Phgdh

Cells were lysed in RIPA buffer (Sigma-Aldrich, R0278) with protease inhibitor cocktail (Genedepot, P3100) and phosphatase inhibitor cocktail (Genedepot, P3200). Protein concentrations were determined by the Bradford protein assay (BIORAD, 500-0006), and equal amounts of protein were loaded and separated in sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Proteins were then transferred to nitrocellulose membranes (BIORAD, 1620177). After blocking with 5% skim milk, the membranes were probed with primary antibodies. Antibodies used in this study were as follows: STAT1 (Cell Signaling Technology, AB_2799965, 1/1000); pSTAT1 Serine-727 (Cell Signaling Technology, AB_2773718, 1/1000); PHGDH (Cell Signaling Technology, AB_2737030, 1/1000); Nestin (Abcam, AB_10859398, 1/1000); FKBP9 (Novus Biologicals, AB_11005959, 1/1000); β-actin (Cell Signaling Technology, AB_2223172, 1/1000); IDH1-R132H (DIANOVA, AB_2335716, 1/1000); IDH1 (Cell Signaling Technology, AB_10950504, 1/1000); Anti-rabbit IgG (Jackson ImmunoResearch, AB_2307391, 1/5000); and Anti-mouse IgG (Jackson ImmunoResearch, AB_2307392, 1/5000). After washing three times, the membranes were incubated with secondary antibodies. Band signals were developed with ECL western blotting substrate kit (Pierce, 32106).

Total cell lysates and nuclear protein-enriched lysates from cultured cells or ectopic bone implants were obtained using the appropriate cell lysis buffers, and Western blot analysis was performed as described previously.^{10 ,12 (link)} Briefly, proteins were separated by SDS-­PAGE, and protein-containing nitrocellulose membranes were incubated overnight with primary antibodies specific for the following proteins: AMPK (#2532; Cell Signaling Technology), p-AMPK^T172 (#2535, Cell Signaling Technology), ATF4 (#11815, Cell Signaling Technology), β-­actin (A5441, Sigma-Aldrich), eIF2α (#9722, Cell Signaling Technology), p-eIF2α^S51 (#9721, Cell Signaling Technology), Lamin A/C (sc-376248, Santa Cruz Biotechnologies), PHGDH (#66350, Cell Signaling Technology) and PSAT1 (NBP1-55368, Bio-Techne). Appropriate HRP-conjugated secondary antibodies were used for chemiluminescent detection of proteins (Western Lightning Plus, PerkinElmer).

All antibodies used in the experiments were purchased. Actin (AF5003, 1:1000) and LMNB1 (AF1408, 1:1000) were purchased from Beyotime; P21 (10355-1-AP, 1:1000) was purchased from Proteintech (Wuhan, China); PHGDH (66350, 1:1000), yH2AX (9718, 1:800) and Ki67 (9129, 1:400) were purchased from Cell Signaling Technology (Danvers, MA, USA); PSAT1 (53804, 1:1000) was purchased from Signalway Antibody (Greenbelt, MD, USA); PSPH (DF12711, 1:1000) was purchased from Affinity Biosciences (Cincinnati, OH, USA); H3 (ab1791, 1:1000), H3K4me3 (ab213224, 1:1000), H3K9me3 (ab176916, 1:1000), H3K27me3 (ab192985, 1:1000), H3K36me3 (ab282572, 1:1000) were purchased from Abcam (Waltham, MA, USA).

Total cell lysates and nuclear protein-enriched lysates were collected using appropriate cell lysis buffers, and western blot analysis was performed as described previously^{40 (link),48 (link)}. Analysis of histone methylation was performed 24 h after induction of osteoclastogenesis, unless otherwise specified. Proteins were separated by SDS–PAGE, transferred to nitrocellulose membranes and incubated overnight with primary antibodies against β-actin (A5441, Sigma-Aldrich), c-MYC (5605, Cell Signaling Technology), H3K4Me3 (9751, Cell Signaling Technology), H3K9Me3 (13969, Cell Signaling Technology), H3K27Me3 (9733, Cell Signaling Technology), H3K36Me3 (4909, Cell Signaling Technology), H3K79Me3 (74073, Cell Signaling Technologies), lamin A/C (sc-376248, Santa Cruz Biotechnologies), NFATc1 (sc-7294, Santa Cruz Biotechnologies), PHGDH (66350, Cell Signaling Technologies) or PSAT1 (NBP1-55368, Bio-Techne). Appropriate anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies were used for chemiluminescent protein detection (Western Lightning Plus, PerkinElmer).

Either whole-cell protein extracts or fractionated cell extracts were separated by SDS-PAGE (10%) and subsequently used for western blotting. Immunoblot analysis was performed using primary antibodies targeting hexokinase II (1:500 dilution; #2106); hexokinase I (1:1000; #2804); N-Myc (1:1000; #9405); c-Myc (1:1000; #9402); PHGDH (1:1000, #13428, all from Cell Signaling Technology). To ensure equivalent protein loading, blots were stained with a primary antibody raised against β-actin (1:2000 dilution; #A5441, Sigma Aldrich Merck, Munich, Germany). Detection and visualization was achieved using HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (GE Healthcare) and the ECL™ Prime Western Blotting Detection Reagent (GE Healthcare, #RPN2236), respectively. Data were analyzed using a FusionFX7 detection device (Vilber Lourmat).