Anti sp1

RIPA lysis buffer (ThermoFisher, MI, USA) was utilized to extract proteins from tissues or cells according to a standard protocol. The protein concentration of each sample was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher, MI, USA). Equal amounts of protein were loaded into SDS-polyacrylamide gels and separated through electrophoresis. Later, the proteins were transferred from the gels to PVDF membranes (Sigma-Aldrich, USA). The membranes were blocked with 3% BSA for half an hour at room temperature and then incubated with primary antibodies overnight at 4 °C. On the next day, the membranes were washed with TBST 3 times before incubation with specific secondary antibodies for 1 h at room temperature. Signals were detected by using a standard ECL kit. The primary antibodies used in the study were as follows: anti-SP1 (1:2000; Cell Signalling, USA); anti-N-cadherin (1:1000; Abcam, USA); anti-vimentin (1:2000; Abcam, USA); anti-E-cadherin (1:1000; Abcam, USA); anti-STMN1 (1:1000; Abcam, USA); and anti-β-actin (1: 5000, Abcam, USA).

Paraffin‐embedded KPC mouse and human pancreatic cancer patient tissues were deparaffinized in xylene and then rehydrated in graded ethanol. Slides were steamed with pH 9 antigen retrieval (Dako). Endogenous peroxidases were blocked with a 3% hydrogen peroxide solution. Tissues were then blocked with Dako protein block and incubated with primary antibody overnight. Slides were stained with anti‐SP1 (Cell Signaling, Cat # 9389) and anti‐GRP78 (Cell Signaling, Cat # 3177) at 1 : 200 dilutions. Slides were washed with PBS, incubated with secondary anti‐rabbit antibody, and conjugated to horseradish peroxidase, for 30 min. Slides were washed again with PBS. Diaminobenzidine Peroxidase Substrate Kit (Vector Laboratories) was then added to slides. Primary antibody was omitted for negative controls. Slides were mounted with permount. Images were obtained on a Leica DM6B with a 20× objective.

RIPA buffer containing Protease Inhibitor Cocktail (Selleck, B14001) was used for cell lysis. After centrifuging the cell extracts at 12,000 × g for 15 min, the supernatant was collected. Approximately 40 µg of total proteins were separated by SDS-polyacrylamide gel electrophoresis before being transferred to nitrocellulose (NC) membranes. Following a 2-h blocking step with 5% defatted milk at room temperature, the membranes were subsequently probed with primary antibodies. Subsequently, the membranes were washed three times and further probed with horseradish peroxidase (HRP)-labeled secondary antibodies (diluted at 1:4000) for 1 h at room temperature, and protein bands were visualized using SuperSignal™ West Pico Plus Chemiluminescent Substrate (ThermoFisher Scientific, TL275133). The primary antibodies used were as follows: anti-TAB182 (Santa Cruz, sc-514517, 1:1000), anti-LDHA (Cell Signaling Technology, 3582, 1:1000), anti-LDHB (Proteintech, 14824–1-AP, 1:1000), anti-c-MYC (Cell Signaling Technology, 18583, 1:1000), anti-SP1 (Cell Signaling Technology, 9389, 1:1000), anti-α-tubulin (Santa Cruz, sc-69969, 1:1000), and anti-actin (Proteintech, 81115–1-RR, 1:1000). The enhanced chemiluminescent reagent (Thermo, MA, USA) was used to detect protein bands.

ChIP was performed on glioma cells treated with PPARγ inhibitor for 48 h by Chip-IT Enzymatic DNA shearing Kit (Active Motif, Carlsbad, CA, USA) as described previously.^{42 (link)} Following treatment, cells were fixed with 1% formaldehyde at room temperature for exactly 8 min, and further processed according to manufacturer’s instruction. Anti-NFκB (Santa Cruz Biotechnology) and anti-SP1 (Cell signaling) were used for immunoprecipitation and non-specific IgG antibody (Abcam) was used as control. After reverse cross-linking and DNA purification, DNA from input (1:10 diluted) or immune-precipitated (IP) samples were quantified by real-time PCR using ABI 7500 real-time thermal cycler with Power SYBR green PCR Master Mix (Life Technologies, Invitrogen) for 40 cycles. Threshold cycle number (Ct) values of IP samples were normalized by corresponding Ct of 1% input DNA. The relative fold change value was analyzed relative to the control. Primer sequences of CIDEA promoter region used for ChIP real-time PCR analysis are listed in Supplementary Table 1.

A total of 2 × 10⁶ cells were seeded in 100 mm dish. Cells were treated with RIPA buffer (Cell Signaling) for lysis. Proteins (100 μg) were separated through SDS-polyacrylamide gel, followed by transfer to polyvinylidene difluoride (PVDF) membrane (Millipore Corp). PVDF membranes were coated with anti-HO-1 (Cat# 5061), anti-Nrf2 (Cat# 12721), anti-Keap1 (Cat# 4678), anti-Bcl-xL (Cat# 2764), anti-Bcl-2 (Cat# 2876), anti-GAPDH (Cat# 5174), anti-cleaved Caspase-3 (Cat# 9661), anti-γH₂AX (Cat# 2577), anti-SOD-2 (Cat# 13141), anti-Sp-1 (Cat# 5931), or anti-β-actin (Cat# 3700) (all from Cell Signaling); and then incubated in the presence of peroxidase-linked (HRP) secondary antibody. Protein bands were visualized through chemiluminescent substrate (Pierce)^{5 (link),15 (link)}.

The cells were harvested and lysed by RIPA buffer. The lysates were boiled at 100 °C for 5 min and centrifuged at 10,000 rpm for 1 min. About 50 ug of total protein were loaded onto SDS-PAGE gel. After that, the proteins were transferred to PVDF membrane at 300 mA for 2.5 h. The membrane was blocked with 5% non-fat milk in 1 × TBST for 1 h at room temperature, then incubated with primary antibodies at 4 °C overnight. Then, the membrane was washed with 1 × TBST for 3 times, 5 min each time, and incubated with secondary antibodies at room temperature for 1 h. Finally, the membrane was incubated with ECL and exposed. The following antibodies were used: anti-SP3 (Santa Cruz, USA), anti-DNMT1 (Cell Signaling Technology, USA), anti-SP1 (Cell Signaling Technology, USA), anti-β-actin (Proteintech).