Journal: Journal of Cancer

Article Title: N6-methyladenosine regulates ATM expression and downstream signaling.

doi: 10.7150/jca.64061

Figure Lengend Snippet: Figure 5. YTHDFs suppress ATM expression in the post-transcriptional levels. (A) SGC7901 cells were transfected with Flag-NC or Flag-YTHDF1 or Flag-YTHDF2 for 36 h, followed by CHX treatment (25 μg/ml) for indicated times. Cellular proteins were collected and Western blot was performed. (B) HEK293 cells were transfected with indicated plasmids (Flag-NC, Flag-YTHDF1 and HA-Ubiquitin), and cells were treated with MG132 (25 μM) for 4 h before harvest. Lysates were immunoprecipitated with anti-HA antibodies and then immunoblotted with anti-ATM antibodies. (C) HEK293 cells were transfected with Flag-NC or Flag-YTHDF1 for 48 h, then cell lysates were subject to immunoprecipitation with anti-Flag antibodies. The immunoprecipitates were subsequently blotted with indicated antibodies. (D and E) SGC7901 cells were transfected with indicated siRNAs for 48 h. RT-qPCR (D) and Western blot (E) were used to assay the indicated molecules. Mean ± SD of three independent experiments. Two-tailed unpaired t test. (F) SGC7901 cells were transfected with indicated siRNAs for 48 h. Immunofluorescence assays were performed to detect the fluorescent expression of p-ATM (Ser1981) and γH2A.X. Scale bar: 50 μm. ***p < 0.001.

Article Snippet: Protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel, transferred to polyvinylidene fluoride membranes, and probed with antibodies against ATM (2873S, Cell Signaling Technology, CST); p-ATM -S1981 (AP0008, ABclonal); METTL3 (15073-1-AP, Proteintech); YTHDF1 (17479-1-AP, Proteintech); YTHDF2 (24744-1-AP, Proteintech); YTHDF3 (25537-1-AP, Proteintech); FTO (TA809392, OriGene); m6A (202003, Synaptic Systems); BRCA1 (20649-1-AP, Proteintech); H2A.X (A11361, ABclonal); γH2A.X (AP0687, ABclonal ); GAPDH(60004-1-Ig, Proteintech); eIF3A (3411T, CST); Flag (F1804, Sigma-Aldrich); anti‐rabbit IgG HRP‐ linked antibody (7074; CST); Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (SA00001-1, Proteintech); α-Tubulin (66031-1-Ig, Proteintech); CHK1(bs-1681R, Bioss); CHK2 (252092, Zenbio); p-CHK2-S19(AP0862, ABclonal); Anti-HA tag monoclonal antibody (TA100012, OriGene); Mouse-IgG (BA1046, Boster); Rabbit IgG (B900610, Proteintech); p21(10355-1-AP, Proteintech); 53BP1 (BA2878, Boster); CDK1 (D160158, BBI life sciences); CDK2 (D199431, BBI life sciences); Cyclin B2 (21644-1-AP, Proteintech).

Techniques: Expressing, Transfection, Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, Immunofluorescence

Journal: Theranostics

Article Title: Therapeutic targeting of the AMPK-Has1 complex formation ameliorates metabolic dysfunction-associated steatohepatitis in mice

doi: 10.7150/thno.120527

Figure Lengend Snippet: Specific Has1 knockdown protects from PO-induced MAFL and MASH in hepatocytes. (A) Representative western blot gels of Has1 protein in livers from mice treated with vehicle or indicated doses of Ele (mean ± SEM, n = 3). (B) Oil red O staining, (C) TG and TC content detection of primary hepatocytes in response to PO stimulation treated with/without Ele or siRNA against Has1 (mean ± SEM, n = 5). (Scale bar, 100 μm.) (D) Representative western blot gels showing Has1, total and phosphorylated AMPK, PPARα, CPT-1A, total and phosphorylated ACC, SREBP-1c, FASN, SCD1, NLRP3, ASC, Caspase-1, Col1a1, and TGFβ proteins in primary hepatocytes in response to the corresponding stimulus treated as described in C . (E) Representative immunofluorescence staining of ASC (green) and NLRP3 (red) in primary hepatocytes with indicated treatments. (Scale bar, 100 μm.) (F) Mitochondrial bioenergetic analysis of primary hepatocytes in response to PO stimulation treated with siRNA against Has1 (mean ± SEM, n = 3). Binding affinity and kinetic parameters for the interactions between Ele and Has1 protein. (G) Docking images showing Has1 and binding position of Ele at Has1 protein, and (H) kinetic analysis using SPR for Ele binding to Has1. (I) ITC assay showing direct interaction of Has1 protein and Ele (mean ± SEM, n = 3). (J) CETSA analysis of the binding specificity of Ele with Has1 protein in PO-induced hepatocytes under the indicated conditions. (K) Western blotting in primary hepatocytes transduced with an AMPK protein expression vector (siAMPK). Cells transduced with empty vector (siNC) served as control. The expression levels of p-AMPK and Has1 were determined after co-treatment with CC. (L) Results by Co-IP assays in primary hepatocytes transfected with Flag-tagged Has1 and HA-tagged AMPK. Anti-Flag and anti-HA antibodies were used as immunoblotting probes. (M) Immunoprecipitation and western blotting analysis indicating the binding of Has1 and AMPK in liver WT mice after a 24-week HFHC, and IgG was served as a control. (N) Representative gels showing the influence of Ele on interaction between Flag-labeled Has1 and HA-labeled AMPK in 293T cells examined by immunoprecipitation and western blot assay ( * P < 0.05, ** P < 0.01; # P < 0.05, ## P < 0.01).

Article Snippet: Primary antibody against Has1 and Pisd supplied by Boster (Wuhan, China).

Techniques: Knockdown, Western Blot, Staining, Immunofluorescence, Binding Assay, Isothermal Titration Calorimetry, Transduction, Expressing, Plasmid Preparation, Control, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Labeling

Journal: Theranostics

Article Title: Therapeutic targeting of the AMPK-Has1 complex formation ameliorates metabolic dysfunction-associated steatohepatitis in mice

doi: 10.7150/thno.120527

Figure Lengend Snippet: 4-MU effectively inhibits MASH progression in diet-induced mice. (A) Flow chart showing strategy for evaluating 4-MU function on mice with spontaneously developed MASH that was exacerbated by HFHC treatment. Representative western blot gels and quantification (B), and IHC staining and quantification (C) of serial liver sections from HFHC-fed mice in indicated groups showing the Has1 protein (mean ± SEM, n = 3 in western blot and n = 6 in IHC). (Scale bar, 100 μm.) (D) Serum hyaluronan content detection in response to HFHC stimulation treated with/without 4-MU (mean ± SEM, n = 5). (E) Body weight, liver weight, and the ratio of liver weight to body weight of wild-type mice treated with 4-MU or vehicle at 24 weeks of HFHC or NC diet administration (mean ± SEM, n = 6). (F) Representative macroscopic and histological images of livers and liver sections (mean ± SEM, n = 6). (G) Representative H&E, oil red O, Masson, and PSR staining of liver sections from mice treated with vehicle or indicated doses of Ele (mean ± SEM, n = 6). (Scale bar, 100 μm.) NAS and quantitative results of oil red O-, Masson- and PSR-positive areas are also shown in (G) (mean ± SEM, n = 6). Hepatic TG and TC (H), and serum ALT and AST (I) in NC- or HFHC-diet-fed mice treated with either vehicle or 4-MU (mean ± SEM, n = 6). ( * P < 0.05, ** P < 0.01 vs. Control+Vehicle; # P < 0.05, ## P < 0.01 vs. HFHC+Vehicle).

Article Snippet: Primary antibody against Has1 and Pisd supplied by Boster (Wuhan, China).

Techniques: Western Blot, Immunohistochemistry, Staining, Control

Journal: Theranostics

Article Title: Therapeutic targeting of the AMPK-Has1 complex formation ameliorates metabolic dysfunction-associated steatohepatitis in mice

doi: 10.7150/thno.120527

Figure Lengend Snippet: The potential factors that MASH and Ele regulate PE on HFHC diet-induced metabolic dysfunction-associated steatohepatitis in mice. (A) The main pathways of intrahepatic PE and PC biosynthesis and metabolism following Ele treatment in HFHC diet-fed mice (mean ± SEM, n = 6). (B and C) Western blot analysis of total and phosphorylated AMPK, SREBP-1c, FASN, PPARα, CPT-1A, NLRP3, ASC, Caspase-1, Col1a1, TGFβ, Pisd, and Pemt proteins in primary hepatocytes in response to PO stimulation treated with PE. (D) Western blot analysis of p-AMPK and Has1 expression following treatment with PE in combination with CC or siHas1. (E) Representative and parametric results of OCR in primary hepatocytes in response to the corresponding stimulus treated with PE (mean ± SEM, n = 3). ( * P < 0.05, ** P < 0.01 vs. DMSO; # P < 0.05, ## P < 0.01 vs. PO+DMSO).

Article Snippet: Primary antibody against Has1 and Pisd supplied by Boster (Wuhan, China).

Techniques: Western Blot, Expressing