Anti lamin a

The cells were immersed in PBS and seeded in glass slides at a concentration of 1 × 10⁴/ml. The slides were fixed in 4% paraformaldehyde for 15 minutes, then immersed in PBS 3 times for 3 minutes each time, and subsequently in 0.5% Triton X‐100 in PBS at room temperature for 20 minutes. Next, the cells were blocked with 5% skim milk powder in PBST for 30 minutes at room temperature, and the following diluted primary antibodies were added: anti‐Lamin B, BOSTER, China; anti‐β‐actin, Abcam, USA; anti‐Lamin A, Abcam, USA. The antibodies used were rabbit anti‐human and diluted at a concentration of 1:200. The cells were incubated overnight at 4°C and subsequently with the fluorescent secondary antibody (1:100, mouse anti‐rabbit antibody, Abcam, USA) for 1 hour at room temperature. Finally, the slides were washed in PBST 3 times for 3 minutes each and observed under a fluorescence microscope.

Formalin‐fixed paraffin‐embedded 4‐μm thick tumour sections were subjected to CCND1 immunostaining, according to the manufacturer's instructions. The antibodies (CCND1a, 1:200; CCND1b, 1:150) were incubated overnight. Negative controls used non‐specific rabbit Immunoglobulin G instead of the primary antibody, and a BC pathologist performed a blind evaluation of the immunostained sections. Slides stained with CCND1a and CCND1b antibodies were evaluated and blinded by an independent BC pathologist. Tumours with >50% CCND1a staining were considered CCND1a positive. Since CCND1b is not generally found in normal cells, tumours were considered positive for CCND1b if any cells showed staining in the nucleus.²⁰, ³⁸, ³⁹ Cells were seeded on glass coverslips, fixed using either 3% paraformaldehyde and blocked for 1 h in 4% BSA/PBS followed by incubation in Anti‐SC35 (1:100, Abcam) for 1 h at room temperature. Cells were washed in PBS and incubated in a secondary FITC conjugated antibody (1:200) for 30 min at room temperature. Cells were washed with PBS and counterstained with Anti‐Lamin A (1:500, Abcam). The specimens were examined with a confocal laser microscope (LSM, Carl Zeiss AG).

Cells were collected, washed, and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing Protease and Phosphatase Inhibitor (Thermo Fisher Scientific). A bicinchoninic acid (BCA) kit (CWBio) was then used to detect the concentration of protein. Equivalent amounts of protein were separated on 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was then blocked with Tris-buffered saline with Tween 20 (TBST) buffer containing 5% skim milk powder and incubated with corresponding primary antibodies at 4 °C overnight. The primary antibodies used were anti-p65 (Cell Signaling Technology), anti-IκBα (Cell Signaling Technology), anti-p50 (Cell Signaling Technology), anti-Histone-H3 (Abcam), anti-β-actin (Abcam), anti-Flag (Cell Signaling Technology), GAPDH (Cell Signaling Technology), anti-L-Lactyl Lysine (PTM Bio Inc), anti-Lactyl-Histone H3 (Lys18) Rabbit mAb (PTM Bio Inc), anti-KRAS (Abcam), anti-KRAS^G12D (Abcam), anti-Lamin A (Abcam). Membranes were then washed with TBST three times and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology) for 1 h at room temperature. Signals were developed with ECL Blotting Detection Reagents (Thermo Fisher Scientific).

Chemicals were obtained from the following sources: cis-Diammineplatinum (II) dichloride (CDDP), N-acetylcysteine (NAC) and MG132 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were obtained from the following sources: anti-AKT (#9272 for western blot), anti-AKT (#2920 for proximity ligation assay, PLA), anti-p-AKT (Ser473), anti-Myc, anti-His, anti-FOXO1, anti-FOXO3, anti-FOXO4, anti-GAPDH, anti-Normal Rabbit IgG, horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG (Cell signaling Technology, Beverly, MA, USA), anti-K48 Ubiquitin, anti-K63 Ubiquitin, anti-α-Tubulin (Millipore, Temecula, CA, USA), anti-HA for western blot, anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MUL1, anti-Lamin A and anti-HA for ChIP assay (abcam, Cambridge, MA, USA), anti-hemagglutinin (HA), anti-GFP (Santa Cruz, Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma-Aldrich), Alexa Flour 488-conjugated goat anti-Mouse and Alexa Flour 546-conjugated goat anti-Rabbit (Invitrogen, Carlsbad, CA, USA)

Western blot analyses were carried out as previously described56 (link). The primary antibodies anti-Phospho-phospholamban (Ser16, Cell Signaling, Beverly, MA, USA), anti-phospholamban (Abcam, Cambridge, MA, USA), anti-Phospho-lamin A (Ser22, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-lamin A (Abcam) and anti-β-actin (Cell Signaling) were used.

To measure protein expression in glioma cells, we prepared cellular extracts using the NE-PER® nuclear, cytoplasmic, and membrane extraction kit (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Protein extracts were separated by SDS-PAGE on a 12% polyacrylamide gel, and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). Membranes were blocked at room temperature with 5% non-fat milk before incubating overnight at 4°C with the following primary antibodies: anti-IDH1 R132H mutation antibody (1:500 dilution; Dianova, Hamburg, Germany), anti-IDH1 antibody (1:500 dilution; Dianova), anti-GADD45A (1:200 dilution;Abcam, San Francisco, California, USA), anti-β-catenin (1:1,000 dilution; Abcam), anti-MMP-9 (1:1,000 dilution; Abcam), anti-E-cadherin (1:50 dilution; Abcam), anti-N-cadherin (1:1,000 dilution; Abcam), and anti-fibronectin (1:100 dilution, Abcam). Anti-β-actin (1:1,000 dilution; Abcam), anti-lamin A (1:500 dilution, Abcam) and anti-flotillin (1:500 dilution; Abcam) were used as controls. After primary antibody incubation, membranes were washed and incubated with HRP-conjugated secondary antibodies. Protein bands were visualized using the enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA).