Nbp1 31327

A total of 0.5–2.0 × 10³ cells/well were cultured for imaging on a Falcon® 96-well Black/Clear Flat Bottom TC-treated Imaging Microplate (Corning, New York, NY, USA) for 72 h. When cells reached the appropriate confluence, their surface was washed with a PBS solution, fixed in freshly prepared 4% (v/v) paraformaldehyde (Avantor Performance Materials Poland, Gliwice, Poland) for 10–15 min, washed with PBS, and permeabilized in 0.25% (v/v) Triton X-100 (Sigma‒Aldrich, Saint-Louis, MO, USA) for 15 min at RT. Then, the cells were washed and blocked for 30 min in 1% (w/v) bovine serum albumin (Sigma‒Aldrich, Saint-Louis, MO, USA) solution in 0.1% (v/v) PBS/Tween 20 (Sigma‒Aldrich, Saint-Louis, MO, USA) at RT. Next, the fixed cells were incubated with primary antibodies against vimentin (NBP1-31327, dilution 1:500; Novus Biologicals, Centennial, CO, USA) in a blocking solution at 4 °C overnight. Next, after washing with PBS, a secondary antibody (anti-rabbit antibody Alexa Fluor 488, ab150077; Abcam, Cambridge, UK) in the blocking solution was used for 1 h at RT. Finally, the samples were rinsed with PBS and photographed using an Olympus IX81 fluorescence microscope (Olympus, Warsaw, Poland) with CellSense software (Olympus, Warsaw, Poland).

Prints were washed three times with 1x PBS and fixed using 10% neutral buffered formalin (Thermo Fisher) for 30 minutes at room temperature. Cells were permeabilized using 0.1% Triton X-100 (Sigma) solution in PBS for 15 minutes at room temperature. The samples were then blocked using 1.5% bovine serum albumin (BSA, Sigma) in PBS for 1 hour at room temperature. Primary antibodies pan cytokeratin (PanCK, 1:100, OriGene Catalog # CF190032, Lot # F003) and vimentin (VIM, 1:200, Novus Biologicals Catalog # NBP1–31327, Lot # 44286) were added in 0.15% BSA and incubated at 4 °C overnight. Primary antibodies were removed, and the samples were washed three times with PBS before adding secondary antibodies (1:250, Invitrogen Alexa Fluor 555 goat anti-mouse, Catalog # A21422, Lot # 2139320, and Invitrogen Alexa Fluor 647 goat anti-rabbit, Invitrogen Catalog # A21244, Lot #2179230). Secondary antibodies were incubated at room temperature for 2 hours, removed, and samples washed twice with 0.1% Tween 20 (Fisher Biotech). Nuclei were then labeled with DAPI (NucBlue Fixed Cell ReadyProbes, Invitrogen) in PBS for 30 minutes at room temperature. Samples were imaged using a spinning disk confocal (Yokogawa CSU-X1 on Zeiss Axio Observer).

Total protein from the cells was extracted using radio-immunoprecipitation assay cell lysis buffer (Cell Signaling Technology) supplemented with the protease inhibitor (Roche Ltd, Basel, Switzerland). The protein concentration was determined using a bicinchoninic acid kit (Thermo Fisher Scientific). Equal amounts of protein samples (30 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Following a 1-h block in 5% non-fat milk at room temperature, the membranes were incubated with the primary antibodies against KDM5C (1:1,000, ab264168, Abcam), PFDN5 (1:1,000, ab129116, Abcam), ATG7 (1:1,000, ab52472, Abcam), P62 (1:1,000; NBP1-48320, Novus Biologicals, Littleton, CO, USA), Beclin1 (1:1,000, #3495, Cell Signaling Technology), LC3 (1:1,000; #3868, Cell Signaling Technology), Vimentin (1:1,000, NBP1-31327, Novus Biologicals), N-cadherin (1:1,000, NBP2-19457, Novus Biologicals), GAPDH (1:1,000; ab181602, Abcam) at 4 °C overnight. Subsequently, the membranes were incubated with the HRP-conjugated IgG (1:20,000, ab6721, Abcam) at room temperature for 2 h. Protein blots were developed using enhanced chemiluminescence reagent and analyzed using Image J.