Journal: Oncogene

Article Title: Androgen regulation of soluble guanylyl cyclasealpha1 mediates prostate cancer cell proliferation.

doi: 10.1038/sj.onc.1209956

Figure Lengend Snippet: Figure 6 sGCa1 is overexpressed in advanced prostate cancer tissues. (a) Total mRNA was isolated from prostate tissues (acquired from CHTN), which are normal (N1), BPH (B1–B3), or MPC (C1, C2) and subjected to semi-quantitative RT-PCR to measure the expression of sGCa1, sGCb1, PSA, EZH2, E-cadherin, and AR. (b) The human prostate cancer tissue array slide (from Imgenex) was prepared and subjected to immunohistochemistry analysis to detect the expression of sGCa1 protein. Staining results from one or more representative tissues, of different stages, are shown. DAPI staining shows cell nuclei in the lower panels. (c) The sGCa1 expression levels of 45 tissue samples (five normal; eight stages 1 and 2; 32 stages 3 and 4) were quantified according to manufacturer’s protocol (Imgenex). Note that sGCa1 expression levels are represented relative to the average expression level of normal tissues, which was set to 1. Data represent mean expression values plus/minus s.d.

Article Snippet: Prostate tissue array slide (Imgenex) was processed according to the manufacturer’s protocol.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Immunohistochemistry, Staining

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