Journal: Genes & Diseases

Article Title: Chloride channel accessory 4 suppresses stem cell-like properties of colorectal cancer and enhances anti-PD-1 immunotherapy

doi: 10.1016/j.gendis.2025.101859

Figure Lengend Snippet: CLCA4 suppressed colorectal cancer stem cell expansion by interacting with vimentin to suppress FAK signaling pathways. (A) Western blotting analysis of FAK and p-FAK protein levels in control and CLCA4-overexpressing colorectal cancer (CRC) cells. Right panels: Quantification of protein expression ratio. (B) Western blotting analysis of stemness-related proteins and p-FAK in CLCA4-overexpressing cells treated with or without FAK agonist. Lower panels: Quantification of protein expression ratio. (C) Tumorsphere formation assay was performed to examine the tumorsphere formation ability in CLCA4-overexpressing cells treated with or without FAK agonist. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation). (D) Immunoprecipitation and IgG samples were analyzed by mass spectrometry. Proteins with unused >1.3 were filtered out, and keratin was removed. A total of 336 proteins were identified, including 334 proteins in immunoprecipitation samples and 4 proteins in IgG samples. (E) The immunoprecipitates of CLCA4 were purified using anti-Flag antibody and separated with SDS-PAGE, and the presence of vimentin was analyzed by Western blotting. Normal IgG was used as the negative control. (F) The immunoprecipitates of vimentin were purified using anti-HA antibody and separated with SDS-PAGE, and the presence of CLCA4 was analyzed by Western blotting. Normal IgG was used as the negative control. (G) The differences in protein levels (vimentin, Bmi-1, and p-FAK) among CRC cells transfected with different plasmids were analyzed by Western blotting. Right panels: Quantification of protein expression ratio. (H) Tumorsphere formation assay was performed to examine the tumorsphere formation ability among CRC cells transfected with different plasmids. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation).

Article Snippet: After 7 days, mice were intraperitoneally treated with either an in vivo blocking antibody against mouse PD-1 (Clone: 29F.1A2, BioXcell, Cat# BP0273) or a rat IgG2a isotype control antibody (Clone: 2A3, BioXcell, Cat# BP0089).

Techniques: Protein-Protein interactions, Western Blot, Control, Expressing, Tube Formation Assay, Standard Deviation, Immunoprecipitation, Mass Spectrometry, Purification, SDS Page, Negative Control, Transfection

Journal: The FASEB Journal

Article Title: Lymphocyte Antigen 6G Mediates Vagotomy‐Associated Reduction in Body Weight

doi: 10.1096/fj.202600151RR

Figure Lengend Snippet: Temporary extracellular Ly6G depletion effect on VX‐associated reduction of eWAT weight. (A) Flow cytometry analysis of the frequency of extracellular Ly6G and CD11b double positive cells following one intraperitoneal injection of either anti‐Ly6G or vehicle (PBS). Bars show the % ± SEM of CD11b + Ly6G + in vehicle ( n = 4–5) and at 1 ( n = 2), 2 ( n = 4), 5 ( n = 4), and 9 ( n = 3) days following injection in eWAT, blood, and bone marrow (one‐way ANOVA, Šídák's multiple comparisons test). (B) Schematic diagram depicting the experimental setup: Wild‐type mice were intraperitoneally injected once with anti‐Ly6G antibody or IgG2a antibody 2 days before sham or VX surgery, and tissue was collected 7 days following surgery. (C) Flow cytometry analysis of CD11b + Ly6G + cells in eWAT ( n = 3) following VX or sham. Bars show the proportion of cells from CD45 + (one‐way ANOVA, uncorrected Fisher's LSD). (D) Correlation between extracellular and intracellular expression of Ly6G in flow cytometry analysis. Circles represent each sample stained for both extracellular and intracellular Ly6G in separate fluorescent channels (Pearson r correlation). (E) The mice were weighed daily. The graph shows the difference in body weight (g) of the mice from day 0 (before surgery) of each experimental group ( n = 3) in g ± SEM (two‐way ANOVA, Tukey's multiple comparisons test—Significant differences between experimental groups at each time point are indicated with a, b, and c, and the detailed description can be found in Table ). (F) eWAT weight ( n = 3) was recorded at 7 days following VX or sham surgery. The bars show the relative eWAT weight to sham eWAT weight in % ± SEM (one‐way ANOVA, uncorrected Fisher's LSD). (G) Mice were kept in separate cages according to experimental groups: Sham+IgG2a, VX + IgG2a, sham+anti‐Ly6G, VX + anti‐Ly6G ( n = 3). The food for each cage was weighed at the same time point daily. The curve shows the grams of food consumed per day per cage in g. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. VX, Vagotomy; eWAT, epididymal white adipose tissue.

Article Snippet: To ligate Ly6G surface epitopes, male C57BL/6 mice were intraperitoneally injected with InVivo Mab anti‐mouse Ly6G (100 ug/100 uL) (Bioxcell, #BE0075) or for control InVivoMAb rat IgG2a (anti‐trinitrophenol) isotype (100 ug/100 uL) (Bioxcell, #BE0089) antibodies 2 days before the sham or VX surgery.

Techniques: Flow Cytometry, Injection, Expressing, Staining

Journal: The FASEB Journal

Article Title: Lymphocyte Antigen 6G Mediates Vagotomy‐Associated Reduction in Body Weight

doi: 10.1096/fj.202600151RR

Figure Lengend Snippet: Extracellular Ly6G depletion attenuated VX‐mediated reduction of body weight. (A) Schematic of the experimental setup: Wild‐type mice were intraperitoneally injected with anti‐Ly6G antibody or IgG2a antibody 2 days before, as well as 3 and 5 days following sham or VX surgery, and tissue was collected 7 days following surgery. (B–C) Flow cytometry analysis of CD11b + Ly6G + cells in eWAT ( n = 6) and blood ( n = 6) following VX or sham surgery, with IgG2a or anti‐Ly6G treatment. Bars show the proportion of cells from CD45 + (One‐way Anova, Uncorrected Fisher's LSD). (D–E) Flow cytometry analysis of intracellular Ly6G + cells in eWAT ( n = 3) and blood ( n = 3) following VX or sham surgery, with IgG2a or anti‐Ly6G treatment. Bars show the proportion of cells from CD45 + (One‐way Anova, Uncorrected Fisher's LSD). (F–G) Flow cytometry analysis of CD11b + (IN)Ly6G − F4/80 + cells in eWAT ( n = 3) and blood ( n = 3) following VX or sham surgery, with IgG2a or anti‐Ly6G treatment. Bars show the proportion of cells from CD45 + (One‐way Anova, Uncorrected Fisher's LSD). (H) eWAT weight ( n = 6) was recorded at 7 days following VX or sham surgery. The bars depict the relative eWAT weight to sham eWAT weight in % ± SEM (one‐way ANOVA, uncorrected Fisher's LSD). (I) The mice were weighed daily. The graph shows the difference in body weight of the mice from day 0 (before initiation of surgery) of each experimental group ( n = 6) in % ± SEM (two‐way ANOVA, Tukey's multiple comparisons test—Significant differences between experimental groups at each time point are indicated with a, b, and c, and the detailed description can be found in Table ). ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. VX, Vagotomy; eWAT, epididymal white adipose tissue; BM, bone marrow; SVCs, stromal vascular cells; intracellular (IN).

Article Snippet: To ligate Ly6G surface epitopes, male C57BL/6 mice were intraperitoneally injected with InVivo Mab anti‐mouse Ly6G (100 ug/100 uL) (Bioxcell, #BE0075) or for control InVivoMAb rat IgG2a (anti‐trinitrophenol) isotype (100 ug/100 uL) (Bioxcell, #BE0089) antibodies 2 days before the sham or VX surgery.

Techniques: Injection, Flow Cytometry