Journal: Bone Research

Article Title: De novo serine synthesis regulates chondrocyte proliferation during bone development and repair

doi: 10.1038/s41413-021-00185-7

Figure Lengend Snippet: The serine synthesis pathway in growth plate chondrocytes. a Phgdh , Psat1 , Shmt1 , Shmt2 , Mthfd1 , and Mthfd2 mRNA levels in cultured skeletal progenitor cells (SPCs) and growth plate chondrocytes (CHs) ( n = 3). b Immunoblot of PHGDH, PSAT1 and β-actin in SPCs and CHs ( n = 3). c Immunoblot of PHGDH, PSAT1 and β-actin in SPCs and CHs after transduction with a lentiviral vector carrying a SOX9 overexpression (OE) plasmid or a shRNA (KD) against SOX9 ( n = 3). Empty vector or scrambled shRNA was used as the respective control. d Schematic of carbon atom (circles) transitions of 13 C 6 -glucose used to detect label incorporation into the shown metabolites. e Fractional contribution (FC) of 13 C 6 -glucose ( 13 C 6 -Glc) to serine (Ser) and glycine (Gly) in SPCs and CHs ( n = 3). Serine ( f ) and glycine ( g ) labeling from 13 C 6 -glucose in SPCs and CHs ( n = 3). Specific mass distribution vectors (MDVs) for each metabolite are shown. h Intracellular Ser and Gly levels in SPCs and CHs ( n = 3). The data are presented as the means ± SDs; * P < 0.05 vs. SPCs, ** P < 0.01 vs. SPCs, *** P < 0.001 vs. SPCs (Student’s t test)

Article Snippet: For SOX9 overexpression, skeletal progenitors were transduced with a lentivirus carrying a SOX9 overexpression plasmid (Addgene #36979; multiplicity of infection (MOI) 150), and for SOX9 knockdown, growth plate chondrocytes were transduced with a lentivirus carrying SOX9 shRNA (Addgene #40645; MOI 50).

Techniques: Cell Culture, Western Blot, Transduction, Plasmid Preparation, Over Expression, shRNA, Control, Labeling

Journal: Bone Research

Article Title: De novo serine synthesis regulates chondrocyte proliferation during bone development and repair

doi: 10.1038/s41413-021-00185-7

Figure Lengend Snippet: ATF4 mediates the cellular response to serine metabolic challenge. Immunoblots of phosphorylated eIF2α (Serine 51; p-eIF2α S51 ) and eIF2α ( a ) and of nuclear ATF4 and Lamin A/C ( b ) in bone ossicles and growth plates (GPs). For ectopic implants, chondrogenically primed wild-type progenitors were used as described in Fig. and implanted in mice fed a normal chow (−) or serine/glycine (S/G)-free diet (+). Growth plates were dissected from wild-type ( Phgdh chon+ ) and chondrocyte-specific PHGDH knockout ( Phgdh chon− ) mice ( n = 3). Immunoblots of p-eIF2α S51 and eIF2α ( c ) and of nuclear ATF4 and Lamin A/C ( d ) in chondrocytes cultured in serine/glycine (Ser/Gly)-free medium for the indicated durations ( n = 3). Immunoblots of p-eIF2α S51 and eIF2α ( e ) and of nuclear ATF4 and Lamin A/C ( f ) in chondrocytes treated with vehicle (veh) or NCT-503 for the indicated durations ( n = 3). g Glut1 , Phgdh , Psat1 , and Shmt1 mRNA levels in chondrocytes cultured in Ser/Gly-free medium for the indicated durations ( n = 3). h Slc1a4 and Slc6a9 mRNA levels in chondrocytes treated with NCT-503 for the indicated durations ( n = 3). i Immunoblot of nuclear ATF4 and Lamin A/C in chondrocytes after transduction with a lentiviral vector carrying a shRNA against ATF4 ( n = 3). A scrambled shRNA (shScr) was used as the control. Control and ATF4-knockout cells were cultured in serine/glycine-free medium or treated with NCT-503 (NCT). j Glut1 , Phgdh , Psat1 , and Shmt1 mRNA levels in control and ATF4-deficient chondrocytes cultured in complete medium (+) or serine/glycine-free medium (−) ( n = 3). k Slc1a4 and Slc6a9 mRNA levels in control and ATF4-deficient chondrocytes treated with or without NCT-503 ( n = 3). NCT- represents vehicle-treated. l Proliferation of control and ATF4-deficient chondrocytes cultured in complete or serine/glycine-free medium or treated with or without NCT-503 ( n = 3), as evidenced by BrdU incorporation. NCT- represents vehicle-treated. m Viability of control and ATF4-deficient chondrocytes cultured in complete or serine/glycine-free medium or treated with or without NCT-503 ( n = 3), as determined by Annexin V (AnxV)-propidium iodide (PI) staining followed by flow cytometry. AnxV - PI - cells were considered viable. NCT- represents vehicle-treated. n Matrix deposition of control and ATF4-deficient chondrocytes cultured in complete or serine/glycine-free medium or treated with or without NCT-503 ( n = 3), as evidenced by Alcian Blue (AB) staining. NCT represents vehicle-treated. o Schematic overview of the role of ATF4 in the regulation of serine metabolism in chondrocytes. The data are presented as the means ± SDs; # P < 0.05 (ANOVA), § P < 0.05 vs. shScr-complete medium or shScr-vehicle (ANOVA), ° P < 0.05 vs. shScr-Ser/Gly-free or shScr-NCT (ANOVA)

Article Snippet: For SOX9 overexpression, skeletal progenitors were transduced with a lentivirus carrying a SOX9 overexpression plasmid (Addgene #36979; multiplicity of infection (MOI) 150), and for SOX9 knockdown, growth plate chondrocytes were transduced with a lentivirus carrying SOX9 shRNA (Addgene #40645; MOI 50).

Techniques: Western Blot, Knock-Out, Cell Culture, Transduction, Plasmid Preparation, shRNA, Control, BrdU Incorporation Assay, Staining, Flow Cytometry