Journal: Cancer gene therapy

Article Title: SEMA7A-mediated juxtacrine stimulation of IGFBP-3 upregulates IL-17RB at pancreatic cancer invasive front.

doi: 10.1038/s41417-024-00849-6

Figure Lengend Snippet: Fig. 2 IL-17RB-modulated transcription factor SNAI2 correlates with tumor invasiveness. Matrigel invasion assays of HPAF-II (A) and MIA PaCa-2 (B) cells from parental or IL-17RBDel clones. Each dot represents the datum from one independent experiment (n = 4). Values are expressed as mean ± SD. ***, P < 0.001 (two-tailed Student’s t-test). C Western blotting analysis of proteins harvested from parental or IL- 17RBDel tumor cells (HPAF-II or MIA PaCa-2). D Western blotting analysis of proteins harvested from HPAF-II or MIA PaCa-2 cells transduced with lentiviral pLAS5w (vector) or pLAS5w-IL17RB-Flag (IL17RB-Flag). E Representative IHC images of IL-17RB (green signal), SNAI2 (red signal) and Hoechst 33342 (blue signal) expression in FFPE samples of PDAC patients. Scale bar, 100 μm. F Relative intensity of SNAI2 in IL- 17RB low and high expressing regions. IL-17RB low expressing regions were defined as IL-17RB staining expressed in <10% tumor cells per high power field, and IL-17RB high expressing regions were defined as IL-17RB staining expressed in ≥50% tumor cells per high power field. Each dot represents the datum from one high-power fields (n = 8). Values are expressed as mean ± SD. **, P < 0.01 (two-tailed Student’s t-test). G Representative IHC images of IL-17RB (green signal), VIM (red signal), Hoechst 33342 (blue signal) expression in FFPE samples of PDAC patients. Scale bar, 100 μm. H Relative intensity of VIM in IL-17RB low and high expressing regions. IL-17RB low expressing regions were defined as IL-17RB staining expressed in <10% tumor cells per high power field, and IL-17RB high expressing regions were defined as IL-17RB staining expressed in ≥50% tumor cells per high power field. Each dot represents the datum from one high-power field n = 10). Values are expressed as mean ± SD. *, P < 0.05 (two-tailed Student’s t-test). I, J Western blotting analysis (I) and Matrigel invasion assays (J) of cells from parental, IL-17RBDel (none), IL-17RBDel transduced with pLAS5w (vector), and IL-17RBDel transduced with pLAS5w-SNAI2 (SNAI2). Each dot represents the datum from one independent experiment (n = 4). Values are expressed as mean ± SD. ****, P < 0.0001 (one-way ANOVA).

Article Snippet: For co-staining of SNAI2 or VIM with IL17RB in human PDAC specimens, anti-SNAI2 antibody (1:30; 9585, Cell Signaling) or anti-vimentin antibody (1:500; 10366-1-AP, Proteintech) and anti-IL-17RB antibody (1:500; A81, homemade) were used.

Techniques: Clone Assay, Two Tailed Test, Western Blot, Transduction, Plasmid Preparation, Expressing, Staining

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-25 (IL-25) has a protective role in atherosclerosis development in the aortic arch in mice

doi: 10.1074/jbc.RA117.000292

Figure Lengend Snippet: IL-25 deficiency increases splenic Th1 cells and decreases Tregs and ILC2s. Shown are gating strategies (the graphs show the gating strategy in one Apoe−/−/IL-25−/− mouse) to identify immune cells in the spleen with flow cytometry and the frequencies in the splenic cells. In a and b, the same CD3+CD4+ flow cytometry plot is shown, because it is the same sample from one representative mouse that has been stained for both INF-γ and IL-5. a, to identify Th1 cells, leukocytes were gated as CD3+CD4+IFN-γ+ T cells in the CD3+CD4+ population. Shown is the frequency of Th1 cells in the spleen of Apoe−/− and Apoe−/−/IL-25−/− mice. b, to identify Th2 cells, leukocytes were gated as CD3+CD4+IL-5+ T cells in the CD3+CD4+ population. Shown is the frequency of Th2 cells in the spleen of Apoe−/− and Apoe−/−/IL-25−/− mice. c, to identify Th17 cells, leukocytes were gated as CD3+CD4+IL-17+ T cells in the CD3+CD4+ population. Shown is the frequency of Th17 cells in the spleen of Apoe−/− and Apoe−/−/IL-25−/− mice. d, to identify Tregs, leukocytes were gated as CD3+CD4+CD25+FoxP3+ T cells in the CD3+CD4+ population. Shown is the frequency of Tregs in the spleen of Apoe−/− and Apoe−/−/IL-25−/− mice. e, to identify ILC2s, leukocytes were gated as CD45+, lineage-negative cells (Lin−) expressing IL-17RB, intermediate (int) IL-7ra, and ICOS in the CD45+ population. Shown is the frequency of ILC2s in the spleen of Apoe−/− and Apoe−/−/IL-25−/− mice. Mann–Whitney test was used; n = 10–19 for Apoe−/− and n = 22 for Apoe−/−/IL-25−/− mice; *, p = 0.022; ****, p < 0.0001. Each dot represents one mouse, and the bars show the mean value ± S.D.

Article Snippet: The extracellular antibodies that were used are CD3-PE/Cy7 (clone 145-2C11), CD4-PB (clone RM4-4), CD8-AF700 (clone YTS156.7.7), CD25-APC (clone 3C7), ICOS-PB (clone C398.4A), CD45-APC/Cy7 (clone 30-F11), IL-7ra-FITC (clone SB/199), and IL-17RB-APC (clone 752101, R&D Systems, Abingdon, UK), whereas the intracellular are Foxp3-PE (clone MF-14), IL-5-APC (clone TRFK5), IFN-γ-PE (clone XMG1.2), and IL-17A-APC (clone TC11-18H10.1).

Techniques: Flow Cytometry, Staining, Expressing, MANN-WHITNEY

Journal: Ear, nose, & throat journal

Article Title: The PI3K-Akt-HIF-1α Pathway Reducing Nasal Airway Inflammation and Remodeling in Nasal Polyposis.

doi: 10.1177/0145561319857697

Figure Lengend Snippet: Figure 1. Lipopolysaccharide (LPS)-induced cytokine expression by activation of the Akt/HIF-1a pathway in human nasal epithelial cells by ELISA. Hypoxia-inducible factor 1a and p-Akt expressed by LPS stimulation for 24 hours in human nasal epithelial cells by Western blot. Lipopolysaccharide induced a dose-dependent increase in HIF-1a (A and B), p-Akt (A and C), IL-25 (D), and IL-17RB (E). Values represent the means (SD) of at least 3 independent experiments. *P < .05; **P < .01. ELISA indicates enzyme-linked immunosorbent assay; HIF-1a, hypoxia- inducible factor 1a; IL, interleukin.

Article Snippet: The primary antibodies against HIF-1a and p-Akt (Abcam, Cambridge, Massachusetts) and against IL-25 and IL-17RB (Santa Cruz Biotechnology, Santa Cruz, California) were used.

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Ear, nose, & throat journal

Article Title: The PI3K-Akt-HIF-1α Pathway Reducing Nasal Airway Inflammation and Remodeling in Nasal Polyposis.

doi: 10.1177/0145561319857697

Figure Lengend Snippet: Figure 2. Hypoxia-inducible factor 1a knockdown reduced cytokine expression in human nasal epithelial cells. Hypoxia-inducible factor 1a and p-Akt protein (A and C) and HIF-1a mRNA (B and D) levels were determined by Western blotting and RT-PCR, respectively. The levels of IL-25 (E) and IL-17RB (F) were measured by ELISA in the supernatants of human nasal epithelial cells. Values represent the means (SD) of at least 3 independent experiments. *P < .05. ELISA indicates enzyme-linked immunosorbent assay; HIF-1a, hypoxia-inducible factor-1a; IL, interleukin; RT-PCR, reverse transcription-polymerase chain reaction.

Article Snippet: The primary antibodies against HIF-1a and p-Akt (Abcam, Cambridge, Massachusetts) and against IL-25 and IL-17RB (Santa Cruz Biotechnology, Santa Cruz, California) were used.

Techniques: Knockdown, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Polymerase Chain Reaction

Journal: Ear, nose, & throat journal

Article Title: The PI3K-Akt-HIF-1α Pathway Reducing Nasal Airway Inflammation and Remodeling in Nasal Polyposis.

doi: 10.1177/0145561319857697

Figure Lengend Snippet: Figure 3. LY294002 interrupted LPS-induced PI3K signaling activation in human nasal epithelial cells. LY294002 significantly decreased the levels of HIF-1a (A), p-Akt (B) and IL-25 (C), and IL-17RB (D) production in epithelial cells in a dose-dependent manner by Western blot and ELISA analysis. Values represent means (SD) of at least 3 independent experiments. *P < .05 versus negative control; **P < .01 versus negative control. ELISA indicates enzyme-linked immunosorbent assay; HIF-1a, hypoxia-inducible factor-1a; IL, interleukin.

Article Snippet: The primary antibodies against HIF-1a and p-Akt (Abcam, Cambridge, Massachusetts) and against IL-25 and IL-17RB (Santa Cruz Biotechnology, Santa Cruz, California) were used.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Ear, nose, & throat journal

Article Title: The PI3K-Akt-HIF-1α Pathway Reducing Nasal Airway Inflammation and Remodeling in Nasal Polyposis.

doi: 10.1177/0145561319857697

Figure Lengend Snippet: Figure 4. Lipopolysaccharide induced IL-25 and IL-17RB expression via the PI3K/Akt/HIF-1a pathway in vivo. Chronic rhinosinusitis mice were stimulated with LPS or saline with or without preadministration of LY294002 (PI3K inhibitor) or 2-methoxyestradiol (HIF-1a inhibitor). A, Hematoxylin and eosin (HE) staining in the nasal mucosa in each group (magnification 400, scale bar ¼ 20 mm). B and D, Hypoxia-inducible factor-1a, IL-25, and IL-17RB expression by Western blot. C, Eosinophil counts from HE staining were significantly increased after LPS stimulation and decreased by LY294002 or 2-methoxyestradiol (n ¼ 3) in the nasal mucosa. Values represent the means (SD) of at least 3 independent experiments. The number of sneezes (E) and frequency of nasal rubbing (F) were counted over a 10-minute observation period immediately following LPS stimulation (n ¼ 3).*P < .05 versus negative control; #P < .05 versus positive control. HIF-1a indicates hypoxia- inducible factor-1a; IL, interleukin.

Article Snippet: The primary antibodies against HIF-1a and p-Akt (Abcam, Cambridge, Massachusetts) and against IL-25 and IL-17RB (Santa Cruz Biotechnology, Santa Cruz, California) were used.

Techniques: Expressing, In Vivo, Saline, Staining, Western Blot, Negative Control, Positive Control

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A Flow diagram of the i.p. HDM sensitization model. Challenge phase: D24, Remission phase: D30. B Airway resistance of mice was determined on day 1 (D1), D8, D15, D24 and D30 under methacholine (0, 6.25, 12.5, 25 and 50 mg/mL in PBS) stimulation. C Serum IgE ( n = 8), D lung IL-4, IL-5 and IL-13 ( n = 8), E eosinophils in peripheral blood ( n = 6), and F cytology of bronchoalveolar lavage fluid (BALF) ( n = 10) were determined at indicated time points. G Gating strategy. In live CD45 + lymphocytes, Th2s are defined as CD4 + GATA3 + , and total ILC2s are defined as lineage – CD90.2 + NK1.1 – NKp46 – Rorγt – GATA3 + . H Kinetics of the frequency and number of ST2 – KLRG1 + IL-17RB + ILC2 subset in the lamina propria of small intestines (siLP) ( n = 10). I , J IL-13 + percentage and MFI I and IL-5 + percentage and MFI J of ST2 – KLRG1 + IL-17RB + ILC2s in siLP after stimulation with PMA, ionomycin, brefeldin A and monensin. K , L Pie charts of IL-13 + K and IL-5 + L cell populations in siLP in remission under stimulation ( n = 10). Mice in control group (Ctrl) were treated with corresponding volume of sterile normal saline. Data points are individual mice pooled from three independent experiments for all panels. Data are shown as the mean ± SD. Statistical comparisons were performed using unpaired one-way ANOVA with Dunnett’s test except in B and F using unpaired two-way ANOVA. p values are shown on the graphs. Source data are provided as a Source Data file.

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Control, Sterility, Saline

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A Donor CD45.1 + mice were sensitized and challenged with HDM follow the i.p. HDM sensitization model protocol. On day 30 (remission), lineage-negative cells were isolated from bone marrow (BM), spleen, lungs and siLP, respectively and cultured in vitro for expansion. Subsequently, 5 × 10 5 CD45 + lineage – CD90.2 + NK1.1 – NKp46 – ST2 – KLRG1 + IL-17RB + ILC2s were sorted and injected into the irradiated CD45.2 + mice via tail vein. Recipient CD45.2 + mice were then treated with 25 μg HDM intranasally for 3 times once daily. 24 hours later, single cells in lungs were prepared for flow cytometry analysis. B – E Representative plots and statistical results (bar graph) showing the percentages and the numbers of ST2 – KLRG1 + IL-17RB + ILC2s at lungs in mice that received cells derived from B BM, C spleen, D lungs, or E siLP, respectively. F , G IgE levels in serum F and IL-13 and IL-5 protein levels in lungs ( G ) of recipient mice in each group were detected by ELISA. H Histological scores and representative images of lungs of recipient mice by hematoxylin-eosin staining. Magnification: ×200, scale bar = 100 μm. Results are representative of three independent experiments. Means ± SD from 8 mice each group. Statistical comparisons were performed using unpaired, two-sided Welch’s t-test except in F – H using unpaired two-way ANOVA. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 2A Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Isolation, Cell Culture, In Vitro, Injection, Irradiation, Flow Cytometry, Derivative Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A CD45.1 + and irradiated CD45.2 + mice were surgically jointed from olecranon to knee joint and recovered for 4 weeks to establish blood chimerism. Subsequently, CD45.1 + mice were treated with HDM as Fig. . B The percentages and the numbers of ST2 – KLRG1 + IL-17RB + ml-ILC2s in siLP of CD45.1 + and CD45.2 + were analyzed on day 30. Cells were gated on CD45.1 + lineage – CD90.2 + NK1.1 – NKp46 – GATA3 + . For in vivo imaging experiments, mice were treated with HDM to establish the asthma relapse model as portrayed in Fig. . C On day 30 (remission), ST2 – KLRG1 + IL-17RB + ILC2s were sorted from siLP and stained with 5 mM Xenolight DiR, and then injected intravenously (5 × 10 5 cells/each) into various groups of mice. Specifically, mice in DiR- group were injected with unstained cells. For the remission + CCX282-B group, recipient mice resting from asthma symptoms were treated with the CCR9 antagonist CCX282-B at 50 mg/kg (daily injection subcutaneously) from day 24 to day 30. For the remission + Etrolizumab group, recipient mice resting from asthma symptoms were treated with the anti-α4β7 monoclonal antibody Etrolizumab at 5 mg/kg (injection intravenously) on day 24, 26, 28 and 30. For the relapse group, mice in remission were injected with DiR-stained cells, followed with treatment of HDM at 50 μg intranasally for 2 times once per hour. Mice in the relapse + FTY720 group received intravenous injection of FTY720 along with 2-time intranasal administration of HDM. Mice in all groups were imaged 2 hours post cell injection, and organs including lungs, spleen, liver, SI and large intestine were then rapidly harvested and imaged. D S1P expression in plasma collected from mice post asthma relapse (D34, D37 and D40) was measured by ELISA. The S1P signaling antagonist FTY720 was administered intraperitoneally to asthmatic mice on days 31 to 33 for 3 times. E The percentages and the numbers of ST2 – KLRG1 + IL-17RB + ILC2s in lungs were detected on D34. F Histological examination of lungs by hematoxylin-eosin staining. Magnification: ×200, scale bar = 100 μm. G Protein levels of IL-13 in lungs was detected by ELISA. Results are representative of three independent experiments. Means ± SD from 6 mice for each group. Statistical comparison was conducted using unpaired, two-sided Welch’s t test for B and unpaired one-way ANOVA with Dunnett’s test for the rest. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 3A Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Irradiation, In Vivo Imaging, Staining, Injection, Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A WT or Rag1 – / – mice were stimulated with HDM, and anti-KLRG-1 mAb or isotype IgG were administered intraperitoneally as indicated. B The percentage and the number of CD45 + lineage – CD90.2 + NK1.1 – NKp46 – GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in lungs were analyzed on day 24. C Pulmonary IL-13 was detected by ELISA. D Airway resistance was measured under stimulation of methacholine. E Pulmonary histological changes were observed by hematoxylin-eosin staining. Magnification: ×200, scale bar = 100 μm. F Rag1 – / – mice were stimulated with HDM, and CD45 + lineage – CD90.2 + ST2 – KLRG1 + IL-17RB + ILC2s were sorted from siLP on D30. In vitro culturing cells were treated with anti-KLRG1 mAb or isotype control, and then adoptively transferred into untreated ILC2 depleted (anti-CD90.2 mAb or isotype control) Rag1 – / – mice via tail vein (5 × 10 5 cells/mouse). Recipient mice were intranasally treated (i.n.) with 25 μg HDM or 500 ng recombinant IL-25 for 3 consecutive days. G The percentage and the number of CD45 + lineage – CD90.2 + NK1.1 – NKp46 – GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in lungs. H , I IL-13 level in lungs homogenates H and airway resistance I was measured. J Pulmonary histological changes were analyzed. Magnification: ×200, scale bar = 100 μm. Results are representative of three independent experiments. Data was shown as Means ± SD. n = 6 in all panels except for n = 3 in D and I . Statistical comparison was conducted using unpaired, two-sided Welch’s t test, except in ( D ) and ( I ) using unpaired two-way ANOVA with the Bonferroni posttest to analyze dose response to methacholine. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 4F Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, In Vitro, Control, Recombinant, Comparison

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A Flow chart of asthma mice model establishment and sample collection. B , C On days 21 to 23, BrdU (0.8 mg/mice) was given intranasally. At indicated time points, single-cell suspension was prepared and subjected to flow cytometry analysis; Dynamic changes of the percentage B and numbers C of CD45 + lineage – CD90.2 + GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in siLP. D The proportions of BrdU + ST2 – KLRG1 + IL-17RB + ILC2s in siLP of HDM-challenged mice were analyzed by flow cytometry. A control histogram (shaded in blue) was generated using cells from ctrl mice stained with BrdU. E During asthma remission (AR) (day 30) of the i.p. HDM sensitization model, three ILC2 subsets including KLRG1 – , KLRG1 + IL-17RB + and KLRG1 + IL-17RB + (marked as AR-KLRG1 – , AR-KLRG1 + IL-17RB + and AR-KLRG1 + IL-17RB + ) were purified from the total CD45 + lineage – CD90.2 + ST2 – cells from siLP. Besides, ST2 – KLRG1 + IL-17RB + ILC2s (marked as naive-KLRG1 + IL-17RB + ILC2s) were sorted from siLP of untreated WT mice. Cells were then cultured (5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 3 /well) with 10 ng/mL IL-2 and 20 ng/mL IL-7, and treated with (alarmins + ) or without (alarmins-) 200 ng/mL IL-25 and 200 ng/mL IL-33 every other day for 14 days. Subsequently, cells were collected for further analysis. (F-H) Ki-67 + percentage F , number ( G ) and fold changes H of expansion of cells were determined. I Representative plots and statistical results (bar graph) showing the percentages of IL-13 + and IL-5 + of these four ILC2 subsets after stimulation with PMA, ionomycin, brefeldin A and monensin. Data are representative of four independent experiments. Means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SD; n = 5 for the naive-KLRG1 + IL-17RB + group, and n = 9 for the other groups. B , C statistical comparisons were performed using unpaired, two-sided Welch’s t test. F – I unpaired two-way ANOVA with the Bonferroni posttest analysis was performed. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 5E Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Suspension, Flow Cytometry, Control, Generated, Staining, Purification, Cell Culture

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A In asthma remission, KLRG1 – , KLRG1 + IL-17RB – , KLRG1 + IL-17RB + subsets (marked as AR-KLRG1 – , AR-KLRG1 + IL-17RB – and AR-KLRG1 + IL-17RB + ) in total CD45 + lineage – CD90.2 + ST2 – ILC2s in siLP were sorted. KLRG1 + IL-17RB + ILC2 (marked as naive-KLRG1 + IL-17RB + ) were isolated from siLP of naive WT mice (5 ~ 7 × 10 5 for every subset, purity > 98%). Total RNA was isolated, and qRT-PCR was performed. Heat map of mRNA expression values of selected genes (see Fig. ). n = 6 for AR-KLRG1 – ILC2, AR-KLRG1 + IL-17RB – ILC2 and AR-KLRG1 + IL-17RB + ILC2, n = 3 for naive-KLRG1 + IL-17RB + ILC2. B – F Sorted CD45 + lineage – CD90.2 + NK1.1 – NKp46 – ST2 – KLRG1 + IL-17RB + ml-ILC2s were transfected with siRNAs targeting Tcf7 or Tox (si Tcf7 or si Tox ) or negative control (Negative Ctrl). Together with AR-KLRG1 – , AR-KLRG1 + IL-17RB – and naive-KLRG1 + IL-17RB + ILC2s (5 ± 10 3 per well), a combination of 200 ng/mL IL-25 and 200 ng/mL IL-33 were added every two days with IL-2 and IL-7 for 8 days. Knockdown efficiency of Tcf7 or Tox was confirmed (see Fig. ). B Cell expansion was calculated relative to day 0. C Protein levels of IL-13 in culture medium were detected by ELISA. n = 8 for ml-ILC2 Ctrl, ml-ILC2 Negative Ctrl, ml-ILC2 si Tcf7 and ml-ILC2 si Tox , n = 5 for AR-KLRG1 – ILC2, AR-KLRG1 + IL-17RB – ILC2 and naive-KLRG1 + IL-17RB + ILC2. D Heat map of mRNA expression values of selected genes (see Fig. ). n = 3. Expression of E CCR9 and F S1PR1 of all cells in each group were detected with flow cytometry. n = 3. G Flow chart of asthma mice model, BrdU administration, RNA interference and sample collection. On days 7–11, BrdU (0.8 mg/mice) was given intranasally daily. Rag1 – / – mice ( n = 5) were injected with Ambion in vivo si-negative control or indicated siRNAs on days 12–18, and were intranasally treated with 25 μg HDM on days 72-74. The percentage of BrdU + in ST2 – KLRG1 + IL-17RB + ILC2s in H siLP on day 71 and in ( I ) lungs on day 75 were detected with flow cytometry. J Flow chart of asthma mice model, adoptive transfer, RNA interference, and sample collection. Rag1 – / – mice were exposed with HDM and rested till AHR vanished as described in Fig. . Mice were injected with Ambion in vivo si-negative control, si Tcf7 or si Tox on days 16-18, and were intranasally treated with 25 μg HDM on days 19–21. For the groups of in vivo si Tcf7 +ml-ILC2 AT + HDM and in vivo si Tox +ml-ILC2 AT + HDM, mice were injected (i.v.) with 5 × 10 5 ml-ILC2s precultured with transfection of si-negative control on day 19 before HDM treatment; For the groups of in vivo si Tcf7 +si Tcf7 -ml-ILC2 AT + HDM and in vivo si Tox +si Tox -ml-ILC2 AT + HDM, mice were injected (i.v.) with 5 × 10 5 ml-ILC2s precultured with transfection of siRNAs on day 19 before HDM treatment. Twenty-four hours after the last HDM treatment, K airway resistance ( n = 3) and L IL-13 level in lung homogenates were measured ( n = 6). M Pulmonary histological changes were analyzed ( n = 6). Magnification: ×200, scale bar = 100 μm. Data was shown as Means ± SD. Results are representative of three independent experiments. Statistical comparison was conducted using unpaired one-way ANOVA with Dunnett’s test, except in ( B ) and ( K ) using unpaired two-way ANOVA with the Bonferroni posttest. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 6G, J Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Transfection, Negative Control, Knockdown, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Injection, In Vivo, Adoptive Transfer Assay, Comparison

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: Flow cytometry was performed to analyze CD45 + lineage – CRTH2 + CD127 + GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in PBMCs purified from asthmatic patients during remission phase (all rested for 50 days or longer) and healthy donors. A Gating strategy, B statistical plots, C IL-13 + percentage and MFI and D IL-5 + percentage and MFI of CD45 + lineage – CRTH2 + CD127 + GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s. E KLRG1 – , KLRG1 + IL-17RB – and KLRG1 + IL-17RB + subsets were purified from the total CD45 + lineage – CRTH2 + CD127 + ST2 – ILC2s in PBMC of 6 asymptomatic asthmatic patients (marked as A-KLRG1 – , A-KLRG1 + IL-17RB – and A-KLRG1 + IL-17RB + ). KLRG1 + IL-17RB + subsets were sorted from total ILC2s in PBMC of 3 healthy donors (marked as H-KLRG1 + IL-17RB + ). Cells were then cultured (5 × 10 3 /well) with 10 ng/mL IL-2 and 20 ng/mL IL-7, and treated with (alarmins+) or without (alarmins−) 200 ng/mL recombinant human IL-17E/IL-25 and 200 ng/mL recombinant human IL-33 every other day for 21 days. Subsequently, cells were collected for further analysis. F Ki-67 + percentage, G absolute number and H fold changes of expansion of cells were determined. Representative plots and statistical results (bar graph) showing the percentages of I IL-13 + and J IL-5 + of three ILC2 subsets after stimulation with PMA, ionomycin, brefeldin A and monensin. KLRG1 – , KLRG1 + IL-17RB – and KLRG1 + IL-17RB + subsets in total CD45 + lineage – CRTH2 + CD127 + ST2 – ILC2s in PBMC of asymptomatic asthmatic patients (marked as A-KLRG1 – , A-KLRG1 + IL-17RB – and A-KLRG1 + IL-17RB + ) and KLRG1 + IL-17RB + subsets in total ILC2s in PBMC of healthy donors (marked as H-KLRG1 + IL-17RB + ) were sorted (4.5 ~ 6.8 × 10 5 for every subset, purity > 98%). K , L Total RNA was isolated, and qRT-PCR was performed. K Heat map of mRNA expression values of selected genes. L Bar plots of mRNA expression values (relative to GAPDH ) for genes significantly changed. Data was shown as Means ± SD. n = 8 H and 15 A for ( A – D ). n = 11 ( H ) and 18 A for E – J . n = 6 (A-KLRG1 – , A-KLRG1 + IL-17RB – and A-KLRG1 + IL-17RB + ) and 3 (H-KLRG1 + IL-17RB + ) for ( K, L ). Statistical comparisons were performed using unpaired, two-sided Welch’s t test for ( B – D ), and unpaired one-way ANOVA with Dunnett’s test for ( L ). p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 7E Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Flow Cytometry, Purification, Cell Culture, Recombinant, Isolation, Quantitative RT-PCR, Expressing