Nanog

Mamospheres treated with SM compound, apoptosis ((BCL-2, 1:1000, #4223, Cell Signaling Technology), (BAX, 1:1000, #41162, Cell Signaling Technology)), stem cell marker ((NANOG, 1:1000, #8822, Cell Signaling Technology), (OCT4, 1:1000, #2750, Cell Signaling Technology), (SOX2, 1:1000, #2748, Cell Signaling Technology)), related signaling pathway components were examined at protein level. Cells treated to 50 μM SM treatment for 24 h and untreated control cells were first lysed to reveal the proteins inside them. Western blot analysis was performed according to the protocol in method 2.6.

Western blot assay was performed as described previously 20 (link). Briefly, total protein from cells was extracted using RIPA buffer (Beyotime, Shanghai, China) supplemented with complete EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland). The extracted protein was separated on SDS polyacrylamide gels and transferred to the PVDF membranes. Western blotting was performed using antibodies against human COL6A1 (Abcam, Milton, Cambridge, UK), N-cadherin, E-cadherin, Vimentin, FAK, Src, p-FAK, p-Src, p-STAT1 (Tyr701), p-STAT1 (Ser727), p-STAT3 (Tyr705), STAT1, STAT3, CD133, ABC2G, SOX2, Nanog, CD63, CD9, TSG101, SOCS5, Flag, GFP, HA, Ubiqution (Ub), GAPDH, p300, c-Jun were purchased from Cell Signaling Technology (CST, USA).

Cells were washed in phosphate buffered saline (PBS) and resuspended at room temperature. After incubation on ice for 30 min, cells lysates were centrifuged at 14,0009 x g at 4°C. Protein concentrations were measured using Bradford protein assay reagent, with bovine serum albumin as a standard. Membranes were incubated overnight at 4°C with primary antibodies (all at 1:1000) against the following molecules: phosphorylated- (p-)STAT3 or caspase 3 (Abcam, Cambridge, MA, USA); STAT3, GAPDH, a-SMA, and E-cadherin, Vimentin, NANOG, OCT4, SOX2, or OPN (Cell Signaling Technology, Danvers, MA, USA). Membranes were then incubated with secondary antibodies (1:5000) at room temperature for 2 h. GAPDH was used as a loading control.

The iPSCs, NPCs, and neurons were cultured on coverslips, washed with PBS three times for 5 min each time, and then fixed with 4% paraformaldehyde (Service, Cat. #G1101, China) for 30 min. They were then washed with PBS three times for 5 min each time with 0.1% Triton X-100 (Solarbio, Cat. #T8200, China), washed again with permeabilized cells for 15 min, PBS-washed again in 5% bovine serum albumin (Solarbio, Cat. #A8010, China), and finally blocked for 30 min at room temperature. Primary antibodies were appropriately incubated overnight at 4°C. The primary antibodies included OCT4 (1:200; Cell Signaling Technology), Nanog (1:1,000; Cell Signaling Technology), TRA-1-60 (1:1,000; Cell Signaling Technology), NESTIN1 (1:1,000; Cell Signaling Technology), PAX6 (1:200; Cell Signaling Technology), GFAP (1:200, Proteintech), MAP2 (1:200, Proteintech), Calnexin (1:200, Sigma-Aldrich), and Kv4.3 (1:500, Omnimabs). Immunoreactivities were visualized with goat anti-mouse antibodies conjugated to Alexa Fluor 594 (1:200; Abcam) and goat anti-rabbit antibodies conjugated to Alexa Fluor 488 (1:200; Abcam). A Nikon laser scanning confocal microscope (ECLIPSE Ni-U) was used to acquire fluorescence images.