Fig 2: ERK1/2 pathway activity in parent and resistant cells. Cell lysates from parent (P) or PLX/AZD-resistant (DR) monolayer (A) or spheroid (B) cultures were immunoblotted for phosphorylated and total ERK1/2, MEK1/2, or p90RSK. ß-actin levels are shown as a protein loading control. Data are representative of two independent experiments. ProteinSimple quantitative analysis of the ratio of phosphorylated/total MEK1/2, ERK1/2, and p90RSK from monolayer (C) or spheroid (D) cell cultures from two additional independent samples.

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: Sensitivity of parent and resistant cells to BRAF and MEK1/2 inhibitors. Dose–response curves for drug-sensitive parent (open squares) or drug-resistant (PLX/AZD-R, closed squares) cells treated with the indicated combined concentrations of PLX4032 and AZD6244 in monolayers (A) or spheroids. (B) Cell viability is expressed as a percentage compared to cells treated with dimethyl sulfoxide vehicle (100%). Data are representative of three independent experiments.

Fig 5: Cellular effects mediated by RET expression in TT cells. a MTT assay for TT cells, treated with an increasing concentration of berberine for 96 h to determine the cell viability. b The phosphorylation status of Akt, MEK1/2 and ERK1/2 were determined in TT cells following the exposure with different concentrations of berberine. c The Bcl-2 and the Capsase-3 activity in TT cells were determined in the presence of increasing concentrations of berberine. d FACS analysis of TT cells treated with various concentrations of berberine for 48 h. Data are mean +/-s.e.m of three separate experiments. e Western Blot analysis to determine the protein expression of pRb, p-pRb, E2F1, Cyclin E in TT cells