Ab200834

The following antibodies were used for western blotting: rabbit monoclonal anti-CD24 (1:500, catalog number ab179821, Abcam, MA, USA), rabbit monoclonal anti-CD44 (1:500, catalog number ab189524, Abcam), rabbit recombinant multiclonal anti-CD133 (1:800, catalog number ab278053, Abcam), mouse monoclonal anti-Nanog (1:1000, catalog number ab173368, Abcam), rabbit monoclonal anti-OCT4 (1:500, catalog number ab200834, Abcam), mouse monoclonal anti-SOX2 (1:1000, catalog number ab79351, Abcam), rabbit polyclonal anti-DLG5 (1:1000, catalog number ab231283, Abcam), rabbit polyclonal anti-PRDM16 (1:1000, catalog number ab106410, Abcam), mouse monoclonal anti-ErbB-2 (1:500, catalog number sc-33684, Santa Cruz Biotechnology, CA, USA), and mouse monoclonal anti-β-actin (1:1000, catalog number A5441, Sigma-Aldrich, MO, USA). Other primary antibodies and secondary antibodies and experimental procedures for immunoblotting are provided in the supplementary experimental procedures^{10 (link)}.

Cells were cultured on a glass slide and fixed in 4% paraformaldehyde for 15 min. The cells were permeabilized with 0.1% Triton X-100 for 10 min, and non-specific antibody interaction sites were then blocked for 15 min. Next, the cells were incubated at 4 °C overnight with primary antibodies, Nanog (ab109250, Abcam, USA), Oct4 (ab200834, Abcam, USA), SSEA4 (ab16287, Abcam, USA), and TRA-1-81 (ab16288, Abcam, USA), to identify ESCs and follicle stimulating hormone receptor (FSHR, 22665-1-AP, Proteintech, China) to identify granulosa cells. On day 2, the cells were incubated with a secondary antibody in a darkroom for 1 h. Finally, the cell nuclei were labeled with Hoechst 33324 for 1 min. After that, the slides were observed and imaged using a fluorescence microscope (DMI 6000, Leica Micro systems, Buffalo Grove, IL, USA).

Antibodies against octamer-binding transcription factor 4 (1:1000, Oct-4; ab200834, abcam), Brachyury (1:1000, sc-166962, Santa Cruz Biotechnology), GATA-binding protein 4 (1:1000, GATA4; sc-25310, Santa Cruz Biotechnology), SRY box transcription factor 17 (1:1000, Sox17; AF1924, R&D system), neuronal differentiation 1 (1:1000, NeuroD1; ab205300, abcam), XRCC5 (1:2000, AF5819, R&D system), IGF2BP1 (1:1000, ab184305, abcam), RBBP4 (1:1000, MAB7416, R&D system), NPM1 (1:1000, sc-271737, Santa Cruz Biotechnology), PCNA (1:1000, sc-56, Santa Cruz Biotechnology), GNAI2 (1:1000, ab137050, abcam), PHB2 (1:1000, sc-133084, Santa Cruz Biotechnology), LAMB1 (1:1000, sc-17810, Santa Cruz Biotechnology), LAMC1 (1:1000, sc-17751, Santa Cruz Biotechnology), β-catenin (1:1000, sc-7963, Santa Cruz Biotechnology), AKT (1:2000, #4691, Cell Signaling Technology), Jun amino-terminal kinases (JNK) (1:1000, #9252, Cell Signaling Technology), phosphorylated (p)-JNK (1:1000, #9255, Cell Signaling Technology), p38 (1:1000, sc-81621, Santa Cruz Biotechnology), p-p38 (1:1000, #9216, Cell Signaling Technology), TGF-β (1:1000, #3711, Cell Signaling Technology), CTGF (1:1000, sc-385970, Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; (1:3000, sc-47724, Santa Cruz Biotechnology) were employed for the western blot analysis.

The proteins of differently treated cervical cancer cells were extracted with RIPA Lysis Buffer (PP110; Protein Biotechnology Co., Ltd, China) according to the manufacturer's instruction. Then, the concentration of the proteins was determined using a BCA protein assay kit (PP202; Protein Biotechnology Co., Ltd, China). Subsequently, 30 µg proteins in each group were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gels were then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated in Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat milk for 1 h. Then, the PVDF membrane was incubated overnight with primary antibodies against NANOG (ab203919; Abcam, USA), OCT4 (ab200834; Abcam, USA), CD133 (ab278053; Abcam, USA), SOX2 (ab92494; Abcam, USA), and β-actin (ab8227; Abcam, USA). Then, the membrane was incubated with HRP-conjugated secondary antibody (ab205718; Abcam, USA) for 2 h at room temperature. The bands of individual proteins were visualized using a chemiluminescence (ECL) kit (PP404; Protein Biotechnology Co., Ltd., China).

Proteins were isolated from LUSC cells and tissues using RIPA buffer (Sigma) and then separated using 10% SDS‐PAGE and transferred to PVDF membranes (Millipore). After being blocked with 5% non‐fat milk, the membranes were probed overnight with, anti‐SOX2 (ab97959, Abcam), anti‐OCT4 (ab200834), anti‐Nanog (ab109250), anti‐PTN (ab79411), and anti‐GAPDH (ab8245, Abcam) antibodies at a dilution of 1:1000. Following 3 times washing, the cells were incubated with HRP anti‐rabbit IgG (ab6721, Abcam, 1:2000). Finally, bands were observed with the enhanced chemiluminescence system (ECL, ThermoFisher).

The in vivo access of CFN and M-CFN to the CSC fractions in tumors was measured in 4T1-induced tumor models. DiI-labeled CFN and M-CFN were intravenously administered to tumor-bearing mice at 1.0 mg/kg DiI. At 8.0 h postinjection, the tumor tissues were removed, frozen and sectioned at 10 µm (Leica 1950, Germany). The tumor sections were stained with primary antibodies against ALDH1A1 (Abcam, ab52492, 1:100), SOX2 (Abcam, ab97959, 1:1000) and Oct4 (Abcam, ab200834, 1:50), followed by Alexa Fluor 488-labeled IgG (H + L) secondary antibody (Beyotime, Jiangsu, China). In contrast, these sections were stained with DAPI for CLSM detection. The access of nanovehicles to these CSC fractions were denoted as the overlap of the red fluorescence signals of nanovehicles and the green signals of typical markers of CSCs in tumor sections. These images were analyzed using Image-Pro Analyzer 3D 7.0 (Media Cybernetics, MD, USA) to monitor the colocalization of nanovehicles with CSCs in tumors and were analyzed using ImageJ software to evaluate the access of nanovehicles to CSC fractions in tumors.