Srebp1

HepG2 cells were fixed by 4% paraformaldehyde for 10 minutes at room temperature after being washed by PBS twice. Then cells were blocked and permeabilized with 0.1% Triton X-100 (MP Biomedicals) and 10% FBS diluted in PBS for 30 minutes at room temperature. After that, cells were incubated with primary antibody SREBP1 (Thermo Fisher Scientific, 1:100), SREBP2 (Abcam, 1:100), INSIG1 (Proteintech, 1:200), CALNEXIN (Enzo, 1:200), and PDI (MilliporeSigma, 1:200) incubated at 4°C overnight. After 5 washes with PBS, cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 568 goat anti-mouse IgG (H+L) secondary antibody (Life Technologies) for 1 hour at room temperature with gentle shaking. At last, the samples were washed with PBS 3 times before staining the nucleus with DAPI for 5 minutes. Immunofluorescence images were obtained and analyzed with Zeiss 710 NLO confocal microscopy.
For fluorescence intensity quantification, ImageJ was applied, and relative intensity was quantified by intensity divided by view area. For colocalization, the rate was quantified using ImageJ with colocalization finder. For each sample, no less than 5 representative images were analyzed to quantify the fluorescence intensity values and calculate an average. Experiments were repeated 3 times individually at least.

Immunofluorescence staining was performed as previously described [16 (link)]. The primary antibodies used were PPAR- γ (Cell Signaling Technology) and SREBP-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies used were Alexa Fluor 488 and/or Alexa Fluor 594.
The SZ95 cells were fixed with 10% formalin for 15 min at room temperature. The cells were subsequently treated with 0.1% Triton X-100 in PBS for 5 min to permeabilize them. Then, the cells were blocked at room temperature for 30 min and incubated with primary antibody against SREBP-1 and PPAR-γ at 37 °C for 1 h. For double staining, a reaction with Alexa Fluor 488 phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) was performed at room temperature for 1 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at 37 °C. The slides were mounted using VECTASHIELD Mounting Medium (VECTOR Laboratories, Burlingame, CA, USA).

JQ1 was purchased from Apexbio (A1910). U18666A was from Sigma-Aldrich (662015). Filipin complex was from Sigma-Aldrich (F9765). ARPE19 cells and HEK293 cells were obtained from American Type Culture Collection. Scrambled and BRD2-, BRD4-, or SREBP1-specific siRNAs were from Thermo Fisher Scientific (scrambled: AM4635; BRD2: AM16708, ID-118266; BRD4: 4457298, ID-s23902; SREBP1: AM51331, ID-5140). Lipofectamine 3000 was from Thermo Fisher Scientific (L3000008).