tbst il 33  (R&D Systems)

Journal: Nature Communications

Article Title: Single-cell multiomics uncovers an endothelial mechanosensitive PIEZO1-IL-33 axis driving pulmonary fibrosis

doi: 10.1038/s41467-026-70193-w

Figure Lengend Snippet: A CellChat analysis of human lung snRNA-seq data. B Venn of 47 shared DEGs from four datasets ( GSE159354 , GSE132771 , GSE128033 , GSE122960 ). C Heatmap of 47-gene expression across cell types. D Correlation analysis (Pearson Correlation) of expression of IGFBP5, IL33, MGP, SPRY1, ACKR1 and mechanical stress scores in ECs. E Correlation analysis (Pearson Correlation) of expression of PIEZO1 and IGFBP5, IL33, MGP, SPRY1, ACKR1 in ECs. F Human snRNA-seq UMAP and violin of IL33 distribution and subpopulations. G Bar chart showed expression of IL33 (mean ± SEM) in IPF (n = 7966 cells) vs control (n = 4174 cells). P value was calculated using the two-tailed unpaired t-test. H Violin of IL33 in PIEZO1 - vs PIEZO1 + ECs. I Bulk-RNA-seq of saline and BLM (7 days and 14 days) induced mice (n = 3 per group). Gene expression levels were Z-score normalized. P value was calculated using the two-tailed unpaired t-test. J qPCR of Il33 in BLM mouse lung ECs. K IL-33 (green) and CD31 (red) co-staining in human IPF vs normal, quantification is shown at right (n = 10). L IL-33 (green) and CD31 (red) co-staining in Yoda1- or GsMTx4-treated fibrotic mouse lungs, quantification is shown at right (n = 3). M IL-33 (green) and CD31 (red)co-staining in Piezo1 WT and Piezo1 ΔEC BLM lungs, quantification is shown at right (n = 3). Error bars represent mean ± SEM, H, K, J, M: two-tailed unpaired t-test; L: one-way ANOVA followed Tukey’s multiple comparwasons test, All n values indicate biologically independent samples. Source data are provided as a Source Data file.

Article Snippet: 30–60 μg of protein was loaded per well on 4–20% SDS-PAGE gels (ACE, #ET15420LGel), transferred to ethanol-activated PVDF membranes (Thermo Fisher Scientific, #88518), blocked with 5% skim milk in TBST, and incubated overnight at 4 °C with primary antibodies diluted in TBST: IL-33 (R&D Systems, #AF3626, 1:1000), PIEZO1 (Proteintech, #15939-1-AP, 1:1 000), CALPAIN2 (Labome, #A03492, 1:1000), P-STAT3 (CST, #9134 s, 1:1000), STAT3 (Abcam, #ab68153, 1:2 000), P-STAT1 (CST, #9171, 1:1000), STAT1 (Proteintech, #10144-2-AP, 1:1000), GAPDH (Servicebio, #GB12002, 1:5000), β-actin (Proteintech, #66009-1, 1:5000), β-tubulin (CST, #2146, 1:5000).

Techniques: Gene Expression, Expressing, Control, Two Tailed Test, RNA Sequencing, Saline, Staining

Journal: Nature Communications

Article Title: Single-cell multiomics uncovers an endothelial mechanosensitive PIEZO1-IL-33 axis driving pulmonary fibrosis

doi: 10.1038/s41467-026-70193-w

Figure Lengend Snippet: A Schematic of BLM-induced fibrosis in Il33 WT vs Il33 ΔEC mice. B Il33 mRNA (n = 4) and IL-33 protein in lung ECs (n = 3). C Representative Masson/PSR images and quantification (n = 5). D αSMA immunofluorescence and quantification (n = 4). E Lung hydroxyproline content (n = 5). F Schematic: EC-specific Il33 overexpression via AAV-Tie1 in Piezo1 WT and Piezo1 ΔEC mice; 4-week pretreatment, BLM at week 4, sacrifice at week 7. G IL-33 expression in ECs, immune and epithelial cells after AAV-OE- Il33 (n = 3). H Masson/PSR images and quantification (n = 5). I αSMA immunofluorescence and quantification (n = 4). J Hydroxyproline content (n = 5). Error bars: mean ± SEM; B,G: two-tailed unpaired t-test; C,D-E,I: two-way ANOVA followed Tukey’s multiple comparisons test; H,J: three-way ANOVA followed Tukey’s multiple comparisons test. All n values indicate biologically independent samples. Source data are provided as a Source Data file.

Article Snippet: 30–60 μg of protein was loaded per well on 4–20% SDS-PAGE gels (ACE, #ET15420LGel), transferred to ethanol-activated PVDF membranes (Thermo Fisher Scientific, #88518), blocked with 5% skim milk in TBST, and incubated overnight at 4 °C with primary antibodies diluted in TBST: IL-33 (R&D Systems, #AF3626, 1:1000), PIEZO1 (Proteintech, #15939-1-AP, 1:1 000), CALPAIN2 (Labome, #A03492, 1:1000), P-STAT3 (CST, #9134 s, 1:1000), STAT3 (Abcam, #ab68153, 1:2 000), P-STAT1 (CST, #9171, 1:1000), STAT1 (Proteintech, #10144-2-AP, 1:1000), GAPDH (Servicebio, #GB12002, 1:5000), β-actin (Proteintech, #66009-1, 1:5000), β-tubulin (CST, #2146, 1:5000).

Techniques: Immunofluorescence, Over Expression, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Single-cell multiomics uncovers an endothelial mechanosensitive PIEZO1-IL-33 axis driving pulmonary fibrosis

doi: 10.1038/s41467-026-70193-w

Figure Lengend Snippet: HUVECs under 20% stretch for 0,6,24 h or 2,12,25 kPa substrates; IL-33 secretion ( n = 3) A and mRNA (n = 3) B . C PIEZO1 knock-down with sh PIEZO1 ( n = 3). HUVECs were transduced with sh PIEZO1 for 48 h, cultured on 25 kPa substrates for 24 h or subjected to 6 h mechanical stretch before measuring IL-33 secretion ( n = 3) D and mRNA ( n = 3) E . HUVECs were cultured on 2, 12, 25 kPa substrates for 24 h or stretched (20%) for 0, 6, 24 h, followed by measurement of calpain activity F and CAPN2 protein G ( n = 3). HUVECs (sh PIEZO1 ) were cultured on 25 kPa substrates for 24 h or subjected to 6 h mechanical stretch, followed by measurement of calpain activity ( n = 3) H and CAPN2 protein levels ( n = 3) I . J CAPN2 knock-down with shCAPN2 ( n = 3). K HUVECs (sh CAPN2 ) were cultured on 25 kPa substrates for 24 h or subjected to 6 h mechanical stretch, IL-33 secretion(left) and mRNA(right) ( n = 3). L Model for paracrine IL-33 regulation. M Cistrome: STAT3 predicted to drive IL-33 regulation. N intersection of top 100 motifs in BLM-treated mouse ECs with top 10 TFs. O The protein levels of P-STAT3 and STAT3 in HUVECs treated with 20% stretch for 0 and 6 hours ( n = 3). P The protein levels of P-STAT3 and STAT3 in HUVECs (sh PIEZO1 or sh CAPN2 ) treated with 20% stretch for 6 h ( n = 3). Q STAT3 knock-down with sh STAT3 ( n = 3). R HUVECs (sh STAT3 ) were cultured on 25 kPa substrates for 24 h or subjected to 6 h mechanical stretch; IL-33 secretion(left) and mRNA(right) ( n = 3). Error bars: mean ± SEM; C , G , J , O – Q : two-tailed unpaired t-test; A , B , F : one-way ANOVA followed Šídák’s multiple comparisons test, D – E , H – I , K , R : two-way ANOVA Tukey’s multiple comparisons test. All n denote biologically independent samples. Source data are provided as a Source Data file.

Article Snippet: 30–60 μg of protein was loaded per well on 4–20% SDS-PAGE gels (ACE, #ET15420LGel), transferred to ethanol-activated PVDF membranes (Thermo Fisher Scientific, #88518), blocked with 5% skim milk in TBST, and incubated overnight at 4 °C with primary antibodies diluted in TBST: IL-33 (R&D Systems, #AF3626, 1:1000), PIEZO1 (Proteintech, #15939-1-AP, 1:1 000), CALPAIN2 (Labome, #A03492, 1:1000), P-STAT3 (CST, #9134 s, 1:1000), STAT3 (Abcam, #ab68153, 1:2 000), P-STAT1 (CST, #9171, 1:1000), STAT1 (Proteintech, #10144-2-AP, 1:1000), GAPDH (Servicebio, #GB12002, 1:5000), β-actin (Proteintech, #66009-1, 1:5000), β-tubulin (CST, #2146, 1:5000).

Techniques: Knockdown, Transduction, Cell Culture, Activity Assay, Two Tailed Test