Fig 2: NF-?B activation significantly mediates the hypoxia–reoxygenation (H-R) challenge-induced increase in the recruitment and occupancy of active RNA polymerase II (RNAPII) at the miR-210 promoter. (A,B) Tandem ChIP-ELOHA analysis showing the relative enrichment of the largest subunit and the catalytic component of RNAPII, RPB1 (A), and the CTD-phosphorylated Ser2/Ser5 RPB1 (B) in the miR-210 proximal promoter in response to H-R challenge-subjected cells ectopically expressing the da-I?Ba mutant. Data from the ChIP-ELOHA analysis are expressed as fold enrichment by first correcting the RPB1-associated miR-210 promoter fragment ELOHA absorbance values to the respective inputs, followed by normalization to fold change values. The ectopic expression of the HA-tagged da-I?Ba mutant was validated using sandwich ELISA immunoassay performed against the HA tag (Supplementary Figure S5A). NF-?B transcriptional activity (Supplementary Figure S5B) was determined in the native lysates to corroborate and validate the translative effects of the ectopic expression of the da-I?Ba mutant. Data from the ChIP-ELOHA analysis are depicted as mean fold enrichment ± S.D. from three technical replicates for each of the four biological replicates belonging to each experimental group (n = 4). (C,D) Representative Western blots (C) and quantitative densitometric analysis (D) showing the relative quantitative abundance of the molecular components of the NELF and DSIF protein complexes associated with the RPB1 subunit recruited at the miR-210 proximal promoter in response to H-R challenge-subjected cells ectopically expressing the da-I?Ba mutant. (E,F) Representative Western blots (E) and quantitative densitometric analysis (F) of the abundance of p-Thr residues in the immunoprecipitated SPT5 as a surrogate measure of the status of RNAPII-mediated transcription elongation in response to H-R challenge-subjected cells ectopically expressing the da-I?Ba mutant. Data from the Western blot and densitometric analysis are expressed as mean fold change ± S.D. from three biological replicates belonging to each experimental group (n = 3). * p = 0.05; **** p = 0.0001; ns: not significant (p > 0.05). S.D.: standard deviation.

Fig 3: E966-0530-45418 diminished M2 macrophage polarization induced by IL6 in PMA-treated THP-1 cells. (A) GSEA of IL6/STAT3 signaling pathway genes in human IPF vs. normal lung samples from the GSE49072 dataset. The P value was computed using the 2-sided permutation test with the Benjamini-Hochberg adjustment for multiple comparisons. FDR: false discovery rate. (B) PPI network validating the interaction of IL6/STAT3/CDK8/RNA polymerase II (POLR2A) signals. Network nodes represent proteins; the colored edges denote evidence of different PPI types. Solid interactions and networks were clustered using the k-means cluster algorithm with default parameters and are indicated by solid lines. (C) PMA-induced THP-1 cells were treated with E966-0530-45418 (5 μM), senexin A (5 μM), pirfenidone (1 mM), or no inhibitor in the presence of IL6 (5 ng/mL) for 3 h and then subjected to nuclear-cytosolic fractionation. The protein levels in the cytosol and nucleus were detected by western blotting analysis and quantified (n = 5 independent samples per group). (D-H) PMA-induced THP-1 cells were treated with the indicated concentration of E966-0530-45418 (μM), senexin A (5 μM), pirfenidone (1 mM), or no inhibitor in the presence of IL6 (5 ng/mL) for 48 h (D and E) or 24 h (F-H). The CD206 levels were evaluated by flow cytometric analysis (n = 4 independent samples per group) (D and E). The mRNA levels of TGFβ1 were determined by RT‒qPCR (n = 3 independent samples per group) (F). The protein levels of arginase I were assessed by western blotting (n = 5 independent samples per group) (G and H). The results are shown as the mean ± SEM. P values were determined using one-way ANOVA followed by Tukey's post hoc test (C, E, F, and H).

Fig 4: E966-0530-45418 markedly mitigated TGFβ1/Smad3/RNA polymerase II signal transduction, subsequently lowering the transcription of EMT and fibrosis-related genes. (A-C, E-G) A549 cells were treated with E966-0530-45418 (5 μM), senexin A (5 μM), pirfenidone (1 mM), or no inhibitor in the presence of TGFβ1 (10 ng/mL) for 3 h and then subjected to nuclear-cytosolic fractionation. The protein levels in the cytosol and nucleus were detected by western blotting and quantified (n = 5 independent samples per group) (A-C). The nuclear proteins were immunoprecipitated with an anti-CDK8 antibody and then subjected to immunoblotting to assess the interactions between CDK8 and p-Smad3 T179, p-RNA Pol II S2/5, MED12, Pin 1, and cyclin C (n = 4 independent samples per group) (E-G). (D) Protein-protein interaction (PPI) network analysis was established by the STRING online database. The network nodes represent proteins, and the colored edges denote evidence of interactions between different proteins in the PPI network. Strong connections and networks were clustered using the k-means cluster algorithm with default parameters and are indicated by solid lines. POLR2A: RNA polymerase II. (H) Schematic illustrating Smad3 binding to the Smad-binding element (sequence logo obtained from the UCSC JASPAR joint website). (I-K) A549 cells were incubated with E966-0530-45418 (5 μM), senexin A (5 μM), pirfenidone (1 mM), or no inhibitor in the presence of TGFβ1 (10 ng/mL) for 6 h and then subjected to a Smad3 ChIP assay with RT‒qPCR analysis of the promoter sequences of N-cadherin, Snail (I), COL1A1, α-SMA (J), TGFβ1 and CTGF (K). The histone H3 antibody (Ab) group was used as a positive control, and the rabbit IgG group was used as a negative control (n = 3 independent samples per group). (L) Schematic illustration of the human E-cadherin and TGFβ1 promoter luciferase reporter genes, the function of which might be affected by TGFβ1 treatment. (M) A549 cells were transfected with 7TFP CDH1 or pGL3-TGFB1 reporter plasmid (1 μg) for 6 h, followed by subsequent treatment with TGF-β1 (10 ng/mL) with E966-0530-45418 (5 µM), senexin A (5 µM), pirfenidone (1 mM), or no inhibitor for 24 h, and luciferase expression was subsequently determined (n = 5 independent samples per group). The results are shown as the mean ± SEM. P values were determined using one-way ANOVA followed by Tukey's post hoc test (B, C, F, G, I-K, and M).