Journal: Nature Communications

Article Title: Autophagy of the m 6 A mRNA demethylase FTO is impaired by low-level arsenic exposure to promote tumorigenesis

doi: 10.1038/s41467-021-22469-6

Figure Lengend Snippet: a Heatmap showing altered expression of the genes in autophagy/lysosome pathways between As-T and control cells. b qPCR analysis of OPTN , p62 , ATG5 , and LAMP1 levels in control, As, and As-T cells ( n = 3). c Immunoblot analysis of FTO in control, As, and four As-T cells. d Immunoblot analysis of HA, NEDD4L, and FTO in As-T cells transfected with empty vector ( EV ) or p62 ( HA tag). e Immunoblot analysis of FTO and HA (p62) in As-T cells as in d treated with CHX over a time course. f Quantification of e . The t 1/2 (half-life) is indicated ( n = 3). g Immunoblot analysis of FTO and HA (p62) in As-T cells with or without FTO deletion in combination with transfection with EV or p62-HA . h Immunoblot analysis of FTO and p62 following immunoprecipitation using species-matched control IgG and an anti-p62 antibody in HaCaT cells. i ubiquitination assay of As-T cells transfected with HA-Ub and Flag-FTO and treated with or without BfnA1 (50 nM) and MG132 (10 μM) for 6 h. j Immunoblot analysis of FTO and HA in As-T cells transfected with plasmids expressing WT and ΔUBA p62 . k Immunoblot analysis of Flag-FTO and HA-p62 (WT or ΔUBA) following immunoprecipitation assay of the binding of Flag-FTO with HA-p62 WT or HA-p62 ΔUBA in HeLa cells transfected with Flag-EV ( empty vector ), Flag-FTO , HA-p62 WT , or HA-p62 ΔUBA . l Immunoblot analysis of FTO and HA in As-T cells transfected with constructs expressing WT p62 or LIR ( W338A ) mutant p62 ( HA ). m Representative images of immune electron microscopy (IEM) analysis for p62 and FTO in control cells. The upper panel shows the negative controls without primary antibodies. The bottom panel shows IEM staining of FTO (10 nm) and p62 (15 nm). n Quantification of the p62 levels in normal human skin ( n = 6) and arsenical keratoses ( n = 3). Related to Supplementary Fig. . a.u.: arbitrary units. o Negative correlation of p62 levels with FTO levels by Spearman’s test in normal human skin ( n = 6) and arsenical keratoses ( n = 3) from Supplementary Fig. . All data were performed on n ≥ 3 biologically independent samples. Error bars are shown as mean ± S.D. ( b , f , n ). p -values by two-tailed unpaired t -test ( b , f , n ); Correlation coefficient r and p- value in o are indicated from Spearman’s Correlation Rank test.

Article Snippet: Flag-EV and FLAG-FTO plasmids were purchased from SinoBiological. pcDNA4-HA-p62 was obtained from Addgene (#28027, kindly provided by Qing Zhong) . p62 with deletion of UBA domain (p62 ΔUBA) was generated as described previously , pLenti- HA-Ub purchased from Addgene (#74218, kindly provided by Melina Fan). p62 LIR-W338A was subcloned into the pcDNA4 vector (Addgene plasmid #28027, kindly provided by Qing Zhong), from the vector expressing the LIR mutant (pBABEpuro- HA-p62-LIR , Addgene plasmid #71306, kindly provided by Jayanta Debnath) using the following primers: Forward, 5‘-CCGGAATTCTATGGCGTCGCTCACCGTGAAGG-3‘; Reverse, 5‘-ATAAGAATGCGGCCGCGCAACGGCGGGGGATGCTTTGAATACTG-3‘).

Techniques: Expressing, Control, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics, Binding Assay, Construct, Mutagenesis, Electron Microscopy, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Autophagy of the m 6 A mRNA demethylase FTO is impaired by low-level arsenic exposure to promote tumorigenesis

doi: 10.1038/s41467-021-22469-6

Figure Lengend Snippet: a GO pathway analysis of downregulated genes shared in both As and As-T cells as compared with control cells. b GSEA of “TNF signaling pathway”-related genes in As-T cells versus control cells. ES: Enrichment score. NES: Normalized enrichment scores, p : p -value, FDR: False discovery rate. c qPCR analysis of NFKB1 , TNF , TNFRSF1A , TNFAIP3 , ATG5 , ATG7 , and p62 in control, As, and four As-T cells ( n = 3). d Luciferase reporter analysis of NF-κB activity in control, As, and four As-T cells ( n = 3). e Luciferase reporter analysis of NF-κB activity in As-T cells treated with or without TNFα (10 and 50 ng/ml) for 20 h ( n = 3). f Immunoblot analysis of NEDD4L, ATG5, ATG7, FTO, and p62 in As-T cells treated with or without TNFα for 20 h. g qPCR analysis of NFKB1 , p62, and NEDD4L mRNA level in cells as in f ( n = 3). h Immunoblot analysis of FTO, RELA, p62, and NEDD4L in As-T cells treated with or without TNF (50 ng/ml) following transfection with or without siRNA targeting p62 or RELA . i qPCR analysis of p62 and NEDD4L mRNA level in cells as in h ( n = 3). j Proximity ligation assay (PLA) of the interaction between FTO and p62 in cells as in h . DAPI is used as a nuclear counterstain (blue). All samples are pretreated with BfnA1 for 6 h. Scale bar: 20 µm. k Quantification of the number of PLA red dots per cell in j ( n = 30 cells from three biologically independent replicates). All data were performed on n ≥ 3 biologically independent samples. Error bars are shown as mean ± S.D. ( c–e , g , i , k ). p -values of all data by two-tailed unpaired t-test are indicated.

Article Snippet: Flag-EV and FLAG-FTO plasmids were purchased from SinoBiological. pcDNA4-HA-p62 was obtained from Addgene (#28027, kindly provided by Qing Zhong) . p62 with deletion of UBA domain (p62 ΔUBA) was generated as described previously , pLenti- HA-Ub purchased from Addgene (#74218, kindly provided by Melina Fan). p62 LIR-W338A was subcloned into the pcDNA4 vector (Addgene plasmid #28027, kindly provided by Qing Zhong), from the vector expressing the LIR mutant (pBABEpuro- HA-p62-LIR , Addgene plasmid #71306, kindly provided by Jayanta Debnath) using the following primers: Forward, 5‘-CCGGAATTCTATGGCGTCGCTCACCGTGAAGG-3‘; Reverse, 5‘-ATAAGAATGCGGCCGCGCAACGGCGGGGGATGCTTTGAATACTG-3‘).

Techniques: Control, Luciferase, Activity Assay, Western Blot, Transfection, Proximity Ligation Assay, Two Tailed Test