Erk1 2

Immunoblotting assays were performed, as previously described and validated by our group described [36 (link),37 (link),38 (link),39 (link),40 (link)] and developed with the Odyssey system (LI-COR Biosciences, Lincoln, NE, USA). The intensity of the bands was quantified by using the FIJI (Fiji Is Just ImageJ) software. Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): CREB (catalog number: #4820), Phospho-CREB (pCREB Ser¹³³; catalog number: #9198), ERK1/2 (catalog number: #9102); and from Santa Cruz Biotechnology (Dallas, TX, USA): p-ERK Antibody (catalog number: #sc-7383).

Flotillin-1 and flotillin-2: mouse monoclonal (BD Biosciences, Heidelberg, Germany) and rabbit polyclonal (Sigma-Aldrich) antibodies; vinculin: mouse monoclonal antibody (Sigma-Aldrich); FAK and pY397-FAK: mouse monoclonal (BD Biosciences); ERK1/2: rabbit polyclonal (C-14, Santa Cruz Biotechnology, Heidelberg, Germany) antibodies; pERK2: mouse monoclonal antibody (E-4, Santa Cruz); GAPDH: mouse monoclonal antibody (Abcam, Cambridge, UK); α-actinin: rabbit polyclonal H-300 antibody (Santa Cruz) for IP, mouse monoclonal antibody (BD Biosciences) for Western blot and mouse monoclonal antibody (BM-75-1, Sigma-Aldrich) for immunofluorescence; His-tag: mouse monoclonal antibody (Novagen, Darmstadt, Germany), myc-tag: mouse monoclonal antibody (Santa Cruz Biotechnology).

Dulbecco’s modified Eagle’s media (DMEM), Dulbecco’s phosphate buffer saline (DPBS), fetal bovine serum (FBS), penicillin–streptomycin, quercetin dihydrate (Qu), 3-(4,5-dimetylthiazol-yl)-diphenyl tetrazolium bromide (MTT), propidium iodide, rhodamine 123, 2,7-dichlorodihydrofluoresceindiacetate (H₂DCF-DA), Fluo-3 AM, proteinase K, ribonuclease A, 1,2-bis-(O-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), β-nicotinamide adenine dinucleotide reduced disodium salt (β-NADH), ascorbic acid, agarose, dimethyl sulfoxide (DMSO) and anti-β-actin antibody were purchased from Sigma–Aldrich Chemicals (St. Louis, MO). Antibodies against Bcl-2, Bax, Bim, AIF, NF-κB, caspase-3/caspase-8, phospho-MEK, MEK1/2, phospho-ERK, ERK 1/2, phospho-p38, p-38, phospho-JNK, JNK, Nrf-2, catalase, Cu/Zn SOD, PI3K, phospho-Akt, Akt and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc,). FITC Annexin V was purchased from BD Pharmingen. Histone H3 antibody was purchased from Cell Signaling Technology.
Stock solution of quercetin (Qu) was prepared in dimethyl sulfoxide (DMSO) and diluted to the desired final concentration with culture medium just before use. The final DMSO concentration did not exceed 0.1% (v/v).

Formalin‐fixed, paraffin‐embedded tissue sections were used for immunohistochemistry (IHC) procedures, as described previously (Calvayrac et al, 2014). Briefly, after rehydration, deparaffinized sections were pretreated by microwave epitope retrieval. Endogenous peroxidase activity was quenched and non‐specific binding was blocked. For IHC of patient tissues, a RHOB monoclonal antibody was used (C‐5, Santa Cruz Biotechnologies, Inc., 1:75). For IHC on mouse lung sections, we used the Ki67 (SP6; Thermo Scientific), ERK1/2 (Santa Cruz Biotechnology), p‐ERK (T202/Y204), p‐AKT (S473), AKT, and cleaved caspase‐3 (Cell Signaling Technology) antibodies, with an Envision kit (DAKO). Sections were lightly counterstained with hematoxylin. Tissues expressing different levels of RHOB were included in each immunohistochemical run to unify any possible discordance in intensity. Two observers (IR, E.C‐T), blinded to the patients' status, independently evaluated the extent and intensity of the staining. For RHOB, the intensity of staining was compared with a known external positive control (0: negative; 1+: mild; 2+: moderate; 3+: intense) as previously described (Calvayrac et al, 2014), and is shown in Fig 1A. Any discordant independent readings were resolved by simultaneous reviews by both observers.

20 µg of whole cell lysates or nuclear extracts of 1 × 10⁶ cells were separated by SDS-PAGE and subsequently transferred onto nitrocellulose membranes (Whatman, Dassel, Germany). The blots were probed with antibodies against bcl-x, cytochrome c, caspase-3, XIAP (all purchased from BD Biosciences, Heidelberg, Germany), ERK 1/2, mcl-1, survivin, Rel-A, Rel-B (all purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, USA), p38, pp38 (all purchased from Cell Signaling Technology^®, MA, USA). GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, USA) was used as a loading control. Signals were detected using the ECL detection system (GE Healthcare, Munich, Germany).

Cells were lysed in Cell Signaling lysis buffer. Western blotting was performed with the following antibodies: Cell Signaling: INPP4B (#8450), phospho-Akt (Ser473) (#4060), Erk1/2 (#4695); Santa Cruz: PTEN (sc-6817-R), Akt 1/2 (sc-1619), BRCA1 (sc-642); Millipore: H2A.X (Ser139) (#05–636), ATM (#07–1286); Bethyl: ATR (A300–138A).