bottom chamber contained serum free rpmi sdf 1 cxcl12  (R&D Systems)

Journal: Advanced Science

Article Title: PDIA6–SCD1 Axis Rewires Lipid Metabolism to Drive Gastric Cancer Progression

doi: 10.1002/advs.75923

Figure Lengend Snippet: CAF‐derived CXCL12 reinforces PDIA6 expression through CXCR4‐STAT3 signaling in GC. (A,B) Immunofluorescence staining and western blot analysis of the CAF markers FAP and α‐SMA in CAFs and paired NFs. (C,D) qRT‐PCR and western blot analyses of PDIA6 mRNA and protein expression in GC cells treated with CAF‐conditioned medium (CAF‐CM) or NF‐conditioned medium (NF‐CM). (E,F) Transwell migration and EdU incorporation assays evaluating the effects of PDIA6 knockdown on CAF‐CM‐induced GC cell phenotypes. (G) Western blot analysis of STAT3 and PDIA6 in GC cells treated with or without CAF‐CM and transfected with control or STAT3 siRNA. (H) RNA‐seq analysis identifying CXCL12 as a highly upregulated secreted factor in CAFs relative to NFs. (I) Correlation analysis of CXCL12 and PDIA6 expression in the TCGA‐STAD cohort. (J,K) Single‐cell RNA‐seq analysis showing fibroblast‐specific expression of CXCL12 in GC tissues. (L,M) Western blot analysis of CXCL12 protein expression and ELISA of CXCL12 secretion in CAFs, NFs, and GC cells. (N) Western blot analysis of p‐STAT3 (Tyr705), total STAT3, and PDIA6 in GC cells pretreated with AMD3100 (2.5 µg/mL) for 30 min and then incubated with or without CAF‐CM for 24 h. (O) Western blot analysis of p‐STAT3 (Tyr705), total STAT3, and PDIA6 in GC cells treated with CAF‐CM together with IgG control or αCXCL12 (10 µg/mL, 24 h). (P) Western blot analysis of p‐STAT3 (Tyr705) and PDIA6 in GC cells treated with CAF‐CM or recombinant human CXCL12 (rhCXCL12, 25/50/100 ng/mL) for 24 h, with or without AMD3100 pretreatment. (Q,R) Cell proliferation and migration assays evaluating the effects of rhCXCL12 stimulation and αCXCL12 neutralization on GC cell behavior. Data are presented as mean ± SD.

Article Snippet: CXCL12 levels in cell culture supernatants were quantified using a human CXCL12 ELISA kit (RK00266, Abclonal, China) according to the manufacturer's instructions.

Techniques: Derivative Assay, Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Migration, Knockdown, Transfection, Control, RNA Sequencing, Single Cell, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Neutralization

Journal: Advanced Science

Article Title: PDIA6–SCD1 Axis Rewires Lipid Metabolism to Drive Gastric Cancer Progression

doi: 10.1002/advs.75923

Figure Lengend Snippet: CAF‐derived CXCL12 reinforces PDIA6 expression through CXCR4‐STAT3 signaling in GC. (A,B) Immunofluorescence staining and western blot analysis of the CAF markers FAP and α‐SMA in CAFs and paired NFs. (C,D) qRT‐PCR and western blot analyses of PDIA6 mRNA and protein expression in GC cells treated with CAF‐conditioned medium (CAF‐CM) or NF‐conditioned medium (NF‐CM). (E,F) Transwell migration and EdU incorporation assays evaluating the effects of PDIA6 knockdown on CAF‐CM‐induced GC cell phenotypes. (G) Western blot analysis of STAT3 and PDIA6 in GC cells treated with or without CAF‐CM and transfected with control or STAT3 siRNA. (H) RNA‐seq analysis identifying CXCL12 as a highly upregulated secreted factor in CAFs relative to NFs. (I) Correlation analysis of CXCL12 and PDIA6 expression in the TCGA‐STAD cohort. (J,K) Single‐cell RNA‐seq analysis showing fibroblast‐specific expression of CXCL12 in GC tissues. (L,M) Western blot analysis of CXCL12 protein expression and ELISA of CXCL12 secretion in CAFs, NFs, and GC cells. (N) Western blot analysis of p‐STAT3 (Tyr705), total STAT3, and PDIA6 in GC cells pretreated with AMD3100 (2.5 µg/mL) for 30 min and then incubated with or without CAF‐CM for 24 h. (O) Western blot analysis of p‐STAT3 (Tyr705), total STAT3, and PDIA6 in GC cells treated with CAF‐CM together with IgG control or αCXCL12 (10 µg/mL, 24 h). (P) Western blot analysis of p‐STAT3 (Tyr705) and PDIA6 in GC cells treated with CAF‐CM or recombinant human CXCL12 (rhCXCL12, 25/50/100 ng/mL) for 24 h, with or without AMD3100 pretreatment. (Q,R) Cell proliferation and migration assays evaluating the effects of rhCXCL12 stimulation and αCXCL12 neutralization on GC cell behavior. Data are presented as mean ± SD.

Article Snippet: CXCL12 levels in cell culture supernatants were quantified using a human CXCL12 ELISA kit (RK00266, Abclonal, China) according to the manufacturer's instructions.

Techniques: Derivative Assay, Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Migration, Knockdown, Transfection, Control, RNA Sequencing, Single Cell, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Neutralization