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 stimulates drug‐induced apoptosis. (A) To assess the correlation of the expression of apoptosis‐related gene signature in resistant cells compared to sensitive cells, GSEA was used to analyse the gene expression profiles in chemoresistant cells (SGC‐R and BGC‐R) and their parental sensitive cells (SGC7901 and BGC823). (B) Apoptosis of SGC7901 and SGC‐R (left) or BGC823 and BGC‐R (right) under DDP (1.2 μg/ml) incubation for 24 h was measured by a PI/Annexin V double staining assay. (C) Apoptosis of SGC‐R (left) or BGC‐R (right) cells with ATF4 over‐expression and DDP treatment for 24 h was measured. (D) Apoptosis of SGC7901 (up) or BGC823 (down) cells with ATF4 knockdown and DDP treatment for 24 h was measured by a PI/Annexin V double staining assay. (E) The expression of apoptosis marker cleave‐PARP1 (C‐PARP1) and ATF4 in SGC7901 (left) or BGC823 (right) cells with ATF4 knockdown and DDP (1.2 μg/ml) treatment for 24 h was detected by western blotting

Article Snippet: The following antibodies were used for western blotting, or immuno‐precipitation (IP): ATF4 (rabbit, 11815), β‐TrCP (rabbit, 4394), β‐actin (rabbit, 4970), CDK1 (rabbit, 9111), CDK2 (rabbit, 18048), p‐eIF2α (rabbit, 3398), Myc (mouse, 2276) and cleaved PARP1 (C‐PARP1) (rabbit, 9541) were purchased from Cell Signaling Technology (Shanghai, China). p‐ATF4(S219) (rabbit, csb‐pa020025) was from CUSABIO (Wuhan, China).

Techniques: Expressing, Gene Expression, Incubation, Double Staining, Over Expression, Knockdown, Marker, Western Blot

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: Stabilisation of ATF4 reverses chemoresistance. (A) Cell viability of SGC‐R cells with DDP treatment, with/without D4476 (0, 25 or 50 μM) combination for 24 h was measured by MTS assay. (B) Apoptosis of SGC‐R cells with DDP treatment, with/without D4476 (25 μM) combination for 24 h was analysed by PI/Annexin V double staining. (C) Expression of apoptosis marker C‐PARP1 and ATF4 in SGC‐R cells with DDP treatment, with/without D4476 combination for 24 h was detected by western blotting. The short‐time and long‐time exposure results were shown as ATF4 (short) and ATF4 (long), respectively. Cell viability (D) and apoptosis (E) of SGC‐R cells with DDP treatment, with/without BTZ (1 μM) combination for 24 h was measured. (F) Expression of apoptosis marker C‐PARP1 and ATF4 in SGC‐R cells with DDP treatment, with/without BTZ (0.5 or 1 μM) combination for 24 h was detected by western blotting. The short‐time and long‐time exposure results were shown as ATF4 (short) and ATF4 (long), respectively. After ATF4 knockdown, cell viability of SGC‐R cells with DDP (5 μg/ml) treatment, with/without D4476 (25 μM) (G) or BTZ (1μM) (H) combination for 24 h was measured by MTS assay. (I) After ATF4 knockdown, expression of C‐PARP1 in SGC‐R cells with DDP (5 μg/ml) treatment, with/without D4476 (up) or BTZ (down) combination for 24 h was detected by western blotting

Article Snippet: The following antibodies were used for western blotting, or immuno‐precipitation (IP): ATF4 (rabbit, 11815), β‐TrCP (rabbit, 4394), β‐actin (rabbit, 4970), CDK1 (rabbit, 9111), CDK2 (rabbit, 18048), p‐eIF2α (rabbit, 3398), Myc (mouse, 2276) and cleaved PARP1 (C‐PARP1) (rabbit, 9541) were purchased from Cell Signaling Technology (Shanghai, China). p‐ATF4(S219) (rabbit, csb‐pa020025) was from CUSABIO (Wuhan, China).

Techniques: MTS Assay, Double Staining, Expressing, Marker, Western Blot, Knockdown

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 is downregulated to promote chemoresistance in gastric cancer. (A) The gene expression profiles were analysed by Gene Set Enrichment Analysis (GSEA) in chemoresistant cells (SGC‐R and BGC‐R) and their parental sensitive cells (SGC7901 and BGC823), assessing the correlation to ATF4 target gene signatures in resistant cells compared to sensitive cells, ‘R’ represent drug‐resistant cells, ‘S’ represent sensitive cells. (B) Expression of ATF4 in chemoresistant cells (SGC‐R and BGC‐R) and their parental sensitive cells (SGC7901 and BGC823) was detected by western blotting, and Actin was used as loading control. (C) Cell viability of SGC‐R cells with ATF4 over‐expression, and DDP treatment for 24 h was measured by MTS assay. (D) The clonal growth of SGC‐R cells with ATF4 over‐expression, and DDP treatment for 7 days was assayed in Matrigel. (E) Cell viability of SGC7901 cells with ATF4 knockdown, and DDP treatment for 24 h was measured by MTS assay. (F) The clonal growth of SGC7901 cells with ATF4 knockdown, and DDP treatment for 7 days was assayed in Matrigel

Article Snippet: The following antibodies were used for western blotting, or immuno‐precipitation (IP): ATF4 (rabbit, 11815), β‐TrCP (rabbit, 4394), β‐actin (rabbit, 4970), CDK1 (rabbit, 9111), CDK2 (rabbit, 18048), p‐eIF2α (rabbit, 3398), Myc (mouse, 2276) and cleaved PARP1 (C‐PARP1) (rabbit, 9541) were purchased from Cell Signaling Technology (Shanghai, China). p‐ATF4(S219) (rabbit, csb‐pa020025) was from CUSABIO (Wuhan, China).

Techniques: Gene Expression, Expressing, Western Blot, Control, Over Expression, MTS Assay, Knockdown

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: βTrCP‐enhanced ubiquitination‐dependent proteasomal degradation of ATF4 in chemoresistant cells. (A) Turnover of endogenous ATF4 in SGC7901 or SGC‐R cells under protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml) incubation was detected by western blotting using anti‐ATF4 antibody. (B) The relative grey value of ATF4 compared to Actin in ‘A’ was analysed by Image J, which was further normalised to the ‘0’ time point sample. The fitted curves were drawn with GraphPad software and the half‐life of ATF4 turnover was analysed and shown. (C) Expression of ATF4 in SGC‐R (up) or BGC‐R (down) with different protein degradation inhibitors (lysosome inhibitor: CQ, proteasome inhibitor: MG132 and calpain inhibitor: PD150606) treatment was determined by western blotting. The short‐time and long‐time exposure results were shown as ATF4 (short) and ATF4 (long), respectively. (D) After pre‐treated with MG132, ubiquitination of endogenous ATF4 in sensitive or resistant cells was detected by anti‐ATF4 immunoprecipitation (IP) and anti‐Ub immunoblot. ATF4 ubiquitination was normalised to immunoprecipitated ATF4, and the normalised ratio was shown. (E) βTrCP1‐mediated ATF4 ubiquitination was determined by an in vitro ubiquitination assay in HEK293T cells. (F) The interaction of ATF4 with βTrCP1 in SGC‐R (up) or BGC‐R (down) cells pre‐treated with MG132 was analysed by anti‐ATF4 co‐immunoprecipitation (co‐IP), followed by anti‐βTrCP1 and anti‐ATF4 immunoblot. (G) Expression of ATF4 in SGC‐R (left) or BGC‐R (right) with βTrCP1 knockdown was determined by western blotting. (H) Expression of ATF4 in SGC‐R (left) or BGC‐R (right) with Cullin1 knockdown was detected by western blotting

Article Snippet: The following antibodies were used for western blotting, or immuno‐precipitation (IP): ATF4 (rabbit, 11815), β‐TrCP (rabbit, 4394), β‐actin (rabbit, 4970), CDK1 (rabbit, 9111), CDK2 (rabbit, 18048), p‐eIF2α (rabbit, 3398), Myc (mouse, 2276) and cleaved PARP1 (C‐PARP1) (rabbit, 9541) were purchased from Cell Signaling Technology (Shanghai, China). p‐ATF4(S219) (rabbit, csb‐pa020025) was from CUSABIO (Wuhan, China).

Techniques: Ubiquitin Proteomics, Incubation, Western Blot, Software, Expressing, Immunoprecipitation, In Vitro, Co-Immunoprecipitation Assay, Knockdown

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: The following antibodies were used for western blotting, or immuno‐precipitation (IP): ATF4 (rabbit, 11815), β‐TrCP (rabbit, 4394), β‐actin (rabbit, 4970), CDK1 (rabbit, 9111), CDK2 (rabbit, 18048), p‐eIF2α (rabbit, 3398), Myc (mouse, 2276) and cleaved PARP1 (C‐PARP1) (rabbit, 9541) were purchased from Cell Signaling Technology (Shanghai, China). p‐ATF4(S219) (rabbit, csb‐pa020025) was from CUSABIO (Wuhan, China).

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