Journal: Frontiers in Immunology

Article Title: Endoplasmic reticulum stress-induced CRELD2 promotes APMAP-mediated activation of TGF-β/SMAD and NF-κB pathways in esophageal squamous cell carcinoma

doi: 10.3389/fimmu.2025.1616201

Figure Lengend Snippet: CRELD2 increases the membrane localization of APMAP. (A) The interaction between CRELD2 and APMAP was detected in the indicated cells by co-immunoprecipitation assay using anti-CRELD2 followed by immunoblot with anti-APMAP or anti-CRELD2. (B) The expression profile of APMAP was systematically analyzed in tumor tissues and paired normal counterparts using the UALCAN database. (C, D) qRT-PCR (C) and Western blot (D) analysis of APMAP expression in HEEC and ESCC cell lines (TE1, KYSE150, and KYSE170). (E) qRT-PCR and Western blot analysis of CRELD2 and APMAP expression in TE1 and KYSE150 cells with CRELD2 overexpression or knockdown. (F) qRT-PCR assay of KYSE150 and TE1 cells treated with Tg (+) or DMSO (–) for 12 h. (G) The subcellular localization of APMAP in KYSE150 cells treated as indicated was measured by immunofluorescence staining. Nuclei are stained with DAPI. Arrows indicate plasma membrane localization of APMAP. Scale bar, 25 μm. (H) Western blot showing APMAP expression in the membrane fraction from each treatment condition. The protein levels were quantified by band densitometry. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, n.s., not significant.

Article Snippet: Antibodies against APMAP (25953-1-AP, Proteintech, Wuhan, China), ZEB2 (67514-1-Ig, Proteintech, Wuhan, China), XBP1s (647501, Biolegend, San Diego, CA, USA), TGFBR1 (ab235578, Abcam, Cambridge, UK), CRELD2 (sc-365168, Santa Cruz Biotechnology, Dallas, Texas, USA), TAB2 (A9867, ABclonal Technology, Wuhan, China), TAB1 (RT1603, HuaBio, Hangzhou, China) were also respectively used.

Techniques: Membrane, Co-Immunoprecipitation Assay, Western Blot, Expressing, Quantitative RT-PCR, Over Expression, Knockdown, Immunofluorescence, Staining, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Endoplasmic reticulum stress-induced CRELD2 promotes APMAP-mediated activation of TGF-β/SMAD and NF-κB pathways in esophageal squamous cell carcinoma

doi: 10.3389/fimmu.2025.1616201

Figure Lengend Snippet: APMAP activates TGF-β/SMAD and NF-κB signaling during ER stress-induced EMT and proliferation. (A) qRT-PCR analysis of EMT and proliferation marker expression in KYSE150 and TE1 cells transfected with control siRNA (si-NC) or si-APMAP. (B) Western blot analysis of the indicated proteins expression in KYSE150 and TE1 cells transfected with si-NC or si-APMAP. (C) The protein expression of key genes of the TGF-β/SMAD, NF-κB, IL6/STAT3, Wnt/β-catenin, and PI3K/AKT signaling pathways in APMAP-knockdown KYSE150 and TE1 cells was examined by Western blot assay. (D) The effect of APMAP knockdown on the protein expression of SMAD2/3 and p-SMAD2/3 in Tg (100 nM, 12 h) treated KYSE150 and TE1 cells. (E, F) Co-immunoprecipitation assay was conducted with anti-TGFBR1 or anti-APMAP antibodies followed by immunoblot with anti-TAK1, anti-TGFBR1 or anti-APMAP in the indicated cells. (G) Western blot analysis of p-TAK1 and TAK1 expression in APMAP-manipulated KYSE150 cells and TE1 cells (overexpression or knockdown). (H) Co-immunoprecipitation assay was conducted with anti-APMAP antibody followed by immunoblot with anti-TAB1, anti-TAB2, or anti-APMAP in the indicated cells. (I) The proposed model for ER stress-induced CRELD2 promotes membrane the localization of APMAP, which increases its interaction with TAK1, TAB1, and TAB2, thereby promoting the activation of the TGF-β/SMAD and NF-κB pathways and inducing the EMT and proliferation of ESCC cells, is shown. The protein levels were quantified by band densitometry. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Antibodies against APMAP (25953-1-AP, Proteintech, Wuhan, China), ZEB2 (67514-1-Ig, Proteintech, Wuhan, China), XBP1s (647501, Biolegend, San Diego, CA, USA), TGFBR1 (ab235578, Abcam, Cambridge, UK), CRELD2 (sc-365168, Santa Cruz Biotechnology, Dallas, Texas, USA), TAB2 (A9867, ABclonal Technology, Wuhan, China), TAB1 (RT1603, HuaBio, Hangzhou, China) were also respectively used.

Techniques: Quantitative RT-PCR, Marker, Expressing, Transfection, Control, Western Blot, Protein-Protein interactions, Knockdown, Co-Immunoprecipitation Assay, Over Expression, Membrane, Activation Assay

Journal: Frontiers in Immunology

Article Title: Endoplasmic reticulum stress-induced CRELD2 promotes APMAP-mediated activation of TGF-β/SMAD and NF-κB pathways in esophageal squamous cell carcinoma

doi: 10.3389/fimmu.2025.1616201

Figure Lengend Snippet: CRELD2 increases the membrane localization of APMAP. (A) The interaction between CRELD2 and APMAP was detected in the indicated cells by co-immunoprecipitation assay using anti-CRELD2 followed by immunoblot with anti-APMAP or anti-CRELD2. (B) The expression profile of APMAP was systematically analyzed in tumor tissues and paired normal counterparts using the UALCAN database. (C, D) qRT-PCR (C) and Western blot (D) analysis of APMAP expression in HEEC and ESCC cell lines (TE1, KYSE150, and KYSE170). (E) qRT-PCR and Western blot analysis of CRELD2 and APMAP expression in TE1 and KYSE150 cells with CRELD2 overexpression or knockdown. (F) qRT-PCR assay of KYSE150 and TE1 cells treated with Tg (+) or DMSO (–) for 12 h. (G) The subcellular localization of APMAP in KYSE150 cells treated as indicated was measured by immunofluorescence staining. Nuclei are stained with DAPI. Arrows indicate plasma membrane localization of APMAP. Scale bar, 25 μm. (H) Western blot showing APMAP expression in the membrane fraction from each treatment condition. The protein levels were quantified by band densitometry. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, n.s., not significant.

Article Snippet: The cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 15 min. After washing with PBS, the cells were blocked with 2% bovine serum albumin, and then incubated with the rabbit anti-human polyclonal antibody APMAP (Cusabio, Wuhan, China, dilution at 1:100) at 4°C for 24 h. Finally, the cells were incubated with a fluorescent secondary antibody for 1 h at room temperature.

Techniques: Membrane, Co-Immunoprecipitation Assay, Western Blot, Expressing, Quantitative RT-PCR, Over Expression, Knockdown, Immunofluorescence, Staining, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Endoplasmic reticulum stress-induced CRELD2 promotes APMAP-mediated activation of TGF-β/SMAD and NF-κB pathways in esophageal squamous cell carcinoma

doi: 10.3389/fimmu.2025.1616201

Figure Lengend Snippet: Silencing of APMAP alleviates the malignant phenotype of ESCC cells. (A) The transfection efficiency of si-APMAP in KYSE150 and TE1 cells was detected by qRT-PCR method. (B, C) The proliferation ability of the indicated cells was assessed by MTS (B) and colony formation (C) assays. (D, E) Cell migration and invasion of the indicated cells were verified through wound healing (D) and transwell invasion (E) assays. Scale bar, 100 μm. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: The cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 15 min. After washing with PBS, the cells were blocked with 2% bovine serum albumin, and then incubated with the rabbit anti-human polyclonal antibody APMAP (Cusabio, Wuhan, China, dilution at 1:100) at 4°C for 24 h. Finally, the cells were incubated with a fluorescent secondary antibody for 1 h at room temperature.

Techniques: Transfection, Quantitative RT-PCR, Migration

Journal: Frontiers in Immunology

Article Title: Endoplasmic reticulum stress-induced CRELD2 promotes APMAP-mediated activation of TGF-β/SMAD and NF-κB pathways in esophageal squamous cell carcinoma

doi: 10.3389/fimmu.2025.1616201

Figure Lengend Snippet: APMAP activates TGF-β/SMAD and NF-κB signaling during ER stress-induced EMT and proliferation. (A) qRT-PCR analysis of EMT and proliferation marker expression in KYSE150 and TE1 cells transfected with control siRNA (si-NC) or si-APMAP. (B) Western blot analysis of the indicated proteins expression in KYSE150 and TE1 cells transfected with si-NC or si-APMAP. (C) The protein expression of key genes of the TGF-β/SMAD, NF-κB, IL6/STAT3, Wnt/β-catenin, and PI3K/AKT signaling pathways in APMAP-knockdown KYSE150 and TE1 cells was examined by Western blot assay. (D) The effect of APMAP knockdown on the protein expression of SMAD2/3 and p-SMAD2/3 in Tg (100 nM, 12 h) treated KYSE150 and TE1 cells. (E, F) Co-immunoprecipitation assay was conducted with anti-TGFBR1 or anti-APMAP antibodies followed by immunoblot with anti-TAK1, anti-TGFBR1 or anti-APMAP in the indicated cells. (G) Western blot analysis of p-TAK1 and TAK1 expression in APMAP-manipulated KYSE150 cells and TE1 cells (overexpression or knockdown). (H) Co-immunoprecipitation assay was conducted with anti-APMAP antibody followed by immunoblot with anti-TAB1, anti-TAB2, or anti-APMAP in the indicated cells. (I) The proposed model for ER stress-induced CRELD2 promotes membrane the localization of APMAP, which increases its interaction with TAK1, TAB1, and TAB2, thereby promoting the activation of the TGF-β/SMAD and NF-κB pathways and inducing the EMT and proliferation of ESCC cells, is shown. The protein levels were quantified by band densitometry. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: The cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 15 min. After washing with PBS, the cells were blocked with 2% bovine serum albumin, and then incubated with the rabbit anti-human polyclonal antibody APMAP (Cusabio, Wuhan, China, dilution at 1:100) at 4°C for 24 h. Finally, the cells were incubated with a fluorescent secondary antibody for 1 h at room temperature.

Techniques: Quantitative RT-PCR, Marker, Expressing, Transfection, Control, Western Blot, Protein-Protein interactions, Knockdown, Co-Immunoprecipitation Assay, Over Expression, Membrane, Activation Assay