Ab52866

Inhibitors were dissolved in DMSO and were used at the following concentrations: 50 nM CENP-E inhibitor (GSK923295, Cambridge Bioscience), 2–3 μM MPS1 inhibitor (NMS-P715, Merck Chemicals) and 0.15–0.45 μM reversine (Sigma-Aldrich).
Antibodies were used as follows: mouse α-tubulin (Thermo Fisher Scientific, 32-2500), 1:5000 (western blotting or WB) and 1:500 (immunofluorescence or IF); rabbit α-tubulin (Abcam, ab52866), 1:500 (IF); mouse CENP-A (Abcam, ab13939), 1:200 (IF); human CREST (Antibodies Incorporated, 15-234-0001), 1:300 (IF); rabbit pericentrin (Abcam, ab4448), 1:2000 (IF); rabbit KIF11 (Novus Biologicals, NB5000-181), 1:1000 (IF); mouse p53 (Cell Signaling Technologies, 48818S), 1:1000 (IF); and rabbit MAD2 (Millipore UK, MABE866), 1:1000 (WB) and 1:500 (IF).

For western blotting, frozen skin tissues were ground in liquid nitrogen and lysed with ice-cold Radio-immunoprecipitation Assay (RIPA) buffer (R0278, Sigma, Shanghai, China). The supernatant was collected after centrifugation of the tissue lysate at 12,000 rpm in a refrigerated centrifuge (5804R, Eppendorf, Shanghai, China) at 4° C for 10 min, followed by the measurement of protein concentrations using a BCA assay kit (Thermo Fisher Scientific, NY, USA). Protein samples (40 μg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Millipore Immobilon, Darmstadt, Germany). The membranes were blocked with 5% skimmed milk in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 2 h, and incubated with primary antibodies diluted in PBS against SMAD2/3 (8685s, CST, Shanghai, China), p-SMAD2/3 (8828s, CST), and β-actin (ab52866, Abcam, Shanghai, China) at 4° C overnight. These membranes were, then, washed three times with TBST for 10 min each and incubated with horseradish peroxidase-conjugated secondary antibody (ab6721, Abcam) at room temperature for 1 h. Protein signals were detected using electro-chemiluminescence (ECL) reagent (Thermo Fisher Scientific).

For immunofluorescence staining, the cells were fixed with 4% paraformaldehyde for 15 min in PBS, permeabilized with 0.2% Triton X-100 in PBS, blocked with blocking buffer and then stained with primary antibodies against α-tubulin (ab52866, Abcam), α-actinin (A7811, Sigma-Aldrich), or cleaved caspase-1 p20 (PRS3459, Sigma-Aldrich) followed by Alexa Fluor 488/546/633 secondary antibodies. ASC was detected with an Alexa Fluor 488 Conjugated antibody (17507, Cell Signaling). Rhodamine phalloidin dye (R415, Invitrogen, Carlsbad, CA, USA) was incubated with the cells for 20 min at 37 °C for F-actin. The stained cells were visualized and analyzed using a laser scanning confocal microscope with a 63×/1.4NA oil immersion objective lens and excitation wavelengths of 488, 543, and 633 nm. All images were acquired using ZEN 2012 software.

Total protein was extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, China). Protein lysates were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies: anti-ICAM-1(1:1000, Abcam, ab282575), anti-α-tubulin (1:2000, Abcam, ab52866) overnight at 4°C, and subsequently incubated with the secondary antibodies for an hour. The protein bands were detected using an ECL chemiluminescence kit (Beyotime, China) and quantified by ImageLab software (USA).

Blots were prepared using a Mini Trans‐Blot Cell (Bio‐Rad, 1660828EDU) and PVDF Immobilon membrane (Millipore, Bedford, MA, USA) using buffer formulations and run times according to the General protocol for WB by Bio‐Rad.
The membrane was blocked for 1 h in 3% BSA in TBST (20 mm pH 7.5 Tris/HCl, 150 mm NaCl, and 0.1% Tween‐20), rinsed with TBST, and incubated overnight at 4 °C with primary antibodies to histone H3 acetyl K9 (ab10812, dilution 1 : 500 in 3% BSA/TBST Abcam, Cambridge, MA, USA) and α‐tubulin (Abcam, ab52866, dilution 1 : 10 000 in 3% BSA/TBST). The membranes were rinsed with TBST and then incubated with secondary antibody goat anti‐rabbit (Abcam, ab97051, dilution 1 : 20 000 in 3% BSA/TBST) for 1 h at room temperature. The membranes were rinsed and treated with a chemiluminescent (Immobilon Western; Millipore) using ChemiDoc XRS+ (Bio‐Rad). The signal was quantified using image lab™ 6.0 (Bio‐Rad). The obtained signal for histone H3 (acetyl K9) was normalized to the α‐tubulin signal as the housekeeping protein for each group, respectively. Obtained values for the VPA‐treated group were further normalized to the control values [76]. Fold minor to 0.5 and major than 2.0 was defined as significant. For technical western blot positive controls, indifferent tissue was used.