Journal: Clinical and Translational Medicine

Article Title: CK1δ stimulates ubiquitination‐dependent proteasomal degradation of ATF4 to promote chemoresistance in gastric Cancer

doi: 10.1002/ctm2.587

Figure Lengend Snippet: CK1δ phosphorylates ATF4 to stimulate its ubiquitination‐dependent proteasomal degradation. (A) The scheme of ATF4 protein was shown, and expression of phosphorylated ATF4‐S219 [p‐ATF4 (S219)], total ATF4 and βTrCP1 in sensitive and resistant cells were determined with western blotting. Expression of ATF4 in SGC‐R (left) or BGC‐R (right) with CK1δ knockdown (B) or inhibition (C) by D4476 was detected by western blotting. (D) Co‐IP was performed with anti‐HA antibody in HEK293T cells with HA‐ATF4 and Flag‐CK1α/δ/ε co‐transfection, and the interaction was detected by western blotting with anti‐Flag and anti‐ATF4 antibodies. (E) p‐ATF4(S219) in SGC7901 (left) or BGC823 (right) with Flag‐CK1δ over‐expression was analysed by western blotting. (F) After Flag‐CK1δ transfection, cells were pre‐treated with MG132, and interaction of ATF4 with βTrCP1 in SGC7901 (left) or BGC823 (right) cells was analysed by anti‐ATF4 co‐IP, followed by anti‐βTrCP1 and anti‐ATF4 immunoblot. (G) Interaction of wild‐type Myc‐ATF4 (Myc‐ATF4‐WT) or Myc‐ATF4‐S219A mutant with Flag‐βTrCP1 with/without Flag‐CK1δ co‐transfection in HEK293T cells was determined with anti‐Myc co‐IP, followed by anti‐Flag and anti‐Myc immunoblot. (H) βTrCP1‐mediated ATF4 ubiquitination in HEK293T cells with/without Flag‐CK1δ over‐expression was measured by in vitro ubiquitination assay. (I) Protein turnover of exogenous Myc‐ATF4‐WT or Myc‐ATF4‐S219A in SGC‐R cells under CHX (50 μg/ml) treatment with indicated times was detected by western blotting using anti‐Myc antibody. The relative grey value of ATF4 compared to Actin was analysed, and the normalised expression ratio was shown

Article Snippet: CK1δ (mouse, sc‐55553) and Ub (Ubiquitin) (mouse, sc‐8017) were from Santa Cruz (Shanghai, China).

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Knockdown, Inhibition, Co-Immunoprecipitation Assay, Cotransfection, Over Expression, Transfection, Mutagenesis, In Vitro

Journal: Clinical and Translational Medicine

Article Title: CK1δ stimulates ubiquitination‐dependent proteasomal degradation of ATF4 to promote chemoresistance in gastric Cancer

doi: 10.1002/ctm2.587

Figure Lengend Snippet: Dynamic ATF4 protein degradation in adaptive chemotherapy. (A) The process of drug withdraw and re‐addition for resistant cells maintaining was shown. Generally, the resistant cells were maintained in DDP free medium for 2–3 weeks to release the stress of DDP, which was named as SGC‐R wash and BGC‐R wash cells. Then DDP containing medium was used to re‐stress SGC‐R wash and BGC‐R wash cells for additional 2–3 weeks, and these cells were named as SGC‐R wash+DDP and BGC‐R wash+DDP. The sensitivity (B) and apoptosis (C) of different medium maintaining resistant cells as indicated to DDP for 24 h was measured. (D) Expression of ATF4 in resistant cells cultured with different medium as indicated was detected by western blotting. (E) Expression of ATF4 in sensitive cells (left for SGC7901 and right for BGC823) with time‐course DDP (1.2 μg/ml) treatment was determined by western blotting. (F) Interaction of Myc‐ATF4 with Flag‐βTrCP1 in HEK293T with/without DDP treatment for 12 h was determined by anti‐Flag co‐IP, and followed with anti‐Myc and anti‐Flag immunoblot. (G) The ubiquitination level of ATF4‐mediated by βTrCP1 with/without DDP treatment for 12 h was measured by in vitro ubiquitination assay. (H) Interaction of ATF4 with βTrCP1 and CK1δ in SGC7901 cells with/without DDP treatment for 12 h was determined by anti‐ATF4 co‐IP, then immunoblot with anti‐βTrCP1 and anti‐CK1δ

Article Snippet: CK1δ (mouse, sc‐55553) and Ub (Ubiquitin) (mouse, sc‐8017) were from Santa Cruz (Shanghai, China).

Techniques: Expressing, Cell Culture, Western Blot, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, In Vitro

Journal: Clinical and Translational Medicine

Article Title: CK1δ stimulates ubiquitination‐dependent proteasomal degradation of ATF4 to promote chemoresistance in gastric Cancer

doi: 10.1002/ctm2.587

Figure Lengend Snippet: ATF4 stabilisation by BTZ or CK1δ inhibitor enhances the efficacy of chemotherapy in gastric cancer. Viability (A) and apoptosis (B) of SGC7901 cells treated by DDP with/without BTZ (0.1μM) for 24 h was measured. (C) Viability of SGC7901 cells treated by DDP and D4476 (0, 12.5 or 25 μM) for 24 h was measured by MTS assay. (D) Apoptosis of BGC823 cells treated by DDP and D4476 (25μM) for 24 h was measured. Xenograft model was performed with SGC7901 cells under indicated treatments, tumour growth curve (E) was plotted according to the tumour volume measured every other day, tumour weight (F) and tumour photos (G) were obtained at the end of the experiment. (H) Expression of ATF4, CHOP and Ki67 in different groups as indicated were analysed by IHC. (I) Proposed model. Generally, chemotherapeutics could promote ATF4 to activate CHOP transcription, thus inducing cell apoptosis. Simultaneously, chemotherapeutics increases ATF4‐S219 phosphorylation via CK1δ, which subsequently enhances βTrCP‐mediated ATF4 poly‐ubiquitination‐dependent proteasomal degradation, eventually resulting in chemoresistance. Therefore, this acquired resistance is a dynamic reversible process. Upon drug withdraw, ATF4 expression gradually recovers and resistant cells will re‐sensitise to chemotherapeutics

Article Snippet: CK1δ (mouse, sc‐55553) and Ub (Ubiquitin) (mouse, sc‐8017) were from Santa Cruz (Shanghai, China).

Techniques: MTS Assay, Expressing, Phospho-proteomics, Ubiquitin Proteomics

Journal: mSystems

Article Title: Network controllability analysis reveals the antiviral potential of Etravirine against hepatitis E virus infection

doi: 10.1128/msystems.00438-25

Figure Lengend Snippet: Antiviral activity of CK1ε and AKT inhibitors in g1- and g3-HEV genomic RNA transfected Huh7 cells. ( A ) Real-time quantitative PCR (RT-qPCR) measurement of g3-HEV RNA level in Huh7 cells expressing g3-HEV and treated with PF670462 or AKTi-1/2, as indicated. Relative g3-HEV RNA values were calculated with respect to the value of DMSO-treated samples, normalized with values of Ribonuclease P (RP), and represented as (mean ± SD) of three independent experiments. ( B ) Immunofluorescence image of ORF2 (red, upper panel) or ORF2 (red) and nucleus (blue; lower panel) in Huh7 cells expressing g3-HEV and treated with the indicated inhibitors for 48 hours. ( C ) Quantification of % ORF2-positive cells in five random fields, as represented in panel B . Values are (mean ± SD) of five random fields. P values were calculated using Student's t -test. P < 0.05 was considered significant. ( D ) RT-qPCR measurement of g1-HEV RNA in Huh7 cells expressing g1-HEV and treated with PF670462 or AKTi-1/2, as indicated. Relative g1-HEV RNA values were calculated with respect to the value of DMSO-treated samples, normalized with values of RP, and represented as (mean ± SD) of three independent experiments. ( E ) Immunofluorescence image of ORF2 (red, upper panel) or ORF2 (red) and nucleus (blue; lower panel) in Huh7 cells expressing g1-HEV and treated with the indicated inhibitors for 48 hours. ( F ) Quantification of % ORF2-positive cells in 10 random fields, as represented in panel E . Values are (mean ± SD) of 10 random fields. P values were calculated using Student's t -test. P < 0.05 was considered significant. *, **, ***, and **** represent P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

Article Snippet: The anti-CK1ε antibody (Catalog No. sc-365259) was from Santa Cruz Biotechnology (Texas, USA).

Techniques: Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Immunofluorescence

Journal: mSystems

Article Title: Network controllability analysis reveals the antiviral potential of Etravirine against hepatitis E virus infection

doi: 10.1128/msystems.00438-25

Figure Lengend Snippet: Antiviral activity of CK1ε and AKT inhibitors in g1-HEV clinical isolate-infected Huh7 cells. ( A ) RT-qPCR measurement of g1-HEV RNA in g1-HEV clinical isolate-infected Huh7 cells and treated with PF670462 or AKTi-1/2, as indicated. Relative g1-HEV RNA values were calculated with respect to the value of DMSO-treated samples, normalized with values of RP, and represented as (mean ± SD) of three independent experiments. ( B ) Immunofluorescence image of ORF2 (red, upper panel) or ORF2 (red) and nucleus (blue; lower panel) in g1-HEV clinical isolate-infected Huh7 cells and treated with the indicated inhibitors for 48 hours. ( C ) Quantification of % ORF2-positive cells in five random fields, as represented in ( A ). Values are (mean ± SD) of five random fields. P values were calculated using Student's t -test. P < 0.05 was considered significant. *, **, ***, and **** represent P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

Article Snippet: The anti-CK1ε antibody (Catalog No. sc-365259) was from Santa Cruz Biotechnology (Texas, USA).

Techniques: Activity Assay, Infection, Quantitative RT-PCR, Immunofluorescence

Journal: mSystems

Article Title: Network controllability analysis reveals the antiviral potential of Etravirine against hepatitis E virus infection

doi: 10.1128/msystems.00438-25

Figure Lengend Snippet: Knockdown of CK1ε inhibits g1- and g3-HEV replication. ( A ) Percent viability in Huh7 cells transfected for 72 hours with NT-siRNA (non-targeting siRNA) or CK1ε siRNA, as indicated. The values of NT-siRNA-transfected samples were considered as 100%. ( B ) Western blot analysis of CK1ε (upper panel), CK1δ (middle panel), and GAPDH (lower panel) protein levels in Huh7 cells, transfected with NT-siRNA or CK1ε siRNA for 48 or 72 hours, as indicated. ( C ) RT-qPCR measurement of g1-HEV RNA in Huh7 cells expressing g1-HEV, transfected with NT-siRNA or CK1ε siRNA for 72 hours and treated with PF670462 or DMSO for 48 hours, as indicated. Relative g1-HEV RNA values were calculated with respect to the value of DMSO-treated samples, normalized with values of RP, and represented as (mean ± SD) of three independent experiments. ( D ) Measurement of Renilla -luciferase activity in Huh7 cells expressing g3-HEV-Luc-replicon and transfected with NT-siRNA or CK1ε siRNA for 72 hours and treated with PF670462 or DMSO for 48 hours, as indicated. Renilla -luciferase values were normalized to the cell viability values and represented as mean ± SD of three independent experiments. *, **, ***, and **** represent P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

Article Snippet: The anti-CK1ε antibody (Catalog No. sc-365259) was from Santa Cruz Biotechnology (Texas, USA).

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