Journal: Inflammation

Article Title: KANK4 Regulates CXCL16 Glycosylation Through TMEM260 to Modulate Microglial Activation in Sepsis-associated Encephalopathy

doi: 10.1007/s10753-026-02481-y

Figure Lengend Snippet: KANK4 regulated CXCL16 glycosylation and microglial activation via TMEM260. Microglia were isolated from the hippocampus of mice in Sham, SAE, SAE + AAV-NC, and SAE + AAV-KANK4 groups, followed by the following analyses: (A) IHC was performed to detect CXCL16 protein expression. Microglia were isolated from the Sham and SAE groups for the following assays: (B) Western blot analysis was conducted to assess CXCL16 glycosylation levels ( n = 3). (C) Co-IP was carried out to examine the interaction between TMEM260 and CXCL16. BV2 microglial cells were treated with LPS to establish a cell model. Control and LPS groups were subjected to the following analyses: (D) Western blot analysis was applied to detect CXCL16 expression using both anti-CXCL16 and anti-His-CXCL16 antibodies ( n = 3). (E) Co-IP was conducted to validate the interaction between TMEM260 and CXCL16. (F) Western blot was used to assess O-mannosylation of CXCL16 protein in LPS-treated microglia ( n = 3). (G) Effect of CXCL16 glycosylation site mutations on its glycosylation level. Wild-type (WT) and site-specific mutant (S137A, S117A, S139A) CXCL16 plasmids were transfected into microglia. CXCL16 glycosylation was assessed by Western blot ( n = 3). (H) Impact of CXCL16 glycosylation site mutation (S117A) on the expression of inflammatory factors. In an LPS-induced microglial inflammation model, cells were transfected with either WT or glycosylation-site mutant (S117A) CXCL16 plasmid. The levels of inflammatory cytokines IL-1β, IL-6, and TNF-α in the cells were measured by ELISA. BV2 cells were transfected for TMEM260 overexpression or knockdown. The groups included Control, si-NC, si-TMEM260, NC-OE, and TMEM260-OE, and the following analyses were performed: (I) Western blot analysis was used to detect the expression of TMEM260 and CXCL16 proteins ( n = 3). (J) CHX assay was performed to evaluate the stability of CXCL16 protein. LPS-treated BV2 cells were used to conduct KANK4 overexpression and TMEM260 knockdown rescue experiments, with six groups: Control, LPS, LPS + OE-NC, LPS+KANK4-OE, LPS+KANK4-OE + si-NC, and LPS+KANK4-OE + si-TMEM260. The following analysis was conducted: (K) Western blot analysis was performed to measure CXCL16 protein expression in BV2 cells ( n = 3). For overexpression of TMEM260 and CXCL16 in LPS-treated BV2 cells, the groups included: Control, LPS, LPS + OE-NC, LPS+TMEM260-OE, LPS+TMEM260-OE + OE-NC, and LPS+TMEM260-OE+CXCL16-OE. The following analyses were carried out: (L) Western blot analysis was conducted to detect TMEM260 and CXCL16 protein levels in each group ( n = 3). (M) CCK-8 assay was applied to evaluate microglial cell viability across groups. (N) IF staining was performed to assess the expression of Iba-1 (green) and CD11b (red), indicating microglial activation status (Scale bar = 50 μm). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. LPS/LPS + OE-NC/LPS+TMEM260-OE + OE-NC; ns, no significant difference vs. LPS/Control

Article Snippet: Protein A/G agarose beads (Beyotime) and anti-CXCL16 (Proteintech, 60123-1-Ig, 1:1000) or anti-His (Abcam, ab9108, 1:1000) antibodies were incubated overnight at 4 °C to immunoprecipitate CXCL16 and His-CXCL16 protein complexes, respectively.

Techniques: Glycoproteomics, Activation Assay, Isolation, Expressing, Western Blot, Co-Immunoprecipitation Assay, Control, Mutagenesis, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown, CCK-8 Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Inflammation-Driven Secretion Potential Is Upregulated in Osteoarthritic Fibroblast-Like Synoviocytes

doi: 10.3390/ijms231911817

Figure Lengend Snippet: Chemokine secretion levels by HFLS and HFLS-OA stimulated with TNFα or LPS measured using the ELISA method. Upregulation of chemokine level secretion measured in media using the ELISA method. Cells were stimulated for 24 h with 10 ng/mL of TNFα ( A , C , E ) or LPS ( B , D , F ). Afterward, the media were collected, and CXCL6 ( A , B ), CXCL10 ( C , D ), and CXCL16 ( E , F ) levels were determined. Data are presented as pg/mL ± SEM and analyzed with two-way ANOVA followed by Tukey’s post hoc test. ** 0.01 > p > 0.001; *** p < 0.001 vs. control group. ## 0.01 > p > 0.001; ### p < 0.001 HFLS vs. HFLS-OA stimulated groups. HFLS, human fibroblast-like synoviocytes; HFLS-OA, osteoarthritic human fibroblast-like synoviocytes; TNFα, tumor necrosis factor alpha; LPS, lipopolysaccharide; ELISA, enzyme linked immunosorbent assay; CXCL6, chemokine (C-X-C motif) ligand 6; CXCL10, chemokine (C-X-C motif) ligand 10; CXCL16, chemokine (C-X-C motif) ligand 16.

Article Snippet: The protein levels of the CXCL6 (Human GCP2 (Granulocyte Chemotactic Protein 2) ELISA Kit, ELK Biotechnology, Wuhan, China), CXCL10 (Human IP-10/CXCL10 ELISA Kit, Elabscience, Wuhan, China), CXCL16 (Human CXCL16 ELISA Kit, Elabscience, Wuhan, China), ANGPTL1 (Human ANGPTL1 ELISA Kit, ELK Biotechnology, Wuhan, China), FGF5 (Human FGF5 ELISA Kit, ELK Biotechnology, Wuhan, China) and IGF-2 (Human IGF-2 ELISA Kit, Elabscience, Wuhan, China) were measured using commercially available enzyme-linked immunosorbent assay kits according to the manufacturers’ instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Molecular medicine reports

Article Title: Erythropoietin ameliorates renal interstitial fibrosis via the inhibition of fibrocyte accumulation.

doi: 10.3892/mmr.2015.3157

Figure Lengend Snippet: Figure 5. CXCL12, CCL21 and CXCL16 expression analysis. (A) Representative western blots for CXCL12, CCL21 and CXCL16. Semi‑quantitative analysis of protein expression levels of (B) CXCL12, (C) CCL21 and (D) CXCL16. Actin was used as an internal control. *P<0.05 vs. Sh; †P<0.05 vs. U+V and ‡P>0.05 vs. U+V. UUO, unilateral ureteral obstruction; Sh, control; U+V, UUO+vehicle; U+E1, UUO treated with 300 U/kg rhEPO; rhEPO, recombinant human erythropoietin; U+E2, UUO treated with 1,000 U/kg rhEPO; CXCL16, CXC chemokine ligand 16; CCL21, CC chemokine ligand 21.

Article Snippet: Proteins were electrophoretically transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) which were subsequently incubated with antibodies specific for α‐SMA (1:500), collagen I (1:1,000), fibronectin (1:400), CXCL12 (1:1,000) , CCL21 (1:600), CXCL16 (1:500) and β-actin (1:1,000), followed by incubation with secondary antibody conjugated with IRDye® infrared dye (Rockland Immunochemicals, Inc.).

Techniques: Expressing, Western Blot, Control, Recombinant

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) CXCL16 mRNA expression in the kidney during MuPyV infection. Expression is shown as fold change relative to sham infected samples. Data are from three independent experiments (n = 10-11). (B) Expression of NKCC2 [marks the ascending loop of Henle ], CD8, and CXCL16 in epithelia in sham-infected and 8 dpi kidneys; bottom right photomicrographs are merged images. Representative of two independent experiments. (C) CXCL16 expression in sham-infected, 4 dpi, and 8 dpi kidney lysates (left). Western blot image is representative of two independent experiments with each lane indicating protein lysate from kidneys of individual mice. Protein band intensity quantification for sCXCL16 was normalized to β-actin and analyzed by ImageLab and normalized to the loading control (right). Data are combined from two independent experiments (n = 3-5). (D) Experimental design of in vivo CXCL16 mAb administration. Mice were administered 250 µg of CXCL16 mAb or control rat IgG every two days from days 4-14 post infection and euthanized at 15 dpi. Numbers of CD45 mAb i.v.-negative, CD8 + CD44 + D b -LT359 tetramer + T cells and CD4 + CD44 + T cells in kidneys and spleens of infected mice given anti-CXCL16 or control rat IgG. Data were analyzed by one-way ANOVA (A and C) and by multiple Mann-Whitney tests (D).

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Infection, Western Blot, Control, In Vivo, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) Dense lymphoplasmacytic infiltrate surrounding and infiltrating cortical tubules (H&E, x400). (B) Immunohistochemical staining for Large T antigen in nuclei of tubular epithelium (x250). (C) Expression of CXCR6 on infiltrating CD8 + cells. Photomicrographs (right) are enlarged images in the white squares (left); bottom right are merged images. No 1 o , no primary antibody. (D) Log-fold change of CXCL16 and CXCR6 across four independent studies, each comparing KTx biopsies from patients with PVAN and stable graft function. The error bars indicate the 95% confidence interval of log-fold change, with horizontal dashed grey line indicating log-fold change = 0, or no difference. GSE120495 is an RNA-seq study, while remaining studies are microarray based. Given the heterogeneity of the data, a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. The combined p-values for CXCL16 = 0.000337 and CXCR6 = 2.72x10 -05 . ns, non-significant at adjusted p-value > 0.05; *, statistical significance at adjusted p-value ≤ 0.05; **, statistical significance at adjusted p-value ≤ 0.01; ***, statistical significance at adjusted p-value ≤ 0.001.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Immunohistochemical staining, Staining, Expressing, RNA Sequencing, Microarray

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: Summary of differential gene expression of CXCL16 and CXCR6 in four studies of KTx biopsies with PVAN. Because of the heterogeneity of the data (i.e., one RNA-seq, three microarray), a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. P-values significant after false discover rate (FDR) correction are bolded and italicized in the Adj. P-value column. Cauchy combined p-values < 0.05 are bolded and italicized.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Gene Expression, Microarray