Confocal petri dish

Cells were cultured on a confocal petri dish (NEST Biotechnology Co. Ltd. China), fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature. The cells were stained with anti-HDAC3 antibody (1:100, Abcam, USA), which had been diluted 1:200 in 5% goat serum, overnight at 4 °C. The cells were subsequently stained with Alexa Fluor 594 (R37119) (1:200 dilution in PBS) (Invitrogen) at room temperature for 1 h, followed by incubation with DAPI (1:1000 dilution in PBS) for 5 min (min). The cells were then examined with a Lecia laser scanning microscope (FV1000, Olympus) at 100× magnification [18 (link)].

A549 cells (4 × 10⁵ cells/well) and CSCs suspension were plated onto confocal petri dish (NEST, China) and cultured for 6 h, respectively. Then, cells were washed with PBS, fixed with 4% paraformaldehyde, and blocked in Tris-buffered saline containing Tween-20 (TBST) with 3% BSA (Solarbio, China). Further, cells were incubated with anti-CD133 (1:250) and anti-CD44 (1:250) primary antibodies (Proteintech, USA) overnight at 4°C. For visualization, cells were then stained with PE-conjugated (1:100) and FITC-conjugated (1:100) secondary antibodies (Proteintech, USA) in the dark at 25°C for 1 h, respectively. Finally, the nuclei were labeled with Hoechst 33342 (Aladdin, China). Images were captured under a fluorescent microscope (Nikon, Japan).

The human osteoblast was isolated and cultured as previously described. In brief, from the American Type Culture Collection (ATCC, CRL-2593) and maintained in DMEM (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco, Life Technologies, Carlsbad, CA, USA), 10 mM HEPES (Sigma Aldrich, Poole, UK), and 0.1% penicillin-streptomycin (Sigma Aldrich, Poole, UK). The CRABP2-pcDNA3 plasmid (oe-CRABP2) was synthesized by Santa Cruz and transfected following the manufacturer’s protocol.
Then, human osteoblasts were plated onto the cover glass of a confocal Petri dish (NEST, Hong Kong, China) for transient transfection. DMEM (1 mL) containing 10% FBS was added to the dish, and the cells were cultured for 24 h prior to transfection. Transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific) and Opti-MEM reduced-serum media (Life Technologies, Waltham, Massachusetts, USA) according to the manufacturer’s instructions. To explore the mechanisms of CRABP2-mediated osteoblast apoptosis, the PI3K inhibitor LY294002 was used to pretreat cells (20 μM, MCE, Shanghai, China) for 2 h followed by stimulation with CRABP2-pcDNA3 for 12 h. The choice of inhibitor concentrations and time course was based on a previous study [20 (link)].

The mouse preosteoblast cell line MC3T3-E1 was obtained from American Type Culture Collection (ATCC, CRL-2593) and maintained in DMEM (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco, Life Technologies, Carlsbad, CA, USA), 10 mM HEPES (Sigma-Aldrich, Poole, UK), and 0.1% penicillin–streptomycin (Sigma-Aldrich, Poole, UK). The FGF-2-pcDNA3 plasmid (oe-FGF-2) was synthesized by GeneChem, Inc. Then, MC3T3-E1 cell suspensions (150 μl, containing 1 × 10⁴ cells) were plated onto the cover glass of a confocal petri dish (NEST, Hong Kong, China) for transient transfection. DMEM (1 mL) containing 10% FBS was added to the dish, and the cells were cultured for 24 h prior to transfection. Transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific) and Opti-MEM reduced-serum media (Life Technologies, Waltham, Massachusetts, USA) according to the manufacturer’s instructions. To explore the mechanisms of FGF-2-mediated MC3T3-E1 cell apoptosis, the PI3K inhibitor LY294002 was used to pretreat cells (20 μM, MCE, Shanghai, China) for 2 h followed by stimulation with FGF-2-pcDNA3 for 12 h. The choice of inhibitor concentrations and time course was based on a previous study [22 (link)].

Human OS cell lines and hFOB1.19 suspensions (1 × 10⁴ cells in 150 μL) were plated on coverslips in a confocal petri dish (NEST, Hong Kong, China) for transient transfection. DMEM (1 mL) containing 10% FBS was added to the dish, and the cells were cultured for 24 h prior to transfection. Transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific) and Opti-MEM reduced-serum medium (Life Technologies, Waltham, Massachusetts, USA) according to the manufacturer’s instructions.
siRNA sequences targeting PENK (si-PENK-1 and si-PENK-2) were purchased from GenePharma, Inc. The siRNA sequences were as follows: si-PENK-1 forward, 5′-GCAATCGAGATGGAACCAT-3′ and si-PENK-1 reverse, 5′-ATGGTTCCATCTCGATTGC-3′; si-PENK-2 forward, 5′-CCATCGGTCTACCTATTAT-3′ and si-PENK-2 reverse, 5′-TACGAACGACCAGCTTACC-3′; and si-con forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and si-NC reverse, 5′-ACGUGACACGUUCGGAGAATT-3′. LY294002 was purchased from MedChemExpress (MCE, Shanghai, China) and used at a concentration of 10 μM as previously described [17 (link)]. MG63 cells were pretreated with LY294002 (10 μM) and were then stimulated with si-PENK.