Ab55391

H2887 cells were cultured in 2% O₂ over 4 weeks and fixed with 2% PFA in PBS for 20 min at 37°C. Afterwards cells were washed with 30 mM Glycine/PBS. Cells were permeablized for 2 min in 0.03% Triton/PBS, then washed once in PBS and blocked for 2 h at room temperature with 10% Goat serum in PBS. Primary Kras antibody (Abcam, ab55391) was diluted 1:50 in 10% Goat serum/PBS and incubated over night at 4°C in a humidified chamber. Afterwards, the samples were washed 3x for 10 min with 5% Goat serum/PBS plus 0.2% Tween-20. Secondary antibody against mouse conjugated with Alexa488 was diluted 1:500 in 10% Goat serum/PBS and incubated for 1 h at room temperature. Finally, the samples were washed 3x for 10 min in PBS/0.2% Tween-20 and once in PBS. Detached spheres were always collected by centrifugation and added back into the staining process. Finally, all cells were detached by Versene (Gibco) treatment and gentle scraping, collected, washed once and re-suspended for analysis in PBS. Kras intensities were analyzed using the BD FACS Aria II SORP (BD Biosciences) with a 488 nm excitation laser and a 530/30 band pass filter and gates were selected for viable cell population. Graphical analysis was performed using FlowJo v.10software (FLOWJO, LLC.).

Seliciclib (CYC202, R-roscovitine) was provided by Cyclacel, Ltd (10mM stock solution in dimethyl sulfoxide, DMSO). The seliciclib dosage used (10μM) is clinically achievable (34 (link)) and biological effects of seliciclib at 10μM were due to CDK2 inhibition rather than CDK7/9 blockade in lung cancer cells (19 (link)). Antibodies used were: α-tubulin (T6199, Sigma Aldrich, (1:1000 immunofluorescence and 1:10000 immunoblot), α-tubulin (YL1/2) (NB600-506, Novus Biologicals, 1:1500), γ-tubulin (T5326, Sigma Aldrich, 1:1000), CP110 (12780-1-AP, Proteintech, 1:750), HA.11 clone 16B12 monoclonal antibody (MMS-101P, Covance, 1:3000), cyclin F (C-20) (SC-952, Santa Cruz Biotechnology, 1:500), CEP76 (A302-326A, Bethyl Laboratories, 1:1000), USP33 (A300-925A, Bethyl Laboratories, 1:1000 in 5%BSA) and KRAS (ab55391, Abcam, 1:1000). Secondary antibodies used were: Texas red anti-mouse IgG (H+L) (TI-2000, Vector Laboratories.), Fluorescein anti-mouse IgG (H+L) (FI-2000, Vector Laboratories.), Alexa fluor 594 donkey anti-rat IgG (H+L) (A21209, Invitrogen), ECL anti-rabbit lgG (NA934V, GE Healthcare), ECL anti-mouse lgG (NA931V, GE Healthcare) and horseradish peroxidase–conjugated donkey anti-goat IgG (sc-2020, Santa Cruz Biotechnology.). Hoechst 33342 (62249, Thermo Scientific, 1:25000) stained DNA. Pro-Long Gold anti-fade reagent (P36934, Invitrogen) preserved immunofluorescence.

Total proteins from tissues were extracted by homogenizing in RIPA buffer premixed with protease inhibitor cocktail (Sigma, St. Louis, MO). Proteins concentrations were determined using a BCA protein assay kit (Thermo Scientific, Rockford, IL). Total protein (50 μg) was separated on 12% mini PROTEAN polyacrylamide gels and then transferred to polyvinylidenefluoride (PVDF) (Life Technologies Carlsbad, CA) using iBlot gel transfer system. Membrane was blocked using Odyssey blocking buffer for 1 h at room temperature. Membranes were incubated overnight with rabbit polyclonal to Gli-1 (SC-20687), rabbit polyclonal to Shh (SC-h160), goat polyclonal to β-actin (SC-1616) (1:1000) (Santa Cruz Biotech., Dallas, TX), and mouse monoclonal antibodies against KRAS (ab-55391) (1:1000) (Abcam, Cambridge, MA). After washing with TBST buffer, the membrane was further incubated with their corresponding antirabbit, antigoat, and antimouse IR dye conjugated secondary antibodies (1:10000) (LI-COR Biosciences, Lincoln, NE) for 60 min and visualized using LI-COR imaging system. Expression levels of desired protein were normalized against β-actin (SC-1616) protein expression levels.