Journal: Autophagy

Article Title: LAMP2-FLOT2 interaction enhances autophagosome-lysosome fusion to protect the septic heart in response to ILC2

doi: 10.1080/15548627.2025.2469207

Figure Lengend Snippet: ILC2 is the major IL4-producing cell subset in septic heart. (A, B) Representative flow cytometry plots (A) and bar graph (B) of percentages of IL4-, IL9-, IL13-, and IL5-positive ILC2 in the hearts at the indicated time points after CLP surgery. (C, F) Representative flow cytometry plots (C) and bar graph (F) of percentages of IL4-positive T cells in the hearts after CLP or sham surgery. (D, E) the expression levels of IL4 on CD3-positive T cells (D) and CD4-positive T cells (E) in the hearts after CLP or sham surgery. (G, H) ILC2 were depleted with anti-THY1 antibody and were analyzed by flow cytometry. (I, J) the expression levels and mean fluorescence intensity (MFI) of IL4 on ILC2 in the hearts. The data are expressed as mean ± SEM ( n = 4–5/group). In B, two-way ANOVA with Turkey’s multiple comparisons tests; in F, unpaired Student’s t-test; in H and J, one-way ANOVA. ns, no significance; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Plasma levels of IL4 (MULTI SCIENCES, EK204), IL5 (MULTI SCIENCES, EK205), IL9 (MULTI SCIENCES, EK209), and IL13 (MULTI SCIENCES, EK213) protein levels were detected using mouse ELISA kits.

Techniques: Flow Cytometry, Expressing, Fluorescence

Journal: Cell Reports

Article Title: Mechanical force receptor Piezo1 regulates TH9 cell differentiation

doi: 10.1016/j.celrep.2024.115136

Figure Lengend Snippet: Figure 1. Piezo1 expression is related to the TH9 cell differentiation in allergic airway inflammation and tumors (A) Piezo1 mRNA expression examined by qPCR analyses in T cells activated by various T cell subset-inducing conditions for 5 days (expression in naive T cells was set to 1). (B) Naive T cells were activated by indicated condition for different time course, and Piezo1 mRNA expression was determined by qPCR. Expressions in naive T cells were set to 1. (C) Expression of Piezo1 examined by western blot in T cells activated by the indicated condition for 5 days. (D) Wild-type (WT) mice were sensitized and challenged intranasally with ovalbumin (OVA). mRNA expression of Il9 and Piezo1 in sorted CD4+ T cells isolated from pulmonary dLN cells in WT mice at the indicated time point. (E and F) B16.F10 (E) and EL-4 (F) tumor cells were implanted subcutaneously in WT mice. mRNA expression of Il9 and Piezo1 in sorted CD4+ T cells isolated from tumor in WT mice at the indicated time point. (G–I) B16.F10 tumor cells were implanted subcutaneously in WT mice for 30 days, tumor size was measured, and the percentage of IL-9+CD4+ T cells was examined by flow cytometry. The correlations between the percentage of IL-9+CD4+ T (G) or tumor volume (H) and Piezo1 mRNA level are shown. The correlations between the tumor volume and Piezo1 mRNA level are shown (I). Data are representative of three or four independent experiments (n = 3–10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001, compared with the indicated groups.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GsMTx4 MedChemExpress, USA Cat#HY-P1410 Ionomycin Sigma-Aldrich, USA Cat#I0634 Yoda1 MedChemExpress, USA Cat#HY-18723 Critical commercial assays Co-IP buffer Thermo Fisher Scientific, USA Cat#88804 CD4+ T cell isolation kit Miltenyi Biotec, USA Cat#130-104-453 ECL kit Beyotime Biotechnology, China Cat#P10018AS Enhanced BCA protein assay kit Beyotime Biotechnology, China Cat#P0009 Foxp3/transcription factor staining buffer set BD Bioscience, USA Cat#560133 Mito stress test kit Seahorse Bioscience, USA Cat#101706-100 NE-PER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific, USA Cat#78835 RIPA lysis buffer Beyotime Biotechnology, China Cat#P0013B RNeasy mini kit Qiagen, Germany Cat#74106 PrimeScriptTM RT reagent kit TakaRa, Japan Cat#TakaRa_RR037A SuperReal preMix plus SYBR Green Tiangen, Germany Cat#FP205-02 Dual-Luciferase assay system Promega, USA Cat#E1910 Mouse IL-9 DuoSet ELISA R&D system, USA Cat#DY409 Human IL-9 DuoSet ELISA R&D system, USA Cat#DY209-05 Deposited data Raw and analyzed data This paper GEO:GSE268658 Experimental models: Cell lines Mouse melanoma cell line B16.F10 China cell bank ATCC, China B16F10 Mouse lymphoma cell line EL-4 China cell bank ATCC, China EL4 HEK293T ATCC, USA Cat#CRL-11268 Phoenic-Eco packaging cells Allele Biotechnology, USA Cat#ABP-RVC-10001 Experimental models: Organisms/strains Mouse: C57BL/6J (CD45.1) Beijing Weitonglihua Experimental Animal Center, China NA Mouse: C57BL/6J (CD45.2) Beijing Weitonglihua Experimental Animal Center, China NA Mouse: C57BL/6J-Rag1 / GemPharmatech, China Cat#T004753 Mouse: Piezo1flox/flox GemPharmatech, China Cat#T009627 Mouse: Cd4-Cre GemPharmatech, China Cat#T067676 Mouse: Piezo1 / GemPharmatech, China Cat#T012739 Mouse: Sirt3flox/flox GemPharmatech, China Cat#T006658 Mouse: Hif1aflox/flox GemPharmatech, China Cat#T007329 Mouse: C57BL/6J (GFP) GemPharmatech, China Cat#T054573 Oligonucleotides siRNA targeting sequence: SDHA siRNA, 50-UAAAUCUUCCCAUCUUCAGUU-30 This paper NA siRNA targeting sequence: Piezo1 siRNA, 50-CACCGGCATCTACGTCAAATA-30 This paper NA Piezo1 (Mm01241549_m1) Applied Biosystems, USA Cat#4331182 Piezo1 (Hs00207230_m1) Applied Biosystems, USA Cat#4331182 IL-9 (Mm00434305_m1) Applied Biosystems, USA Cat#4331182 IL-9 (Hs00174125_m1) Applied Biosystems, USA Cat#4331182 Sirt3 (Mm00452131_m1) Applied Biosystems, USA Cat#4331182 Sirt3 (Hs00202030_m1) Applied Biosystems, USA Cat#4331182 (Continued on next page) Cell Reports 44, 115136, January 28, 2025 17

Techniques: Expressing, Cell Differentiation, Western Blot, Isolation, Cytometry

Journal: Cell Reports

Article Title: Mechanical force receptor Piezo1 regulates TH9 cell differentiation

doi: 10.1016/j.celrep.2024.115136

Figure Lengend Snippet: Figure 3. Piezo1 deficiency inhibits TH9 cell differentiation in allergic airway inflammation (A–C) WT and Piezo1/ mice were sensitized and challenged intranasally with OVA on day 0. (A) Pathologic photo of H&E staining (left) and immunohistochemical staining with anti-CD4 mAb (medium) and anti-IL-9 mAb (right) in lungs from mice challenged 48 h after the final OVA challenge. Original magnification, 3400. (B) Flow cytometry of Siglec F in CD11b+ cells from BALF in WT and Piezo1/ mice 48 h after the final OVA challenge. Representative dotplots from flow cytometry are shown (left) and data are summarized (right). (C) Intracellular staining of IL-9 in CD4+ T cells from BALF in WT and Piezo1/ mice 48 h after the final OVA challenge. Representative dotplots from flow cytometry are shown (left), and data are summarized (right). (D–G) Rag1/ mice given an intravenous transfer of WT or Piezo1/ CD4+ T cells (stimulated via anti-CD3 and anti-CD28, together with TGF-b1 and IL-4 for 3 days) were sensitized and challenged intranasally with OVA on day 0, then treated with mouse anti-IL-9 (a-IL-9; R&D Systems) or isotype-matched control antibody (Ctrl Ab; IgG2b; R&D Systems) 20 mg, intravenously, 30 min before the challenges, every day from day 20 to day 24. (D) Flow cytometry of Siglec F in CD11b+ cells from BALF in mice 48 h after the final OVA challenge and data summarized. (E) Il9 mRNA expression and (F) secretion in sorted CD4+ T cells from BALF in indicated mice 48 h after the final OVA challenge and stimulated for 24 h with a-CD3. Expression in control group in WT mice was set to 1. (G) Intracellular staining of IL-9 in CD4+ T cells in BALF from indicated mice 48 h after the final OVA challenge. Representative dotplots from flow cytometry are shown (left) and data are summarized (right). (H) Mixed chimeras were generated by transferring 1 3 107 BM cells (BMs) from either WT (CD45.1+) and WT (CD45.2+) or Piezo1/ (CD45.2+) mice at a ratio of 1:1 to lethally irradiated recipient C57BL/6 GFP+ mice. Intracellular staining of IL-9 in CD4+ T cells among CD45.2+ or CD45.1+ donor cells in the BALF from mixed chimeras reconstituted with BMs from the WT and WT or Piezo1/ mice 48 h after the final OVA challenge. Representative dotplots from flow cytometry are shown (left) and data are summarized (right). Data are representative of three to four independent experiments (n = 4–10 mice per group). ***p < 0.001, compared with the indicated groups.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GsMTx4 MedChemExpress, USA Cat#HY-P1410 Ionomycin Sigma-Aldrich, USA Cat#I0634 Yoda1 MedChemExpress, USA Cat#HY-18723 Critical commercial assays Co-IP buffer Thermo Fisher Scientific, USA Cat#88804 CD4+ T cell isolation kit Miltenyi Biotec, USA Cat#130-104-453 ECL kit Beyotime Biotechnology, China Cat#P10018AS Enhanced BCA protein assay kit Beyotime Biotechnology, China Cat#P0009 Foxp3/transcription factor staining buffer set BD Bioscience, USA Cat#560133 Mito stress test kit Seahorse Bioscience, USA Cat#101706-100 NE-PER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific, USA Cat#78835 RIPA lysis buffer Beyotime Biotechnology, China Cat#P0013B RNeasy mini kit Qiagen, Germany Cat#74106 PrimeScriptTM RT reagent kit TakaRa, Japan Cat#TakaRa_RR037A SuperReal preMix plus SYBR Green Tiangen, Germany Cat#FP205-02 Dual-Luciferase assay system Promega, USA Cat#E1910 Mouse IL-9 DuoSet ELISA R&D system, USA Cat#DY409 Human IL-9 DuoSet ELISA R&D system, USA Cat#DY209-05 Deposited data Raw and analyzed data This paper GEO:GSE268658 Experimental models: Cell lines Mouse melanoma cell line B16.F10 China cell bank ATCC, China B16F10 Mouse lymphoma cell line EL-4 China cell bank ATCC, China EL4 HEK293T ATCC, USA Cat#CRL-11268 Phoenic-Eco packaging cells Allele Biotechnology, USA Cat#ABP-RVC-10001 Experimental models: Organisms/strains Mouse: C57BL/6J (CD45.1) Beijing Weitonglihua Experimental Animal Center, China NA Mouse: C57BL/6J (CD45.2) Beijing Weitonglihua Experimental Animal Center, China NA Mouse: C57BL/6J-Rag1 / GemPharmatech, China Cat#T004753 Mouse: Piezo1flox/flox GemPharmatech, China Cat#T009627 Mouse: Cd4-Cre GemPharmatech, China Cat#T067676 Mouse: Piezo1 / GemPharmatech, China Cat#T012739 Mouse: Sirt3flox/flox GemPharmatech, China Cat#T006658 Mouse: Hif1aflox/flox GemPharmatech, China Cat#T007329 Mouse: C57BL/6J (GFP) GemPharmatech, China Cat#T054573 Oligonucleotides siRNA targeting sequence: SDHA siRNA, 50-UAAAUCUUCCCAUCUUCAGUU-30 This paper NA siRNA targeting sequence: Piezo1 siRNA, 50-CACCGGCATCTACGTCAAATA-30 This paper NA Piezo1 (Mm01241549_m1) Applied Biosystems, USA Cat#4331182 Piezo1 (Hs00207230_m1) Applied Biosystems, USA Cat#4331182 IL-9 (Mm00434305_m1) Applied Biosystems, USA Cat#4331182 IL-9 (Hs00174125_m1) Applied Biosystems, USA Cat#4331182 Sirt3 (Mm00452131_m1) Applied Biosystems, USA Cat#4331182 Sirt3 (Hs00202030_m1) Applied Biosystems, USA Cat#4331182 (Continued on next page) Cell Reports 44, 115136, January 28, 2025 17

Techniques: Cell Differentiation, Staining, Immunohistochemical staining, Flow Cytometry, Cytometry, Control, Expressing, Generated, Transferring, Irradiation

Journal: Cell Reports

Article Title: Mechanical force receptor Piezo1 regulates TH9 cell differentiation

doi: 10.1016/j.celrep.2024.115136

Figure Lengend Snippet: Figure 4. Piezo1 deficiency inhibits TH9 cell differentiation in tumor (A–C) B16.F10 tumor cells were implanted subcutaneously in WT and Piezo1/ mice (n = 10), and tumor size was measured every 5 days for 30 days. (A) Tumor growth curve. (B) Pathologic photo of H&E staining (left) and immunohistochemical staining (right) with anti-CD4 mAb and anti-IL-9 mAb in tumor on day 30. (C) Intracellular staining of IL-9 in CD4+ T cells in tumor from WT and Piezo1/ mice on day 30. Representative dotplots from flow cytometry are shown (left) and data are summarized (right). (D–G) Rag1/ mice given an intravenous transfer of WT or Piezo1/ CD4+ T cells (stimulated via anti-CD3 and anti-CD28, together with TGF-b1 and IL-4 for 3 days) along with simultaneous subcutaneous injection of B16.F10 melanoma cells on day 0, then intravenously treated with anti-IL-9 (a-IL-9; R&D Systems) or isotype-matched control antibody (Ctrl Ab; IgG2b; R&D Systems) 20 mg, every 3 days from day 0. (D) Tumor growth curve. (E) Il9 mRNA expression and (F) secretion in sorted CD4+ T cells from indicated mouse tumor and stimulated for 24 h with a-CD3. Expression in control group in WT mice was set to 1. (G) Intracellular staining of IL-9 in CD4+ T cells in tumor from indicated mice. Representative dot-plots from flow cytometry are shown (left) and data are summarized (right). (H) Mixed chimeras were generated by transferring 1 3 107 BM cells (BMs) from either WT (CD45.1+) and WT (CD45.2+) or Piezo1/ (CD45.2+) mice at a ratio of 1:1 to lethally irradiated recipient C57BL/6 GFP+ mice. Intracellular staining of IL-9 in CD4+ T cells among CD45.2+ or CD45.1+ donor cells in tumor from mixed chimeras mice on day 30. Representative dotplots from flow cytometry are shown (left) and data are summarized (right). Data are representative of three to four independent experiments (n = 4–12 mice per group). ***p < 0.001, compared with the indicated groups.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GsMTx4 MedChemExpress, USA Cat#HY-P1410 Ionomycin Sigma-Aldrich, USA Cat#I0634 Yoda1 MedChemExpress, USA Cat#HY-18723 Critical commercial assays Co-IP buffer Thermo Fisher Scientific, USA Cat#88804 CD4+ T cell isolation kit Miltenyi Biotec, USA Cat#130-104-453 ECL kit Beyotime Biotechnology, China Cat#P10018AS Enhanced BCA protein assay kit Beyotime Biotechnology, China Cat#P0009 Foxp3/transcription factor staining buffer set BD Bioscience, USA Cat#560133 Mito stress test kit Seahorse Bioscience, USA Cat#101706-100 NE-PER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific, USA Cat#78835 RIPA lysis buffer Beyotime Biotechnology, China Cat#P0013B RNeasy mini kit Qiagen, Germany Cat#74106 PrimeScriptTM RT reagent kit TakaRa, Japan Cat#TakaRa_RR037A SuperReal preMix plus SYBR Green Tiangen, Germany Cat#FP205-02 Dual-Luciferase assay system Promega, USA Cat#E1910 Mouse IL-9 DuoSet ELISA R&D system, USA Cat#DY409 Human IL-9 DuoSet ELISA R&D system, USA Cat#DY209-05 Deposited data Raw and analyzed data This paper GEO:GSE268658 Experimental models: Cell lines Mouse melanoma cell line B16.F10 China cell bank ATCC, China B16F10 Mouse lymphoma cell line EL-4 China cell bank ATCC, China EL4 HEK293T ATCC, USA Cat#CRL-11268 Phoenic-Eco packaging cells Allele Biotechnology, USA Cat#ABP-RVC-10001 Experimental models: Organisms/strains Mouse: C57BL/6J (CD45.1) Beijing Weitonglihua Experimental Animal Center, China NA Mouse: C57BL/6J (CD45.2) Beijing Weitonglihua Experimental Animal Center, China NA Mouse: C57BL/6J-Rag1 / GemPharmatech, China Cat#T004753 Mouse: Piezo1flox/flox GemPharmatech, China Cat#T009627 Mouse: Cd4-Cre GemPharmatech, China Cat#T067676 Mouse: Piezo1 / GemPharmatech, China Cat#T012739 Mouse: Sirt3flox/flox GemPharmatech, China Cat#T006658 Mouse: Hif1aflox/flox GemPharmatech, China Cat#T007329 Mouse: C57BL/6J (GFP) GemPharmatech, China Cat#T054573 Oligonucleotides siRNA targeting sequence: SDHA siRNA, 50-UAAAUCUUCCCAUCUUCAGUU-30 This paper NA siRNA targeting sequence: Piezo1 siRNA, 50-CACCGGCATCTACGTCAAATA-30 This paper NA Piezo1 (Mm01241549_m1) Applied Biosystems, USA Cat#4331182 Piezo1 (Hs00207230_m1) Applied Biosystems, USA Cat#4331182 IL-9 (Mm00434305_m1) Applied Biosystems, USA Cat#4331182 IL-9 (Hs00174125_m1) Applied Biosystems, USA Cat#4331182 Sirt3 (Mm00452131_m1) Applied Biosystems, USA Cat#4331182 Sirt3 (Hs00202030_m1) Applied Biosystems, USA Cat#4331182 (Continued on next page) Cell Reports 44, 115136, January 28, 2025 17

Techniques: Cell Differentiation, Staining, Immunohistochemical staining, Cytometry, Injection, Control, Expressing, Generated, Transferring, Irradiation