgG as controls. RT-PCR was performed to detect Robo3 .1 mRNA in elutes. ( C ) Verification of m 6 A sites in Robo3.1 mRNA. Anti-m 6 A IP failed to pull down Robo3.1 mRNA from COS-7 cells expressing Robo3.1 with m 6 A sites mutated ( Robo3.1-MT m6A ) compared with Robo3.1-WT . ( D ) Knockdown of METTL3 in commissural neurons. Dissociated commissural neurons from E10.5 mouse dorsal spinal cord was infected with lenti virus expressing shMettl3 , marked by eGFP labeling. Knockdown by shMettl3 for 48 h resulted in dramatic decrease of METTL3 protein levels in neurons, compared with shCtrl . ( E ) Knockdown of METTL3 led to significant decreases of Robo3.1 protein levels in commissural axons compared with shCtrl . Scale bar, 10 μm. ( F ) Quantification of relative Robo3.1 IF to eGFP in (E). All data are mean ± S.E.M. and are represented as box and whisker plots: **** P = 1.18E–5 ( n = 15 axons for shCtrl ; n = 19 axons for shMettl3 ), by unpaired Student's t test. ( G ) RNA IP (RIP) pulled down Robo3.1 mRNA from mouse embryonic spinal cord lysate with YTHDF1 antibody, but not with control IgG. ( H ) Binding of YTHDF1 with Robo3.1 mRNA is m 6 A-dependent. RIP using YTHDF1 antibody failed to pull down Robo3.1 mRNA from COS-7 cells co-expressing YTHDF1 and Robo3.1 with m 6 A sites mutated ( MT m6A ) compared with WT Robo3.1 .

Article Snippet: The dilutions and sources of antibodies are as following: Robo3.1 (1:500), GFP (1:500, Abcam), YTHDF1 (1:1000, Proteintech), Isl1/2 (1:500, DSHB), Lhx2 (1:500, Santa Cruz Biotechnology), Lhx2 (1:500, Abcam), Lhx9 (1:200, Santa Cruz Biotechnology), TAG1 (1:1000, R&D Systems), TAG1 (1:200, DSHB).

Techniques: Modification, Reverse Transcription Polymerase Chain Reaction, Expressing, Knockdown, Infection, Virus, Labeling, Whisker Assay, Control, Binding Assay

Journal: Nucleic Acids Research

Article Title: The m 6 A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

doi: 10.1093/nar/gkz157

Figure Lengend Snippet: The m 6 A reader YTHDF1 controls translation of Robo3.1 mRNA in an m 6 A-dependent manner. ( A ) YTHDF1 could enhance translation of Robo3.1 . Co-expression of WT Robo3.1 with YTHDF1 in COS-7 cells resulted in a dramatic increase of Robo3.1 protein level by IF, compared with an empty vector expressing eGFP only. However, YTHDF1 failed to increase translation of Robo3.1 with m 6 A sites mutated ( Robo3.1-MT m6A ). Scale bar, 25 μm. ( B ) Quantification of relative Robo3.1 IF to eGFP in (A). ( C ) Western blotting analysis showing regulation of Robo3.1 protein levels by YTHDF1 is through translational control. Protein synthesis inhibitor CHX blocked translation of Robo3.1 in HEK293T cells expressing HA-Robo3.1. Similar effects were found with cells co-expressing HA-Robo3.1 and YTHDF1. ( D ) Quantification of relative HA-Robo3.1 levels to β-actin in (C). All data are mean ± S.E.M. Data of IF quantification (B) are represented as box and whisker plots: *** P = 5.11E–04, ‘ Robo3.1-WT + IRES-eGFP ’ ( n = 16 cells) versus ‘ Robo3.1-WT + Ythdf1-IRES-eGFP ’ ( n = 17 cells); ns, not significant ( P = 0.41), ‘ Robo3.1-WT + IRES-eGFP ’ ( n = 15 cells) vs ‘ Robo3.1-MT m6A + Ythdf1-IRES-eGFP ’ ( n = 18 cells); by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. WB quantification data (D, n = 3 replicates) are represented as dot plots: ** P = 0.002 (‘HA-Robo3.1’ versus ‘HA-Robo3.1 + CHX’); ** P = 0.002 (‘HA-Robo3.1’ versus ‘HA-Robo3.1 + YTHDF1’); *** P = 1.41E-04 (‘HA-Robo3.1 + YTHDF1’ versus ‘HA-Robo3.1 + YTHDF1 + CHX’); by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.

Article Snippet: The dilutions and sources of antibodies are as following: Robo3.1 (1:500), GFP (1:500, Abcam), YTHDF1 (1:1000, Proteintech), Isl1/2 (1:500, DSHB), Lhx2 (1:500, Santa Cruz Biotechnology), Lhx2 (1:500, Abcam), Lhx9 (1:200, Santa Cruz Biotechnology), TAG1 (1:1000, R&D Systems), TAG1 (1:200, DSHB).

Techniques: Expressing, Plasmid Preparation, Western Blot, Control, Whisker Assay, Comparison

Journal: Nucleic Acids Research

Article Title: The m 6 A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

doi: 10.1093/nar/gkz157

Figure Lengend Snippet: YTHDF1 regulates translation of endogenous Robo3.1 in commissural neurons and is controlled by floor plate. ( A ) Knockdown of YTHDF1 in commissural neurons. Dissociated commissural neurons from E10.5 mouse spinal cord was infected with lenti virus expressing shYthdf1-2 and shYthdf1-3 , respectively, and marked by eGFP labeling. Knockdown by shYthdf1 for 48 h resulted in dramatic decrease of YTHDF1 protein levels in commissural axons, compared with shCtrl . ( B ) Knockdown of YTHDF1 led to significant decreases of Robo3.1 protein levels in commissural axons compared with shCtrl . ( C ) Quantification of relative Robo3.1 IF to eGFP in (B). n = 16 axons for shCtrl ; n = 17 axons for shYthdf1-2 ; n = 16 axons for shYthdf1-3 . ( D ) Overexpression of YTHDF1 in commissural neurons. Dissociated commissural neurons from E10.5 mouse spinal cord was infected with lenti virus expressing YTHDF1, marked by eGFP labeling. Overexpression of YTHDF1 resulted in dramatic increase of YTHDF1 protein levels in commissural axons, compared with control. ( E ) Overexpression of YTHDF1 led to significant increases of Robo3.1 protein levels in commissural axons compared with eGFP control. ( F ) Quantification of relative Robo3.1 IF to eGFP in (E). n = 14 axons for IRES-eGFP ; n = 16 axons for Ythdf1-IRES-eGFP . ( G ) Regulation of YTHDF1 expression by floor plate. DSC explants from E10.5 mouse embryonic spinal cords were cultured with FP-CM or Ctrl-CM. WB analysis was carried out to measure YTHDF1 protein levels. ( H ) Quantification of WB signals in (G). All data are mean ± S.E.M. Data of IF quantification (C, F) are represented as box and whisker plots: For C, **** P = 8.05E-7 ( shYthdf1-2 versus shCtrl ), **** P = 3.83E-7 ( shYthdf1-3 versus shCtrl ), ns, not significant ( P = 0.76, shYthdf1-2 versus shYthdf1-3 ); for F, **** P = 3.35E–6; by unpaired Student's t test. WB quantification data (H, n = 5 replicates) are represented as dot plots: **** P = 1.68E–8; by paired Student's t test. Scale bars, 10 μm (B and E).

Article Snippet: The dilutions and sources of antibodies are as following: Robo3.1 (1:500), GFP (1:500, Abcam), YTHDF1 (1:1000, Proteintech), Isl1/2 (1:500, DSHB), Lhx2 (1:500, Santa Cruz Biotechnology), Lhx2 (1:500, Abcam), Lhx9 (1:200, Santa Cruz Biotechnology), TAG1 (1:1000, R&D Systems), TAG1 (1:200, DSHB).

Techniques: Knockdown, Infection, Virus, Expressing, Labeling, Over Expression, Control, Cell Culture, Whisker Assay

Journal: Nucleic Acids Research

Article Title: The m 6 A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

doi: 10.1093/nar/gkz157

Figure Lengend Snippet: Ythdf1 cKO embryos exhibit defects in pre-crossing commissural axon guidance. ( A ) Misprojection of pre-crossing commissural axons into motor columns in Ythdf1 cKO embryos. TAG1 marks commissural axons in E11.5 embryonic sections and there are significantly more misprojecting axons into motor columns (arrowheads) in Ythdf1 cKO embryos compared with their littermate controls. Representative images are shown from three Ythdf1 fl/fl and three Atoh1-Cre +/- ; Ythdf1 fl/fl embryos, respectively. ( B ) Quantification of commissural axons intruding into motor columns. All data are mean ± S.E.M. and represented as box and whisker plots: Ythdf1 fl/fl ( n = 15 sections) versus Atoh1-Cre +/- ; Ythdf1 fl/fl ( n = 12 sections), ** P = 0.0096; by unpaired Student's t test. ( C and D ) DiI tracing of commissural axons with open-book preparations. Pre-crossing axon guidance defects including stalling (arrowheads) and pre-crossing turning (arrows) were observed in Ythdf1 cKO embryos. Representative images of E10.5–11 (C) and E11.5 (D) were shown. ( E ) Quantification of phenotypes in (C and D). Total 31 DiI injections with four Ythdf1 fl/fl embryos and 40 DiI injections with three Atoh1-Cre +/- ; Ythdf1 fl/fl embryos were analyzed. Percentage of observations with phenotypes was calculated. Note that the summed percentage for Atoh1-Cre +/- ;Ythdf1 fl/fl is >100 because some of DiI injections were found with both stalling and pre-crossing turning phenotypes. Scale bars, 50 μm (A, C and D).

Article Snippet: The dilutions and sources of antibodies are as following: Robo3.1 (1:500), GFP (1:500, Abcam), YTHDF1 (1:1000, Proteintech), Isl1/2 (1:500, DSHB), Lhx2 (1:500, Santa Cruz Biotechnology), Lhx2 (1:500, Abcam), Lhx9 (1:200, Santa Cruz Biotechnology), TAG1 (1:1000, R&D Systems), TAG1 (1:200, DSHB).

Techniques: Whisker Assay