Fig 2: Cell specific effects of STS and LPS on SERPINA1 gene expression and AAT protein. (A) (H1975 cells) and (D) (H661 cells) were cultured for 18 h alone or in the presence of 10 µg/ml LPS, 50 nM STS, or 1.5 mg/ml AAT separately and in combinations. Graphs show SERPINA1 gene expression relative to housekeeping gene POLR2A. Bars represent mean (SD) of three independent experiments. p < 0.05 was considered as significant. (B,C) H1975 cells were cultured for 18 h in the medium containing HBSS (controls), 10 µg/ml LPS or 50 nM STS. Supernatants and cell lysates were analysed by Western blot. ß-actin was used for loading control. Representative blots are shown out of three independent experiments. Densitometric data presented as mean (SD) of four (B) and three (C) independent experiments for supernatants and for lysates. 10 ng AAT was added to the blot as a positive control. (E,F) H661 cells were cultured for 18 h in the medium containing HBSS (controls), 10 µg/ml LPS, or 50 nM STS. Supernatants and cell lysates were analysed by Western blot. Representative blots are shown out of three independent experiments. Densitometric data presented as mean (SD) of three independent experiments for supernatants and two for lysates. 10 ng AAT was added to the blot as a positive control.

Fig 3: Exogenously added AAT does not interfere with chloroquine (CQ) or streptonigrin (STN)-induced apoptosis in H1975 cells independent on the presence of LPS. Cells were treated with 200 µM CQ or 200 nM STN alone or together with AAT (1.5 mg/ml) and LPS (10 µg/ml) for 18 h. Percentages of living cells were determined by flow cytometry using PE Annexin V Apoptosis assay. Each bar represents mean (SD) of three independent experiments. p < 0.05 was considered as significant.

Fig 4: Effects of stattic (STAT3 inhibitor) on LPS-induced IL-6 production and STS-induced apoptosis in H1975. Cells were treated with 5 µM stattic for one hour prior to addition of HBSS (control), 10 µg/ml LPS, or 1.5 mg/ml AAT separately and in combination for 18 h. (A) IL6 gene expression was related to housekeeping gene POLR2A. Each bar represents mean (SD) from six independent experiments. (B) Supernatants were analysed for IL-6 release by ELISA. Each bar represents median (25–75 percentile) of four independent experiments. (C) Apoptosis was analyzed by flow cytometry using PE Annexin V Apoptosis assay to detect living cells. Each bar represents mean (SD) of living cells (percentage) from two independent experiments for stattic. p < 0.05 was considered as significant.

Fig 5: CCN1 binds and activates TLR2 and TLR4.a Solid-phase-binding assays between CCN1 and TLR2/4. Recombinant TLR2 or TLR4 proteins (200 ng per well) were added to 96-well plates pre-coated with serially diluted CCN1 protein at indicated amounts. Specific interaction was detected and quantified using polyclonal anti-hTLR2 or anti-hTLR4 antibodies. Recombinant CD14 (200 ng per well) was used as a control. b SPR analyses of CCN1 binding to TLR2. TLR2 was immobilized on CM5 chip and various concentrations of CCN1 was injected as analyte. c Sensorgrams of CCN1 binding to TLR4 analyzed by SPR as in b. d Dot blot analyses of CCN1 and mutant proteins binding to TLR2/4. CCN1-WT, CCN1-D125A, or CCN1-DM proteins (1 µg each) were spotted onto nitrocellulose membrane and incubated with TLR2 or TLR4 proteins (2 µg each in PBS) for 4 h. Binding was detected using polyclonal anti-hTLR2 or anti-hTLR4 antibodies. A representative image is shown. e Tnfa and Il6 mRNAs were quantified in BMDMs from either B6 or MyD88-/- mice treated with CCN1-WT, CCN1-D125A, and CCN1-DM mutant proteins using qRT-PCR analysis. f Tnfa and Il6 mRNAs from BMDMs of B6, Tlr2-/-, or Tlr4-/- mice treated with CCN1 proteins (CCN1-WT, -D125A, -DM, 2 µg per ml each) were quantified using qRT-PCR analyses. All data are represented as mean ± s.d. acquired in triplicate determinations. Statistical evaluation was performed by one-sided, two-sample with equal variance t-tests. *p < 0.05, **p < 0.01, and n.s. = not significant. Source data are provided as a Source Data file.