Ba1105

The control group was cultured in normal conditions and the GPS group was cultured with 400 mg/l GPS for 48 h. K562 cells were centrifuged at 1,000 × g and 4°C for 5 min and then placed on slides. Them, slides were fixed with 4% paraformaldehyde at 4°C for 20 min and washed three times with PBS for 5 min each. After blocking with 0.5% bovine serum albumin (Beyotime Institute of Biotechnology, Haimen, China) at room temperature for 30 min, slides were incubated with mouse anti-p-ERK, p-P38, p-JNK and rabbit anti-NF-κB p65 and cyclin D1 (all at 1:150) overnight at 4°C. Then, slides were washed three times with PBS, and treated with fluorescein isothiocyanate goat anti-mouse (1:100; BA1105) or rabbit (1:100; BA1101; both Wuhan Boster Biological Technology, Ltd.) IgG for 40 min at room temperature in the dark. After being mixed with PI for 1 min, slides were again rinsed with PBS three times, mounted with 50% glycerol and stored in the dark. Immunofluorescence was examined with a Leica Sp2 confocal microscope.

The PCMs and BMMSCs were either separately cultured or co-cultured on coverslips for 7 days in growth medium, followed by differentiation in induction medium for 21 days, as aforementioned. Cells were rinsed in PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were permeabilized by 0.5% Triton X-100 (in PBS) for 20 min at room temperature and then blocked in 5% normal goat serum (NBP2-23475; Novus Biologicals, Ltd.) for 30 min at room temperature. Primary rat monoclonal antibodies, including anti-rod photoreceptor rhodopsin (1:200; NBP2-25159; Novus Biologicals, Ltd.), anti-bipolar neurons visual system homebox 2 (CHX10; 1:500; NBP1-84476; Novus Biologicals, Ltd.) and anti-Müller glia heparin sulfate (1:300; MAB2040; EMD Millipore, Billerica, MA, USA) were diluted and incubated with cells overnight at 4°C. The secondary antibodies (fluorescein isothiocyanate-conjugated) were diluted 1:32 (BA1101 and BA1105; Wuhan Boster Biological Technology, Ltd.) and incubated for 30 min at room temperature in the dark. Following washing thoroughly with PBS, the coverslip was observed by an inverted fluorescence microscope (Olympus Corporation). The positive rate was determined as the percentage of green cells by ImageJ 1.48v (https://imagej.nih.gov/ij/).

At room temperature the cells were fixed for 40 min using 4% buffered paraformaldehyde.
Slides were permeabilized for 10 min using 0.5% Triton X-100, then blocked with 2% BSA
(Wuhan Boster Biological Technology, Ltd.) for 1 h. Primary antibodies rabbit anti-rat
neuron-specific enolase (NSE; 1:50; PA1061, Wuhan Boster Biological Technology, Ltd.),
rabbit anti-rat microtubule-associated proteins 2 (MAP2, Abcam, ab32454, Shanghai, China),
rabbit anti-rat glial fibrillary acidic protein (GFAP; 1:50; A00213, Wuhan Boster
Biological Technology, Ltd.), mouse anti-rat Oligodendrocyte Marker O4 (O4; 1: 250;
MAB345, Chemicon, Temecula, CA, USA), and rabbit anti-rat Myelin Basic Protein (MBP,
Abcam, ab7349) were added to the tissues and incubated at 4°C for 12 h followed by
incubation with the following secondary antibodies: FITC-conjugated anti-rabbit IgG
(1:100; BA1105, Wuhan Boster Biological Technology, Ltd.), CY3-coupled anti-mouse IgG
(1:100; BA1031, Wuhan Boster Biological Technology, Ltd.), and CY3-coupled anti-rabbit IgG
(1:100; BA1032, Wuhan Boster Biological Technology, Ltd.). The nuclei were stained using
Hoechst 33258. The images were captured using an Olympus confocal microscope.