Journal: Cancer research

Article Title: Mobilizing transit-amplifying cell-derived ectopic progenitors prevents hair loss from chemotherapy or radiation therapy

doi: 10.1158/0008-5472.CAN-17-0667

Figure Lengend Snippet: Remodeling of ORS cells for the late regenerative attempt after 5.5Gy of IR. A, Lineage tracing in K5CreER; R26LSLtdTomato mice. B, Cell proliferation mapped by pulse BrdU labeling. The p-cadherin+ lower tip cells started to proliferate at 84hrs (white arrowheads) and regenerated new hair bulb at 96hrs. C, Analysis of BgSC apoptosis by TUNEL. D, Continuous BrdU labeling showed that BgSCs did not proliferate until day 5 (white arrowheads). E, Lineage tracing of BgSCs in K19CreER; R26LSLtdTomato mice. Labeled BgSC progeny did not move out of the bulge until day 5. F, Immunofluorescence of p-cadherin and Sox9. Similar to SHG (yellow arrowheads), p-cadherin+ lower tip cells were also positive for Sox9 (white arrowheads). G, Lgr5 promoter-driven EGFP expression in Lgr5EGFP-Ires-CreERT2 mice. The lower tip cells were positive for EGFP. H, Expression of ShgSC specific genes. Similar to activated ShgSCs, several specific genes were upregulated in lower tip cells at 72hrs. I, Possible cell origin for late regenerative attempts. J, Lineage tracing in Lgr5EGFP-Ires-CreERT2; R26LSLtdTomato mice. Error bars represent mean ± S.E.M. Scale bar=25µm.

Article Snippet: The following antibodies were used: CD34-FITC (eBioscience, 11-0341, 1:50), Sca1-PE-Cy7 (eBioscience, 25-5981, 1:50) and p-cadherin-PE (R&D systems, FAB761P, 1:100).

Techniques: Labeling, TUNEL Assay, Immunofluorescence, Expressing

Journal: eLife

Article Title: Concerted IL-25R and IL-4Rα signaling drive innate type 2 effector immunity for optimal helminth expulsion

doi: 10.7554/eLife.38269

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Arginase-1 (Polyclonal) , R and D Systems , IC5868P.

Techniques: Recombinant, Staining, Control

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A , a diagram of the ex vivo mtROS and phagocytosis assay. B , Total amount of aortic F4/80 + /Trem2 + macrophages were quantified and shown in the bar graph; n=4-7 individual mice per group. C , MitoNeoD MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5 individual mice per group. D , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-6 individual mice per group. E , a diagram of the in vivo phagocytosis assay. F , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-7 individual mice per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Ex Vivo, Phagocytosis Assay, In Vivo

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A-D , Mouse scRNA-seq data were re-analyzed from a previous publication . Uniform manifold approximation and projection (UMAP) representation of 11 aortic CD45 + immune cell clusters were shown in A . Trem2 gene expression pattern ( B ) and Pkm gene expression pattern ( C ) were shown in the UMAP. D , Violin plots show the Pkm and Trem2 expression distribution among aortic macrophage subpopulations. ResMac: resident macrophages; InflaMac: inflammatory macrophages. E , HMDMs transfected with PKM siRNA were treated with 50 μg/ml oxLDL for 24 h before subjected to mtROS assay (left panel) or phagocytosis assay (right panel). MFI was quantified and shown in the bar graph; n=4-5 per group. F , Representative confocal images of macrophages immunostained for PKM2 (green) and Tom20 (red). Nuclei were stained by DAPI (blue). Scale bar: 5 μm. G , HMDMs treated with 20 μg/ml LDL (control) or oxLDL for 3 h were lysed, subjected to cell fractionation into mitochondrial and cytosol fractions. PKM2 and ATP5A (mitochondria fraction loading control) blot images from mitochondrial fractions were shown on the left. PKM2 and β-actin (cytosol fraction loading control) blot images from cytosol fractions were shown on the right. Images were quantified, normalized to each loading control, and expressed as fold change of control. n=4 per group. H , WT or Cd36 -null peritoneal macrophages treated with 20 μg/ml LDL (control) or oxLDL for 3 h and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and ATP5A and blot images were shown. Images were quantified, normalized, and expressed as fold change of control. n=4 per group. I , WT macrophages treated with 20 μg/ml oxLDL or pre-treated 1 or 5μM shikonin before addition of oxLDL, incubating for 3 h, and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and Tom20 and blot images were shown. Images were quantified and expressed as fold change of control. n=3 per group. J , WT macrophages pre-treated with 20 μg/ml oxLDL or in combination with 1 μM shikonin for 24 h before mtROS or phagocytosis assay. The MitoNeoD (left) or pHrodo (right) MFI was quantified and shown in the bar graph; n=3-4 per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Gene Expression, Expressing, Transfection, Phagocytosis Assay, Staining, Control, Cell Fractionation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: Induction of HNSCC in Nlrp3-deficient mice by 4NQO. a Haematoxylin and eosin (H&E) staining showed the pathological changes in mouse tongues after 4NQO exposure. The dotted line indicates the epithelial basement membrane. Bar, 200 μm. b Schematic of the 4NQO tumorigenesis protocol. c Representative pictures of oral lesions in control and Nlrp3−/− mice at 24 weeks. d Representative H&E histological sections of 4NQO induced HNSCC. Bar, 50 μm. e Tumor incidence in the Nlrp3−/− (n = 10) and control (n = 10) groups. ns, no significant difference. f The proportion of oral lesion types in the two groups at week 24. g Representative image of tongue SCC in the two groups of mice. The red dotted circle denotes the lesion area. h Quantitative analysis of tumor size and i number in the Nlrp3−/− (n = 7) and control groups (n = 8) at the experimental endpoint. *, P < 0.05. j The body weights of 4NQO-treated control and Nlrp3−/− mice from the beginning to the end of this experiment. *, P < 0.05; ***, P < 0.001. Error bar, SD

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Staining, Membrane, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: NLRP3 is overexpressed in the stromal cells of human HNSCC. a Representative immunofluorescence images of NLRP3 (red) and CK-14 (green) in human HNSCC tissue. Bar, 50 μm. b Flow cytometry to detect NLRP3 expression (mean ± SD) in HNSCC TILs, and cells were gated by CD14+. c, d Representative IHC images and corresponding quantification data of NLRP3 expression in oral mucosa (n = 20), dysplasia (n = 61) and HNSCC (n = 157) (***, P < 0.001). Bar, 50 μm. e Representative IHC staining of NLRP3 in HPV-negative (n = 142) and HPV-positive HNSCC tissue (n = 15) and f quantification analysis (**, P < 0.01). Bar, 50 μm. G Kaplan–Meier analysis using overall survival (OS) of NLRP3 expression in HNSCC patients. median cut-off, n = 148. Error bar, SD

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Immunofluorescence, Flow Cytometry, Expressing, Immunohistochemistry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: Increased NLRP3 expression in TAMs predicts poor prognosis in HNSCC. a The ratio of M1 (CD64, CD68) and M2-like macrophage (MRC1, CD163) signatures in the low-expression (n = 259) and high-expression NLRP3 groups (n = 259) in the TCGA HNSCC database (***, P < 0.001). b Co-immunofluorescence staining of NLRP3 (red) and CD206 (green) in human HNSCC tissue. The white arrow shows co-expressed cell (yellow). T, tumor area; S, stromal area. Bar, 50 μm. c Flow cytometry to detect NLRP3 expression (mean ± SD) in CD206+ TAMs in human HNSCC PBMCs (n = 6) and TILs (n = 5). d Quantitative statistical analysis of NLRP3- and CD206-positive populations on gated cells (**, P < 0.01; ***, P < 0.001). e Correlation analysis of CD14+/CD206+/NLRP3+ and CD14+/CD206+ cell proportions in HNSCC patient PBMC and TIL, n = 11. f Representative IHC staining of NLRP3, CD206 and CD163 in human HNSCC using serial sections. Bar, 50 μm. g Correlation analysis between NLRP3, CD206 and CD163 protein expression in HNSCC tissue microarray using histoscore, n = 157. h Correlation analysis between NLRP3, MRC1 (CD206 encoding gene) and CD163 RNA expression in the TCGA HNSCC database. unit, expression (RSEM, Log2(Val + 1)), n = 520. i Hierarchical clustering plot of NLRP3, CD11b, CD206 and CD163 in HNSCC based on histoscore. n = 74. j Survival curves of high NLRP3 and CD206 expression vs. low NLRP3 and CD206 expression patients; high NLRP3 and CD163 expression vs. low NLRP3 and CD163 expression patients; low NLRP3 on high CD206 expression vs. high NLRP3 on CD206 high expression patients; and low NLRP3 on high CD163 expression vs. high NLRP3 on CD163 high expression patients. Error bar, SD

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry, Immunohistochemistry, Microarray, RNA Expression

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: NLRP3 overexpression is positively correlated with TAMs in mouse HNSCC. a Representative IHC images of NLRP3, CD206 and F4/80 in Tgfbr1/Pten 2cKO and 4NQO-induced mouse HNSCC tumor tissues. Bar, 50 μm. b Correlation analysis between NLRP3, CD206 and F4/80 expression histoscores in mouse HNSCC tissues. n = 27. c Co-immunofluorescence staining of CD206 (green) and NLRP3 (red) in mouse HNSCC tissue. Bar, 50 μm. d, e Representative flow cytometry plots and quantification analysis showed the population change of NLRP3+ TAMs in the blood of normal wild-type and tumor-bearing individuals in two mouse models (n = 6 in 4NQO; n = 4 in 2cKO) (***, P < 0.001). Error bar, SD

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Over Expression, Expressing, Immunofluorescence, Staining, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: Knockdown of NLRP3 in THP-1 cells inhibits M2-like macrophage polarization. a THP-1 cells were treated with CAL27 cultured medium (CM) or IL-4 and IL-13 after treatment with PMA; NLRP3 and Caspase-1 expression were analyzed by western blot, and b IL-1β amounts in the supernatant were detected by ELISA (n = 3 per group; ***, P < 0.001). c Western blotting analysis of NLRP3 expression in NLRP3-knockout or control THP-1 cells. d, e Flow cytometry plots and quantification analysis of CD206 in NLRP3-deficient and control THP-1 macrophages after CAL27 CM culture (n = 3 per group; ***, P < 0.001). f ELISA results of IL-1β amounts in control and siNLRP3 THP-1 macrophage cultured medium (n = 3 per group; ***, P < 0.001). g, h NLRP3-deficient THP-1 macrophages were exposed to IL-1β (20 ng/ml) for 48 h after CAL27 CM treatment, and CD206 was analyzed by flow cytometry (n = 3 per group; ***, P < 0.001). i Growth curves of CAL27 cells in different groups as measured by a CCK-8 assay (n = 3 per group; ***, P < 0.001). Error bar, SD

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Knockdown, Cell Culture, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Knock-Out, Control, Flow Cytometry, CCK-8 Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: NLRP3 deficiency reduced the proportion of TAMs and MDSCs. a Representative IHC images and quantification of IL-1β in tumor tissues from the two groups of mice. ***, P < 0.001, Bar, 50 μm. b ELISA results of IL-1β amounts in the blood of normal WT (n = 5), 4NQO-treated control (n = 8) and Nlrp3−/− (n = 7) tumor burden mice. **, P < 0.01; ***, P < 0.001; ns, no significant difference. c The Luminex liquid suspension chip analysis result. *, P < 0.05; **, P < 0.01, three mice in each group. d Flow cytometry to detect the percentage of CD11b+/F4/80+/CD206+ TAMs in the spleen of the two groups and e quantitative statistical analysis. *, P < 0.05. f Representative IHC images and quantification of CD206 in control and Nlrp3−/− mouse HNSCC tissues. **, P < 0.01, Bar, 50 μm. g Flow cytometry to detect the percentage of M-MDSCs and PMN-MDSCs in the spleen of the two groups and h quantitative statistical analysis. *, P < 0.05. i Representative IHC images and quantification of Gr-1 in control and Nlrp3−/− mouse HNSCC tissues. **, P < 0.01, Bar, 50 μm

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Luminex, Suspension, Flow Cytometry

Journal: Frontiers in physiology

Article Title: Sex differences in apoptosis do not contribute to sex differences in blood pressure or renal T cells in spontaneously hypertensive rats.

doi: 10.3389/fphys.2022.1006951

Figure Lengend Snippet: FIGURE 6 Treatment with ZVAD-FMK for 2 weeks decreases Th17 cells in male SHR. T cells were measured in kidneys isolated from male and female spontaneously hypertensive rats (SHR) treated with vehicle (veh) or ZVAD-FMK (ZVAD) from 13 to 15 weeks of age (n = 5–6). The following T cells were measured: total CD3+ T cells [panel (A)], CD4+ T helper cells [panel (B)], T regulatory cells [Tregs; CD3+CD4+FOXP3+; panel (C)] and T helper [Th17 cells (CD3+CD4+ROR- γ+; panel (D)]. CD3+ T cells were expressed as % total renal cells. CD4+ T cells were expressed as % total CD3+

Article Snippet: Cells were then washed, fixed and permeabilized (eBioscience; 00-5523-00) and then stained with intracellular antibodies for FoxP3 (1:100; eBiosciences; 17-5773-82), or RARrelated orphan receptor-γ (RORγ; 1:100; R&D Systems; IC6006P) to identify Tregs (CD3+CD4+FoxP3+) and T helper (Th)17 cells (CD3+CD4+RORγ+), respectively.

Techniques: Isolation

Journal: Frontiers in physiology

Article Title: Sex differences in apoptosis do not contribute to sex differences in blood pressure or renal T cells in spontaneously hypertensive rats.

doi: 10.3389/fphys.2022.1006951

Figure Lengend Snippet: FIGURE 9 Treatment with ZVAD-FMK for 6 weeks did not change either splenic or renal T cell profile. T cells were measured in kidneys isolated from male and female spontaneously hypertensive rats (SHR) treated with vehicle (veh) or ZVAD-FMK (ZVAD) from 6 to 12 weeks of age (n = 7). The following T cells were measured: total CD3+ T cells [panel (A)], CD4+ T helper cells [panel (B)], T regulatory cells [Tregs; CD3+CD4+FOXP3+; panel (C)] and T helper (Th17 cells [CD3+CD4+ROR- γ+; panel (D)]. CD3+ T cells were expressed as % total renal cells. CD4+ T cells were expressed as % total CD3+ T cells. Tregs and Th17 cells were expressed as % CD3+CD4+ T cells. Data were compared using 2-Way ANOVA followed by Tukey’s multiple comparisons test; *** indicates p < 0.001; * indicated p < 0.05; NS indicates not significant.

Article Snippet: Cells were then washed, fixed and permeabilized (eBioscience; 00-5523-00) and then stained with intracellular antibodies for FoxP3 (1:100; eBiosciences; 17-5773-82), or RARrelated orphan receptor-γ (RORγ; 1:100; R&D Systems; IC6006P) to identify Tregs (CD3+CD4+FoxP3+) and T helper (Th)17 cells (CD3+CD4+RORγ+), respectively.

Techniques: Isolation