Bs 1064r

The cells were rinsed briefly with phosphate-buffered saline (PBS) and fixed for 20 min in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature. The cells were permeabilized for 10 min with 0.1% Triton X-100 in PBS, and blocked for 45–60 min with 4% bovine serum albumin in PBS at room temperature. Cells were incubated overnight at 4 °C with one of the following antibodies: anti-Oct4 (1:500; Abcam, Cambridge, MA, USA), anti-Sox2 (1:500; NB110-37235, Novus Biologicals, Littleton, CO, USA), anti-Nanog (1:500; Abcam, Cambridge, MA, USA), anti-c-Myc (1:250; bs-4963R, Bioss, Woburn, MA, USA), anti-Klf4 (1:250, bs-1064R, Bioss, Woburn, MA, USA). This was followed by incubation with the following secondary antibody: Alexa Fluor 488-labeled anti-rabbit IgG (1:500; Invitrogen, Carlsbad, CA, USA). Nuclei were counterstained using DAPI (1 mg/mL PBS; Invitrogen, Carlsbad, CA, USA).

Western blot analysis was performed as previously described [44 (link)]. The primary antibodies used were anti-FLAG (AF519, Beyotime,1:1,000), anti-MYOD (ABP53067, Abbkine, 1:500), anti-MyHC (B103, DHSB, 0.5 mg/mL), anti-FASN (10624-2-AP, Proteintech, 1:200), anti-CPT1 (bs-23779R, Bioss, 1:500), anti-ULK1 (bs-3602R, Bioss,1:500), anti-LC3B (NB100-2220, Novus, 2.0 mg/mL), anti-P62 (18420-1-AP, Proteintech, 1:1,000), anti-TBP (44059, Cell signaling, 1:1000), anti-KLF4 (bs-1064R, Bioss,1:500), anti-GPI (GTX113203, GeneTex, 1:500), anti-TNNI2 (bs-10617R, Bioss, 1:500), anti-CDKN1A (GTX112898, GeneTex, 1:500), and anti-GAPDH (60004-1-Ig, Proteintech, 1:5,000). ProteinFind goat anti-mouse IgG(H + L), HRP conjugate (HS201-01, TransGen, 1:1,000) and ProteinFind goat anti-rabbit IgG(H + L), HRP conjugate (HS101-01, TransGen, 1:500) were used as secondary antibodies. The original images of western blot are shown in Additional file 1.