Journal: Frontiers in Genetics

Article Title: Activation of CCL21-GPR174/CCR7 on cardiac fibroblasts underlies myocardial ischemia/reperfusion injury

doi: 10.3389/fgene.2022.946524

Figure Lengend Snippet: mRNA and protein expression of key genes and related receptors. (A) mRNA expression of CCL21, XCR1, CXCL13, CASR, and EDN1. (B) Protein expression of CCL21, CXCL13, and XCR1. (C) Protein expression of GPR174/CCR7 (the receptors of CCL21) and CXCR5 (the receptors of CXCL13). n = 3–6. * p < 0.05, ** p < 0.01 vs. the sham group. I/R, ischemia reperfusion.

Article Snippet: The primary antibodies were as follows: goat anti-CCL21 (1:1000; R&D Systems, Minneapolis, MN), rabbit anti-XCR1 (1:1000; Novus Biologicals, Littleton, CO), goat anti-CXCL13 (1:1000; R&D Systems, Minneapolis, MN), goat anti-CCR7 (1:1000; Novus Biologicals, Littleton, CO), rabbit anti-GPR174 (1:1000; Biorbyt, Cambridge, United Kingdom), rabbit anti-CXCR5 (1:1000; Absin, Shanghai, China), and rabbit anti-β-tubulin (1:5000; Cell Signaling Technology, Beverly, MA).

Techniques: Expressing

Journal: iScience

Article Title: Tertiary lymphoid structure and decreased CD8 + T cell infiltration in minimally invasive adenocarcinoma

doi: 10.1016/j.isci.2022.103883

Figure Lengend Snippet:

Article Snippet: Rabbit anti-CXCR5 monoclonal antibody (D6L3C) , CST , Cat#: 72172S.

Techniques: Recombinant, Software

Journal: iScience

Article Title: Tertiary lymphoid structure and decreased CD8 + T cell infiltration in minimally invasive adenocarcinoma

doi: 10.1016/j.isci.2022.103883

Figure Lengend Snippet: TLS formation in MIA tumors (A) The schema of TLS formation. The first stage is B & T cell aggregation. The second stage is follicle formation with T-zone and B-zone. The third stage is ectopic germinal center formation with formation with follicular dendritic cell in light-zone. (B) Hierarchical clustering of Z score normalized gene expression of B cell infiltration markers (CD19 and CD20), follicle phenotype markers (CXCR5 and CXCL13), and lymphocyte homing signal markers (CCR7 and CCL19). Color of the top bar represents tumor (red) or adjacent normal (blue) tissue. Correlations between expression levels from these six TLS markers and immune cell abundance corrected by tumor purity are shown in the right bar. p Values are calculated using Spearman correlation; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns p > 0.05. (C) Typical observation field of MIA six-plex TLS panel staining. The merged-channel multispectral image was captured after 5-cycle TSA staining and DAPI counterstain, revealing a classic lymphoid structure residing in MIA stromal tissue. Single channels are shown for the area bounded by the white rectangle. Fake colors are assigned as DAPI: blue, CD8: yellow, CXCR5: pink, CD4: green, CD20: red, CD35: cyan. Tfh cells are highlighted with white arrows (lower right corner images). (D–G) Paired comparisons of CD20 + B cell density, CD4 + T cell density (E), CD35 + follicle dendritic cell density (F), and CD8 + T cell density (G) between non-follicle (Non-Fol) and TLS areas

Article Snippet: Rabbit anti-CXCR5 monoclonal antibody (D6L3C) , CST , Cat#: 72172S.

Techniques: Expressing, Staining

Journal: iScience

Article Title: Tertiary lymphoid structure and decreased CD8 + T cell infiltration in minimally invasive adenocarcinoma

doi: 10.1016/j.isci.2022.103883

Figure Lengend Snippet: Treg recruitment as potential mechanism of decreased CD8 + T cell infiltration in tertiary lymphoid structures (A) Comparison of immune suppressive cell infiltration estimated from XCELL between MIA tumor and adjacent normal tissues. p values are calculated using Mann-Whitney U test; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns p > 0.05. (B) Plot of Tfr/Tfh marker ratio vs CD8 + T cell infiltration. FoxP3, PDCD1, and IL2RA are markers of Tfr cells, while BCL6, ICOS, and IL7R are markers of Tfh cells. See text. p values are calculated using Spearman correlation test; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns p > 0.05. (C) Typical observation field of MIA 7-plex panel staining for Tfr and Tfh. Merged multispectral image was captured after 6-cycle TSA staining and DAPI counterstain and reveals a mature TLS architecture. Single channels are shown for the area bounded by the white rectangle. Fake colors are assigned as DAPI: blue, CD8: yellow, CXCR5: pink, CD4: green, FOXP3: red, BCL6: cyan and CD3: orange. Sub-populations of CD4 + T cells are highlighted by arrows (Tfr: red, Treg: purple, Th: green and Tfh: cyan). (D) Paired comparison of CD8 + T cell density between non-follicle (Non-Fol) and TLS areas. (E) Paired comparison of ratio of regulatory T cells to helper T cells between TLS and non-follicle areas. (F) Comparison of CD8 + T cell density between groups with high and low Tfr/Tfh ratio

Article Snippet: Rabbit anti-CXCR5 monoclonal antibody (D6L3C) , CST , Cat#: 72172S.

Techniques: MANN-WHITNEY, Marker, Staining

Journal: iScience

Article Title: Tertiary lymphoid structure and decreased CD8 + T cell infiltration in minimally invasive adenocarcinoma

doi: 10.1016/j.isci.2022.103883

Figure Lengend Snippet:

Article Snippet: Rabbit anti-CXCR5 monoclonal antibody (D6L3C) , CST , Cat#: 72172S.

Techniques: Recombinant, Software

Journal: Journal of Neuroinflammation

Article Title: CXCR5 down-regulation alleviates cognitive dysfunction in a mouse model of sepsis-associated encephalopathy: potential role of microglial autophagy and the p38MAPK/NF-κB/STAT3 signaling pathway

doi: 10.1186/s12974-021-02300-1

Figure Lengend Snippet: Schematic illustration of the experimental design. a Timeline of WT mice that underwent sham surgery or CLP. b Timeline of WT or CXCR5 −/− mice that underwent sham surgery or CLP. c Timeline of LPS-treated microglial cells treated with CXCR5 siRNA with or without pretreatment with the p38MAPK inhibitor SB203580 or agonist P79350. d Timeline of LPS-treated microglial cells treated with CXCR5 siRNA with or without pretreatment with the autophagy inhibitor 3-MA or agonist rapamycin. CLP cecal ligation and puncture, CXCR5 C-X-C chemokine receptor type 5, LPS lipopolysaccharide, siRNA short interfering RNA, 3-MA 3-methyladenine, WT wild-type

Article Snippet: For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4 °C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594.

Techniques: Ligation, Small Interfering RNA

Journal: Journal of Neuroinflammation

Article Title: CXCR5 down-regulation alleviates cognitive dysfunction in a mouse model of sepsis-associated encephalopathy: potential role of microglial autophagy and the p38MAPK/NF-κB/STAT3 signaling pathway

doi: 10.1186/s12974-021-02300-1

Figure Lengend Snippet: Cognitive deficits and up-regulation of CXCR5 in a mouse model of sepsis-associated encephalopathy. Adult male C57BL/6 J mice were subjected to sham surgery or CLP. a Escape latency time, b time spent in the target quadrant, and c number of crossings over the target quadrant in the Morris water maze beginning on day 14 after CLP. d Freezing time in the fear conditioning test on day 19 after CLP ( n = 16 per group). e, f Western blot and densitometry of hippocampal CXCR5 on days 3, 7 and 14 after CLP ( n = 4 per group). GAPDH was used as an internal control. g Double staining of CXCR5 with microglia marker Iba-1 in the hippocampus at 14 days after CLP. Arrows indicate double-stained cells. Magnification, 200 × . Scale bar, 100 μm. h Quantification of immunofluorescent cells co-staining positive for CXCR5 and Iba-1. * p < 0.05 vs. sham. CLP cecal ligation and puncture, CXCR5 C-X-C chemokine receptor type 5, GAPDH glyceraldehyde-3-phosphate dehydrogenase, Iba-1 ionized calcium binding adaptor molecule-1

Article Snippet: For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4 °C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594.

Techniques: Western Blot, Double Staining, Marker, Staining, Ligation, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: CXCR5 down-regulation alleviates cognitive dysfunction in a mouse model of sepsis-associated encephalopathy: potential role of microglial autophagy and the p38MAPK/NF-κB/STAT3 signaling pathway

doi: 10.1186/s12974-021-02300-1

Figure Lengend Snippet: CXCR5 knockout ameliorated sepsis-induced cognitive dysfunction in mice with sepsis-associated encephalopathy. WT mice or mice lacking the CXCR5 gene (CXCR5 −/− ) were subjected to sham surgery or CLP. a Western blot and b densitometry of hippocampal CXCR5 on day 14 after CLP. GAPDH was used as an internal control ( n = 4 per group). Mice were assessed in the Morris water maze on day 14 after CLP, followed by the fear conditioning test. c Escape latency time, d time spent in the target quadrant, and e number of crossings over the target quadrant in the Morris water maze. f Freezing time in the fear conditioning test ( n = 16 per group). * p < 0.05 vs. WT + sham; # p < 0.05 vs. WT + CLP. CLP cecal ligation and puncture, CXCR5 C-X-C chemokine receptor type 5, GAPDH glyceraldehyde-3-phosphate dehydrogenase, WT wild-type

Article Snippet: For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4 °C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594.

Techniques: Knock-Out, Western Blot, Ligation

Journal: Journal of Neuroinflammation

Article Title: CXCR5 down-regulation alleviates cognitive dysfunction in a mouse model of sepsis-associated encephalopathy: potential role of microglial autophagy and the p38MAPK/NF-κB/STAT3 signaling pathway

doi: 10.1186/s12974-021-02300-1

Figure Lengend Snippet: CXCR5 knockout restored hippocampal autophagy in mice with sepsis-associated encephalopathy. WT mice or mice lacking the CXCR5 gene (CXCR5 −/− ) were subjected to sham surgery or CLP. a Representative transmission electron micrographs of the hippocampus show zoomed-in views (magnification, 5000 × ; Scale bar, 500 nm) of full image of a cell (magnification, 2000 × ; Scale bar, 2000 nm). Arrows indicate autophagosome. b Representative immunofluorescence micrographs showing LC3 in the hippocampal CA1 region. Magnification, 200 × . Scale bar, 100 μm. c Quantification of immunofluorescent cells staining positive for LC3. d – h Western blot and densitometry of hippocampal LC3, beclin-1, Atg-5 and p62, and the ratio of LC3-II/LC3-I. GAPDH was used as an internal control ( n = 4 per group). * p < 0.05 vs. WT + sham; # p < 0.05 vs. WT + CLP. Atg-5 autophagy-related gene-5, CLP cecal ligation and puncture, CXCR5 C-X-C chemokine receptor type 5, GAPDH glyceraldehyde-3-phosphate dehydrogenase, LC3 microtubule-associated protein 1 light chain 3, WT wild-type

Article Snippet: For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4 °C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594.

Techniques: Knock-Out, Transmission Assay, Immunofluorescence, Staining, Western Blot, Ligation

Journal: Journal of Neuroinflammation

Article Title: CXCR5 down-regulation alleviates cognitive dysfunction in a mouse model of sepsis-associated encephalopathy: potential role of microglial autophagy and the p38MAPK/NF-κB/STAT3 signaling pathway

doi: 10.1186/s12974-021-02300-1

Figure Lengend Snippet: CXCR5 knockout reversed the alteration in hippocampal microglial M1/M2 polarization in mice with sepsis-associated encephalopathy. WT mice or mice lacking the CXCR5 gene (CXCR5 −/− ) were subjected to sham surgery or CLP. a Western blot and densitometry of hippocampal Iba-1 on day 14 after CLP. b Representative micrographs of immunohistochemistry for Iba-1 in the hippocampal CA1 and the number of Iba-1-positive cells. Magnification, 200 × . Scale bar, 100 μm. Representative immunofluorescence micrographs and quantitation ( n = 4 per group) after staining for c M1 marker iNOS and d M2 marker Arg-1 in the hippocampal CA1 region. Magnification, 200 × . Scale bar, 100 μm. * p < 0.05 vs. WT + sham; # p < 0.05 vs. WT + CLP. Arg-1 arginase-1, CLP cecal ligation and puncture, CXCR5 C-X-C chemokine receptor type 5, Iba-1 ionized calcium binding adaptor molecule-1, iNOS inducible nitric oxide synthase, WT wild-type

Article Snippet: For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4 °C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594.

Techniques: Knock-Out, Western Blot, Immunohistochemistry, Immunofluorescence, Quantitation Assay, Staining, Marker, Ligation, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: CXCR5 down-regulation alleviates cognitive dysfunction in a mouse model of sepsis-associated encephalopathy: potential role of microglial autophagy and the p38MAPK/NF-κB/STAT3 signaling pathway

doi: 10.1186/s12974-021-02300-1

Figure Lengend Snippet: CXCR5 knockout decreased hippocampal p-p38MAPK, IL-1β and IL-6 levels in mice with sepsis-associated encephalopathy. WT mice or mice lacking the CXCR5 gene (CXCR5 −/− ) were subjected to sham surgery or CLP. a – d Western blot and densitometry of hippocampal p-p38MAPK, p38MAPK, IL-1β and IL-6 ( n = 4 per group). * p < 0.05 vs. WT + sham; # p < 0.05 vs. WT + CLP. CLP cecal ligation and puncture, CXCR5 C-X-C chemokine receptor type 5, IL-1β interleukin (IL)-1β, IL-6 interleukin (IL)-6, p38MAPK mitogen-activated protein kinase, p-p38MAPK phosphorylated p38MAPK, WT wild-type

Article Snippet: For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4 °C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594.

Techniques: Knock-Out, Western Blot, Ligation

Journal: Journal of Neuroinflammation

Article Title: CXCR5 down-regulation alleviates cognitive dysfunction in a mouse model of sepsis-associated encephalopathy: potential role of microglial autophagy and the p38MAPK/NF-κB/STAT3 signaling pathway

doi: 10.1186/s12974-021-02300-1

Figure Lengend Snippet: CXCR5 knockdown restored p38MAPK-dependent autophagy in LPS-treated microglial cultures. Microglial cells were treated with CXCR5 siRNA. a Western blot and densitometry of CXCR5 in microglia at 24 h after CXCR5 siRNA administration. $ p < 0.05 vs. cells treated without siRNAs. Microglial cells were treated with CXCR5 siRNA at 24 h before LPS treatment. Some cultures were also treated with p38MAPK inhibitor SB203580 or agonist P79350 at 1 h before siRNA administration. b Representative micrographs of immunohistochemistry for LC3 in microglia at 24 h after LPS treatment. Magnification, 200 × . Scale bar, 100 μm. c Calculation of LC3-positive area. d – h Western blot and densitometry of LC3, beclin-1, Atg-5 and p62, and the ratio LC3-II/LC3-I ( n = 4 per group). * p < 0.05 vs. control; # p < 0.05 vs. LPS; & P < 0.05 vs. LPS + CXCR5 siRNA. Atg-5 autophagy-related gene-5, GAPDH glyceraldehyde-3-phosphate dehydrogenase, LC3 microtubule-associated protein 1 light chain 3

Article Snippet: For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4 °C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594.

Techniques: Western Blot, Immunohistochemistry

Journal: Journal of Neuroinflammation

Article Title: CXCR5 down-regulation alleviates cognitive dysfunction in a mouse model of sepsis-associated encephalopathy: potential role of microglial autophagy and the p38MAPK/NF-κB/STAT3 signaling pathway

doi: 10.1186/s12974-021-02300-1

Figure Lengend Snippet: CXCR5 knockdown reversed the alteration of p38MAPK-dependent microglial M1/M2 polarization and production of inflammatory cytokines in LPS-treated microglial cultures. a Representative micrographs of immunohistochemistry for Iba-1 in the microglia. Magnification, 200 × . Scale bar, 100 μm. b Calculation of Iba-1-positive area. c – h Western blot and densitometry of Iba-1, CD86, CD206, IL-1β and IL-6 ( n = 4 per group). * p < 0.05 vs. control; # p < 0.05 vs. LPS; & P < 0.05 vs. LPS + CXCR5 siRNA. GAPDH glyceraldehyde-3-phosphate dehydrogenase, Iba-1 ionized calcium binding adaptor molecule-1, IL-1β interleukin-1β, IL-6 interleukin-6, LPS lipopolysaccharide

Article Snippet: For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4 °C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594.

Techniques: Immunohistochemistry, Western Blot, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: CXCR5 down-regulation alleviates cognitive dysfunction in a mouse model of sepsis-associated encephalopathy: potential role of microglial autophagy and the p38MAPK/NF-κB/STAT3 signaling pathway

doi: 10.1186/s12974-021-02300-1

Figure Lengend Snippet: CXCR5 knockdown inhibited activation of p38MAPK-dependent NF-κB/STAT3 signaling in LPS-treated microglial cultures. a – d Western blot and densitometry of p-p38MAPK, p38MAPK, p-NF-κB, NF-κB, p-STAT3 and STAT3 and relevant ratios ( n = 4 per group). * p < 0.05 vs. control; # p < 0.05 vs. LPS; & P < 0.05 vs. LPS + CXCR5 siRNA. GAPDH glyceraldehyde-3-phosphate dehydrogenase, LPS lipopolysaccharide, NF-κB nuclear factor kappa-B, p38MAPK p38 mitogen-activated protein kinase, p- phosphorylated, STAT3 signal transducer and activator of transcription

Article Snippet: For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4 °C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594.

Techniques: Activation Assay, Western Blot

Journal: Journal of Neuroinflammation

Article Title: CXCR5 down-regulation alleviates cognitive dysfunction in a mouse model of sepsis-associated encephalopathy: potential role of microglial autophagy and the p38MAPK/NF-κB/STAT3 signaling pathway

doi: 10.1186/s12974-021-02300-1

Figure Lengend Snippet: Autophagy inhibition attenuated the effects of CXCR5 knockdown on microglial polarization and inflammatory cytokines production in LPS-treated microglial cultures. a – g Western blot and densitometry of LC3, Iba-1, CD86, and CD206, IL-1β, IL-6 and the ratio of LC3-II/LC3-I ( n = 4 per group). * p < 0.05 vs. control; # p < 0.05 vs. LPS; & P < 0.05 vs. LPS + CXCR5 siRNA. CXCR5 C-X-C chemokine receptor type 5, GAPDH glyceraldehyde-3-phosphate dehydrogenase, Iba-1 ionized calcium binding adaptor molecule-1, IL-1β interleukin (IL)-1β, IL-6 interleukin (IL)-6, LC3 microtubule-associated protein 1 light chain 3, LPS lipopolysaccharide, 3-MA 3-methyladenine

Article Snippet: For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4 °C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594.

Techniques: Inhibition, Western Blot, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: CXCR5 down-regulation alleviates cognitive dysfunction in a mouse model of sepsis-associated encephalopathy: potential role of microglial autophagy and the p38MAPK/NF-κB/STAT3 signaling pathway

doi: 10.1186/s12974-021-02300-1

Figure Lengend Snippet: Schematic diagram depicting the possible mechanisms through which CXCR5 regulates autophagy. MAPK signaling downstream of CXCR5 induced by LPS promotes the up-regulation of NF-κB/STAT3 axis. Consequently, activation of NF-κB/STAT3 pathway contributes to incomplete activation of autophagy and production of inflammatory cytokines, thereby impairs learning and memory function. CXCR5 down-regulation results in inhibition of MAPK/NF-κB/STAT3 axis and activation of autophagy in brain, which leads to the improved cognitive function in mice with sepsis-associated encephalopathy. Atg-5 autophagy-related gene-5, CXCR5 C-X-C chemokine receptor type 5, LC3 microtubule-associated protein 1 light chain 3, LPS lipopolysaccharide, NF-κB nuclear factor kappa-B, p38MAPK mitogen-activated protein kinase, p- phosphorylated, STAT3 signal transducer and activator of transcription

Article Snippet: For co-labeling of CXCR5 and Iba-1, sections were incubated overnight at 4 °C with primary antibodies (Invitrogen) against CXCR5 (1:300) and Iba-1 (1:300), then incubated with an anti-mouse or anti-rabbit IgG (1:1000; Cell Signaling Technology) conjugated with Alexa Fluor® 488 and Alexa Fluor® 594.

Techniques: Activation Assay, Inhibition