Fig 2: NAC treatment inhibited cell senescence, and TIME signaling mediated the EMT in Bmi-1-/- cells more than in Bmi-1-/-p16-/- AT2 cells.Pulmonary AT2 cells from 7-week-old WT, Bmi-1-/-, Bmi-1-/-p16-/-, and NAC-treated Bmi-1-/- (Bmi-1-/- + NAC) mice. a Representative micrographs of cells stained cytochemically for SA-ß-gal. b Percentage of SA-ß-gal-positive areas relative to the total. c Second-passage AT2 cell proliferation were determined by CCK-8 assays, and spectrophotometry at 450 nm. Cell number was determined at the indicated hours relative to the cell number at hour 0. d Western blots for SFTPC, a-SMA, pERK1/2 (Thr202/Tyr204), ERK1/2, p16, p53, p21, mature TGF-ß1, IL-11, and Snail. ß-actin was used as the loading control. e Protein expression relative to ß-actin was assessed by densitometric analysis. f Pulmonary AT2 cells were treated with TGF-ß1 (5 ng/ml, 48 h), TGF-ß1 (5 ng/ml, 48 h), and anti-IL-11 antibody (2 µg/ml, 48 h), IL-11 (5 ng/ml, 48 h), or IL-11 (5 ng/ml, 48 h) and the MEK inhibitor (PD98059) (10 µm, 48 h). Western blots for SFTPC, a-SMA, pERK1/2 (Thr202/Tyr204), ERK1/2, p16, p53, and p21. ß-actin was used as the loading control. g Protein expression relative to ß-actin was assessed by densitometric analysis. h Representative micrographs of cells immunofluorescently stained for a-SMA and SFTPC, with DAPI staining the nuclei. i The percentage of a-SMA- or SFTPC-positive areas was relative to the total. j WT AT2 cells treated with CM from third-passage pulmonary fibroblasts from the WT, Bmi-1-/-, Bmi-1-/-p16-/- or NAC-treated Bmi-1-/- (Bmi-1-/- + NAC) groups. k The percentage of a-SMA- or SFTPC-positive areas relative to the total. Six biological replicates were used per experiment. Values are the means ± SEM of six determinations. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the Bmi-1-/- group; &P < 0.05, &&P < 0.01 compared with the Bmi-1-/-p16-/- group. ^P < 0.05, ^^P < 0.01, ^^^P < 0.001 compared with the vehicle group; +P < 0.05, ++P < 0.01, +++P < 0.001 compared with the TGF-ß1 treatment group; @P < 0.05 compared with the TGF-ß1 and anti-IL-11 antibody treatment group; -P < 0.05, --P < 0.01 compared with the IL-11 treatment group.

Fig 3: NAC treatment inhibited TIME signaling in Bmi-1-/- mice more than in Bmi-1-/-p16-/- mice.Western blots of pulmonary extracts showing a SFTPC, collagen 1, a-SMA, mature TGF-ß1, TGF-ßRII, Smad2, pSmad2 (Ser465/467), pSmad2/3 (Ser423/425); b IL-11, IL-11Ra1, MEK1/2, pMEK1/2(Ser217/221), ERK1/2, pERK1/2(Thr202/Tyr204), elF4E, p-elF4E(Ser209), RSK, and p-RSK(Ser380). c, d Protein expression relative to ß-actin (graph a) was assessed by densitometric analysis. Six mice per group were used for experiments. Values are the means ± SEM of six determinations. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the Bmi-1-/- group; &P < 0.05, &&P < 0.01 compared with the Bmi-1-/-p16-/- group. e Pulmonary fibroblasts from 7-week-old WT mice treated with TGF-ß1 (5 ng/ml, 48 h), TGF-ß1 (5 ng/ml, 48 h) and anti-IL-11 antibody (2 µg/ml, 48 h), IL-11 (5 ng/ml, 48 h), or IL-11 (5 ng/ml, 48 h) and the MEK inhibitor (PD98059) (10 µm, 48 h). Representative micrographs immunofluorescently labeled for IL-11 and vimentin, with DAPI staining the nuclei. f Percentage of IL-11-positive areas relative to the total. g Western blots showing p16, IL-11, and collagen 1. ß-actin was used as the loading control. h Protein expression relative to ß-actin was assessed by densitometric analysis. Six biological replicates were used per experiment. Values are the means ± SEM of six determinations. ^P < 0.05, ^^P < 0.01, ^^^P < 0.001 compared with the vehicle group; +P < 0.05, ++P < 0.01 compared with the TGF-ß1 group; @P < 0.05, @@P < 0.01 compared with the TGF-ß1 and anti-IL-11 group; --P < 0.01 compared with the IL-11 group.

Fig 4: NAC treatment inhibited cell senescence and TIME signaling in Bmi-1-/- more than in Bmi-1-/-p16-/- pulmonary fibroblasts.Pulmonary fibroblasts from 7-week-old WT, Bmi-1-/-, Bmi-1-/-p16-/-, and NAC-treated Bmi-1-/- (Bmi-1-/- + NAC) mice. a Representative micrographs of cells stained cytochemically for SA-ß-gal. b Percentage of SA-ß-gal-positive areas relative to the total areas. c Third-passage fibroblast proliferation was determined by CCK-8 assays and spectrophotometry at 450 nm. Cell number was determined at the indicated hours relative to the cell number at hour 0. d TGF-ß1 and IL-11 concentrations were detected in conditioned medium (CM) by ELISA and assessed by densitometric analysis relative to that of WT CM. e, f Western blots for mature TGF-ß1, IL-11, p19, p53, p21, a-SMA, p16, pERK1/2 (Thr202/Tyr204), and ERK1/2. ß-actin was used as the loading control. g Protein expression relative to ß-actin was assessed by densitometric analysis. h Representative micrographs showing immunofluorescence for IL-11 and p53, with DAPI staining the nuclei. i Percentage of IL-11-positive areas (in p53-positive cells) or p53-positive areas relative to the total area assessed. Six biological replicates were used per experiment. Values are the means ± SEM of six determinations. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the Bmi-1-/- group; &P < 0.05, &&P < 0.01 compared with the Bmi-1-/-p16-/- group. j Western blots of pulmonary fibroblasts from the NAC (1 mm, 48 h)-treated Bmi-1-/- (Bmi-1-/- + NAC) group and the NAC (1 mm, 48 h) & MG132 (5 µm, 48 h)-treated Bmi-1-/- (Bmi-1-/- + NAC + MG132) group. k Protein expression relative to ß-actin was assessed by densitometric analysis. Six biological replicates were used per experiment. Values are the means ± SEM of six determinations. **P < 0.01, ***P < 0.001 compared with the NAC-treated Bmi-1-/- group.

Fig 5: NAC treatment improved pulmonary fibrosis in Bmi-1-/- mice more than in Bmi-1-/-p16-/- mice.a Representative micrographs of paraffin-embedded pulmonary sections stained histochemically for Masson’s trichrome (Masson), immunohistochemically for a-smooth muscle actin (a-SMA), type ? collagen (collagen 1), fibronectin, TGF-ß1 and IL-11, and immunofluorescently for IL-11 receptor a1 (Ra1) and SFTPC, with DAPI staining the nuclei. b Percentage of cells positive for Masson, a-SMA, collagen 1, fibronectin, TGF-ß1, IL-11, IL-11Ra1, or SFTPC or positive areas relative to the total cells or areas. c Col1a1, Col1a2, ACTA2, plasminogen activator inhibitor-1 (PAI-1), TGF-ß1, matrix metalloproteinases (MMP9), MMP12, IL-11 and IL-11Ra1 and SFTPC mRNA levels in lungs by real-time RT-PCR, calculated as the ratio to ß-actin mRNA and expressed relative to that of WT. Six mice per group were used for experiments. Values are the mean ± SEM from six determinations per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the Bmi-1-/- group; &P < 0.05, &&P < 0.01 compared with the Bmi-1-/-p16-/- group.