Fig 1: TSLP promotes accumulation of progenitor factor DDX6(A and B) qRT-PCR analysis of DDX6, KLF4, and FLG of RNA isolated from primary human keratinocytes treated with sfTSLP or lfTSLP for (A) 16 h or (B) 24 h.(C) Immunostaining of DDX6 (red) in primary human keratinocytes stimulated with human TSLP (100 ng/mL) for 20 h. Scale bars: 20 μm.(D) Quantification of DDX6. Graph represents average ± SEM DDX6 intensity from n = 17–28 images/group; ∗∗∗∗p < 0.0001.(E) Representative histogram of Ki67 staining of primary human keratinocytes stimulated with vehicle control, 3 nM lfTSLP, or sfTSLP for 40 h. Isotype IgG used for control.(F and G) Quantification of Ki67+ (F) cell abundance and (G) Ki67 MFI in primary human keratinocytes stimulated with 3 nM lfTSLP and sfTSLP for 24 h. Data represent averages ± SEM of 3 independent experiments using 2 or 3 different human donors in technical triplicates.∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.See also Table S1.
Fig 2: A. fumigatus–stimulated DCs activates JAK/STAT through TSLP. (A) DCs were treated with A. fumigatus and then co-cultured with CD4+ T cells for 4 days, a Western blot analysis was performed to determine the protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin in the CD4+ T cells. (B) CD4+ T cells were cultured with rmTSLP (100 ng/mL) for 3 days, protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin were analyzed by western blot. Quantification of relative protein levels in (A) and (B) were shown in (C) and (D), respectively. Cells were divided into four groups and were treated as described in Figure 4C. (E) Western blot analysis of the protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin in CD4+ T cells. (F) CD4+ T cells were incubated with TLSPR siRNA (80 nM) or NC siRNA for 24 hours, then co-cultured with A. fumigatus–stimulated DCs for 4 days. A Western blot was performed to detect the protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin in CD4+ T cells. Quantification of relative protein levels in (E) and (F) were shown in Supplementary Figure S4C and D. (Data are mean ± SEM, *P < 0.05, **P < 0.01, n = 3).
Fig 3: TSLP expands CD34+ITGα6lo transit amplifying cells (TACs)(A) Experimental timeline for hair growth analysis following s.c. TSLP (250 ng).(B) Quantification of skin area that has entered anagen. Data are from n = 3 mice per group.(C) Representative photos of mouse back skin after s.c. TSLP treatment.(D) Experimental timeline for analysis of TSLP-driven cell proliferation.(E) Representative flow cytometry plots of CD34+ cells.(F) Quantification of CD34+ cells pre-gated on live, single cells.(G) Quantification of total, live, EdU+ cells.(H) IFlow cytometry plots representing ITGα6 expression pre-gated on CD34+ cells.(I) Quantification of CD34+ITGα6 subpopulations.(J) Flow cytometry histogram overlay of CD34+ITGα6lo cells from TSLP- and vehicle-treated groups.(K) Quantification of EdU+CD34+ITGα6lo. Graphs represent means of 2 experiments using 6–8 mice per group ± SEM.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. See also Figure S4.
Fig 4: TSLP promotes FK progression through the JAK/STAT signaling pathway. C57BL/6 mice were pretreated by subconjunctival injection with 5 µL BSA, 5 µL rmTSLP (100 ng/µL), 5 µL NC siRNA, or 5 µL TSLP siRNA 1 day before infection with A. fumigatus hyphae (5 µL) for 1 day. (A) Slit-lamp examination was used to assess the clinical manifestation; fluorescein staining was performed to assess the denuded epithelium at 1 day after infection. (B) The average clinical scores were calculated to assess the clinical manifestation at 1 day after infection. (C and D) qRT-PCR was performed to detect the mRNA levels of TSLP, IL-17A, IL-17F, and IL-22 in mouse corneas at 1 day post infection. (E) ELISA was performed to detect the protein levels of IL-17A, IL-17F, and IL-22 in mouse corneas at 1 day after infection. The mouse eyes were subconjunctivally injected with PBS, 5 µL ruxolitinib (0.1 mmol/L), or 5 µL BBI608 (3 mmol/L) 1 day before infection with A. fumigatus hyphae for 1 day. (F) Slit-lamp examination was used to assess the clinical manifestations and fluorescein staining was performed to assess the denuded epithelium at 1 day after infection. (G) The average clinical scores were calculated to assess the clinical manifestation at 1 day after infection. (H) qRT-PCR and (I) ELISA were performed to analyze the mRNA and protein levels of IL-17A, IL-17F, and IL-22 in mouse corneas at 1 day post infection. (Data are mean ± SEM, *P < 0.05, **P < 0.01, n = 6).
Fig 5: TSLP promotes CD4+ T cell proliferation and Th17 cytokine expression. (A) CD4+ T cells were stained with CFSE and then cultured with or without 100 ng/mL rmTSLP for 4 days, flow cytometry was performed to detect the proliferation of CD4+ T cells. (B) Quantification of cell proliferation rate in (A). CD4+ T cells were cultured with rmTSLP (100 ng/mL) for 1, 2, and 3 days. (C) qRT-PCR and (D) ELISA were performed to detect the mRNA and protein levels of IL-17A, IL-17F, and IL-22 in CD4+ T cells. (E) Flow cytometry was conducted to analyze the protein levels of IL-17A in CD4+ T cells. (F) Quantification of IL-17A levels in (E). Ctr, control. (Data are mean ± SEM, *P < 0.05, **P < 0.01, n = 3).
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Mouse TSLP Protein