Journal: bioRxiv
Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis
doi: 10.64898/2026.03.23.713256
Figure Lengend Snippet: A. Diagram of the Canonical and Non-canonical STING signaling (created with BioRender.com ). B. Constitutive STAT3 and STAT1 activation in SAVI iECs. Phospho-STAT3 Y705 and phospho-STAT1 Y701 (normalized to total protein) were measured in iECs from 3 HC and 3 SAVI donors across passages 1, 3, and 5. SAVI iECs showed increased baseline STAT3 activation (mean ± SEM; ***p < 0.001, **p < 0.01, *p < 0.05, unpaired t-test). C. STING/TBK1 inhibition blocks acute cGAMP-induced STAT3 activation in HC iECs. iECs from 3 HC donors were stimulated with 2’3’-cGAMP (20µg/ml) for 30 min–24 h. STING inhibitor IFM35883 (STINGi, 2.5 μM) and TBK1 inhibitor MRT67307 (TBK1i, 5 μM) were pre-treated one hour before cGAMP. STAT3 activation at 4h was prevented by STING/TBK1 inhibition (mean ±SEM, ***p < 0.001, **p < 0.01, Mann-Whitney test). D. STING/TBK1 inhibition reduces constitutive STAT3 activation in SAVI iECs. SAVI iECs were treated with IFM35883 (2.5 μM) from P2–P5 or with TBK1i (5 μM) for 48 h. Quantification is from four experiments in iEC line from patient SAVI1 (mean ±SEM, ***p < 0.001, unpaired t-test). E. STAT3 shRNA knockdown (KD), not STAT1 KD preserves CD144 (VE-cadherin) in SAVI iECs. iECs from 3 individual SAVI patients infected with control, STAT3, or STAT1 shRNA at P2 were analyzed by flow cytometry at P5 (mean ±SEM, **p < 0.01, paired t-test). Representative flow cytometry profiles are shown in Figure S4C. F. Constitutive protein expression of SLUG/ SNAI2 is elevated in SAVI iECs and suppressed by STING inhibition. Protein from 3 HC, 3 SAVI, and 3 iso-SAVI iECs at P5 showed increased SLUG (normalized by GAPDH) in SAVI; IFM35883 (2.5 μM) treatment from P2–P5 prevented SLUG upregulation in SAVI iECs (SAVI 1) (mean ±SEM, ***p < 0.001, **p < 0.01, unpaired t-test). See also Figure S4E. G. STAT3 shRNA knockdown reduces SLUG mRNA expression (normalized by internal control 18S) in SAVI iECs at P3. Data are summarized from 3 individual SAVI patients iEC lines. *p < 0.01 as determined by two-tailed unpaired t-test.
Article Snippet: After blocking with LI-COR blocking buffer, the membranes were incubated with primary antibodies for VE-cadherin (1:1000, CST, Cat#2500S), CD31(1:1000, DAKO, Cat#M0823), SMA (1:2000, Abcam, Cat#ab7817), SM22 (1:1000, Abcam, Cat#ab14106), SOX18 (1:100, Santa Cruz, Cat#sc-166025), SLUG (1:1000, CST, Cat#9585S), P-STAT3 Y705 (1:1000, CST, Cat#9145S), STAT3 (1:1000, CST, Cat#9139S), P-STAT1 Y701 (1:1000, CST, Cat#9167S), STAT1(1:1000, CST, Cat#9172S), P-STING S366 (1:1000, CST, Cat#19781S), STING (1:1000, CST, Cat#13647S), P-TBK1 S172 (1:1000, CST, Cat#5483S), TBK1 (1:1000, CST, Cat#3504S), P-IRF3 S396 (1:1000, CST, Cat#4947S), and IRF3 (1:1000, CST, Cat#11904S) overnight at 4°C; these antibodies are listed in the Key Resource table.
Techniques: Activation Assay, Inhibition, MANN-WHITNEY, shRNA, Knockdown, Infection, Control, Flow Cytometry, Expressing, Two Tailed Test
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