Dm6000b

Normal human nucleus pulposus (HNP) tissues were gently separated from lumbar trauma patients under aseptic conditions, washed with D-Hank’s solution 3–5 times, and then cut into small pieces with ophthalmic scissors(< 1 mm³). Subsequently, tissues were placed overnight in 5 ml of 0.1% type II collagenase (GIBCO, NY, USA) at 37 °C for 8 h. The digested fluid was filtered through 200 meshes filters, followed by filtration and centrifugation at 1000 rpm for 5 min. The supernatant was removed, and the precipitate was suspended in 3 ml of medium and centrifuged at 1000 rpm for 5 min. The supernatant was removed again, and the NPCs were seeded into a culture flask in DMEM/F12 medium (GIBCO, NY, USA) containing 15% fetal bovine serum (FBS, GIBCO, NY, USA), 100 μg/ml streptomycin and 100 U/ml penicillin under 5% CO₂ and saturated humidity at 37 °C. The culture medium was changed three times a week, and NPCs were subcultured at a ratio of 1:3 after reaching 80% confluence. Cell morphology was observed under an inverted microscope (DM6000B; Leica Microsystems, Japan). The concentration of HNPCs were adjusted to 1 × 10⁴/ml, and the cells were seeded into 24-well plates with glass coverslips for 48 h. Cells were fixed in formaldehyde for 20 min, followed by three washes with phosphate-buffered saline (PBS).

Selected plant cell components were visualized by immunocytochemical reactions on resin-free sections with specific primary antibodies provided by Agrisera (AHB2, Cat. No. AS132 745) or PlantProbes (Univeristy of Leeds, Leeds, UK), as follows: JIM5 (Cat. No. JIM5), JIM7 (Cat. No. JIM7), JIM11 (Cat. No. ELD030), JIM20 (Cat. No. ELD033), JIM8 (Cat. No. ELD024), JIM13 (Cat. No. JIM13-050), LM5 (Cat. No. LM5-050), LM6 (Cat. No. LM6-050), LM8 (Cat. No. LM8), LM24 (Cat. No. LM24). Antibodies were diluted (1:20) in 1% BSA in 1× PBS buffer (pH 7.2). The sections were incubated overnight at 4 °C. Next, sections were washed 3 times with 1× PBS buffer (pH 7.2) and incubated with secondary antibodies (goat anti-rat conjugated with FITC, Abcam, Cambridge, UK; goat anti-rabbit IgG DyLight 488 conjugated against AHB2, AS09 633, Agrisera, Vännäs, Sweden), 1:500 diluted in 1× PBS buffer. All reaction steps were conducted following our optimized protocol [109 (link)]. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) according to the producer′s recommendation. The results were documented using a fluorescent microscope DM6000B (Leica Microsystems GmbH, Wetzlar Germany). Control reactions were performed by omitting the incubation step with primary antibodies (Supplementary Figure S2).

Cells from the LAMA-84 cell line (1 × 10⁵) treated with PTPRG siRNA or scramble sequence, were subject to cytospin (Thermo Fisher Scientific, Milan, Italy) and immediately fixed in 4% paraformaldehyde for 10 min at 4 °C. After three washing steps, slides were incubated overnight at 4 °C with primary antibodies: 5 µg/mL phospho-β-catenin (Tyr 654) (ECM Biosciences, Versailles, KY, USA), 5 µg/mL total- β-catenin (Santa Cruz Biotechnology Inc, Heidelberg Germany) and 5 µg/mL anti-PTPRG [44 (link)], in a buffer containing PBS/Tween20 0.05%/BSA 1%/NaN₃ 4mM, followed by the proper secondary Alexa Fluor-conjugated antibodies (1:2000, Thermo Fisher Scientific, Milan, Italy), for 1 h at room temperature. After mounting with an anti-fading solution, samples were analyzed by fluorescence microscopy (DM6000B, Leica Microsystems, Wetzlar, Germay) and confocal microscopy (TCS-SP5, Leica Microsystem, Wetzlar, Germay).

Following HE or immunofluorescence staining, images were captured using an automated microscope (DM6000B, Leica Microsystems, Wetzlar, Germany) with a 340–380 nm filter for DAPI; a 450–490 nm filter for PAX7, MYOD, actin or CD163; and a 590 nm filter for desmin or CD80. DISKUS software (Leica) was used to process and merge the images. The mean number of positive cells in each sample was obtained from at least two randomly selected fields of 0.24 mm² within cross-sectional areas of muscle fibers. The fields containing adipose tissue, glands, or vessels were strictly excluded.

Cells (30,000–80,000) were seeded over glass coverslips placed into 24-well plates prior to treatment. At desired end-points, cells were fixed by 30 min incubation in 4% (w/v) paraformaldehyde (Sigma-Aldrich) in PBS. Cells were sequentially permeabilized with 0.1% (w/v) SDS in PBS (USB Corp.) and subjected to antigen blocking with 10% (v/v) FBS in PBS (both for 20 min). Primary antibodies employed were Bax, p21^Waf1/Cip1, p16^INK4a and phospho(Ser10)-Histone H3 (Table 2). Secondary antibodies were Goat anti-Mouse or anti-Rabbit IgG (H + L) Secondary Antibodies, conjugated either with Alexa Fluor^® 488 or 594 (ThermoFisher Scientific: #A-11001, #A-11005, #A-11008 and #A-11012). DNA counterstaining was performed upon 10 min incubation with 5 μg/mL solution of DAPI (Molecular Probes). Photographs were taken with either an Axio Observer.Z1 (Carl Zeiss AG) or a DM6000B (Leica Microsystems) epifluorescence microscope. Images were analyzed with Image J software (freely available from the National Institutes of Health at the address https://imagej.nih.gov/ij/).