Journal: Genes & Diseases

Article Title: FTO-mediated m6A modification alleviates autoimmune uveitis by regulating microglia phenotypes via the GPC4/TLR4/NF-κB signaling axis

doi: 10.1016/j.gendis.2022.09.008

Figure Lengend Snippet: Downregulated FTO was involved in microglia-elicited inflammation and migration in vitro . ( A , B ) RT-qPCR and Western blot for detecting the expression of iNOS, TNFα, and IL6 with or without LPS + IFN-γ treatment for 24 h ( n = 3). ( C ) Griess assay for detecting NO production in the supernatant with or without LPS + IFN-γ treatment for 24 h ( n = 3). ( D ) Transwell images and corresponding quantifications in the control group and the LPS + IFN-γ group ( n = 3). Scale bar, 100 μm. ( E ) Representative photomicrographs of the wound-healing assay performed on HMC3 cells before and after scratching. ( F ) The overall m 6 A% level of the control group and the LPS + IFN-γ group ( n = 3). ( G ) RT-qPCR for testing the relative mRNA expression of m 6 A methylase, demethylase, and readers in the control group and the LPS + IFN-γ group ( n = 3). ( H ) FTO protein expression in the control group and the LPS + IFN-γ group ( n = 3). Values are analyzed using the unpaired Student's t -test. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: The primary antibodies used in this study were as follows: iNOS (ab178945, Abcam, 1:800), TNFα (ab183218, Abcam, 1:500), IL6 (ab233706, Abcam, 1:500), FTO (ab126605, Abcam, 1:800), GPC4 (sc-517403, Santa Cruz, 1:800), NF-κB p65 (ab32536, Abcam, 1:1,000), NF-κB p-p65 S536 (3033, Cell Signaling Technology, 1:1,000), TLR4 (ab22048, Abcam, 1:1,000), MYD88 (ab133739, Abcam, 1:1,000), CD14 (221678, Abcam, 1:1,000), CXCL10 (14969, Cell Signaling Technology, 1:1,000), and β-actin (20536-1-AP, Proteintech, 1:3,000).

Techniques: Migration, In Vitro, Quantitative RT-PCR, Western Blot, Expressing, Griess Assay, Control, Wound Healing Assay

Journal: Genes & Diseases

Article Title: FTO-mediated m6A modification alleviates autoimmune uveitis by regulating microglia phenotypes via the GPC4/TLR4/NF-κB signaling axis

doi: 10.1016/j.gendis.2022.09.008

Figure Lengend Snippet: FTO knockdown upregulated inflammation and promoted microglial migration. ( A , B ) RT-qPCR and Western blot for determining FTO knockdown efficiency ( n = 3, unpaired Student's t -test). ( C ) Percentage of m 6 A content of the total RNA in the vehicle group and the shFTO group ( n = 3, unpaired Student's t -test). ( D ) CCK-8 assay for determining the proliferation of the vehicle and shFTO groups ( n = 3; unpaired Student's t -test). ( E , F ) The mRNA and protein levels of iNOS, TNFα, and IL6 in the vehicle- and shFTO-treated cells with or without LPS + IFN-γ stimulation ( n = 3; one-way ANOVA). ( G ) ELISAs for testing TNFα expression in the vehicle- or shFTO-treated cells with or without LPS + IFN-γ stimulation ( n = 3; one-way ANOVA). ( H ) Transwell images and corresponding quantifications of the vehicle group, vehicle + LPS&IFN-γ group, shFTO group, and shFTO + LPS&IFN-γ group ( n = 3; one-way ANOVA). ( I ) Representative images of the wound-healing assay performed in the vehicle group, vehicle + LPS&IFN-γ group, shFTO group, and shFTO + LPS&IFN-γ group ( n = 3; one-way ANOVA). ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: The primary antibodies used in this study were as follows: iNOS (ab178945, Abcam, 1:800), TNFα (ab183218, Abcam, 1:500), IL6 (ab233706, Abcam, 1:500), FTO (ab126605, Abcam, 1:800), GPC4 (sc-517403, Santa Cruz, 1:800), NF-κB p65 (ab32536, Abcam, 1:1,000), NF-κB p-p65 S536 (3033, Cell Signaling Technology, 1:1,000), TLR4 (ab22048, Abcam, 1:1,000), MYD88 (ab133739, Abcam, 1:1,000), CD14 (221678, Abcam, 1:1,000), CXCL10 (14969, Cell Signaling Technology, 1:1,000), and β-actin (20536-1-AP, Proteintech, 1:3,000).

Techniques: Knockdown, Migration, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Expressing, Wound Healing Assay

Journal: Genes & Diseases

Article Title: FTO-mediated m6A modification alleviates autoimmune uveitis by regulating microglia phenotypes via the GPC4/TLR4/NF-κB signaling axis

doi: 10.1016/j.gendis.2022.09.008

Figure Lengend Snippet: GPC4 knockdown reversed the effects of FTO on microglia. ( A , B ) RT-qPCR and Western blot for detecting GPC4 expression in the FTO-deficient HMC3 cells transfected with vehicle lentivirus, shGPC4-1, shGPC4-2, or shGPC4-3 lentivirus. ( C , D ) RT-qPCR and Western blot for showing the expression of iNOS, TNFα, and IL6 in vehicle 1 (corresponding to shFTO) +LPS&IFN-γ, shFTO + LPS&IFN-γ, shFTO + shGPC4+LPS&IFN-γ, and shFTO + vehicle 2 (corresponding to shGPC4) +LPS&IFN-γ groups. ( E , F ) CXCL10 mRNA and protein expression in different groups with the abovementioned stimulation. ( G ) The number of human CD4 + T cells that migrated into lower HMC3 cells in the abovementioned groups. ( H ) Expression levels of key proteins involved in the TLR4 pathway (TLR4, CD14, and CXCL10) in the abovementioned groups ( n = 3). Values are analyzed using the one-way ANOVA. ∗ P < 0.05; ∗∗ P < 0.01.

Article Snippet: The primary antibodies used in this study were as follows: iNOS (ab178945, Abcam, 1:800), TNFα (ab183218, Abcam, 1:500), IL6 (ab233706, Abcam, 1:500), FTO (ab126605, Abcam, 1:800), GPC4 (sc-517403, Santa Cruz, 1:800), NF-κB p65 (ab32536, Abcam, 1:1,000), NF-κB p-p65 S536 (3033, Cell Signaling Technology, 1:1,000), TLR4 (ab22048, Abcam, 1:1,000), MYD88 (ab133739, Abcam, 1:1,000), CD14 (221678, Abcam, 1:1,000), CXCL10 (14969, Cell Signaling Technology, 1:1,000), and β-actin (20536-1-AP, Proteintech, 1:3,000).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Transfection

Journal: Genes & Diseases

Article Title: FTO-mediated m6A modification alleviates autoimmune uveitis by regulating microglia phenotypes via the GPC4/TLR4/NF-κB signaling axis

doi: 10.1016/j.gendis.2022.09.008

Figure Lengend Snippet: FTO inhibition aggravated EAU progression in vivo . ( A ) Retinal FTO mRNA and protein expression in the FB23-2-treated mice and the PBS + DMSO-treated mice ( n = 3). ( B ) Experimental flowchart describing the modeling process and drug injection. ( C ) Representative images and quantification of the ocular anterior chamber (upper) and eyeball sections (lower) ( n = 5). Black arrows indicate inflammatory cells. Red arrows indicate conjunctival or ciliary hyperemia, or vasculitis. White arrows indicate posterior synechiae, or retinal folds. Scale bar, 100 μm. ( D , E ) RT-qPCR and Western blot for showing the expression levels of iNOS, TNFα, and IL6 in the EAU + vehicle and EAU + FB23-2 groups ( n = 3). ( F ) Protein expression levels of GPC4, TLR4, p-p65, and p65 in the EAU + vehicle and EAU + FB23-2 groups ( n = 3). Values are analyzed using the unpaired Student's t -test. ∗ P < 0.05; ∗∗ P < 0.01. ( G ) Graphical summary of this study.

Article Snippet: The primary antibodies used in this study were as follows: iNOS (ab178945, Abcam, 1:800), TNFα (ab183218, Abcam, 1:500), IL6 (ab233706, Abcam, 1:500), FTO (ab126605, Abcam, 1:800), GPC4 (sc-517403, Santa Cruz, 1:800), NF-κB p65 (ab32536, Abcam, 1:1,000), NF-κB p-p65 S536 (3033, Cell Signaling Technology, 1:1,000), TLR4 (ab22048, Abcam, 1:1,000), MYD88 (ab133739, Abcam, 1:1,000), CD14 (221678, Abcam, 1:1,000), CXCL10 (14969, Cell Signaling Technology, 1:1,000), and β-actin (20536-1-AP, Proteintech, 1:3,000).

Techniques: Inhibition, In Vivo, Expressing, Injection, Quantitative RT-PCR, Western Blot