Additional file 1 of LOX overexpression programming mediates the osteoclast mechanism of low peak bone mass in female offspring rats caused by pregnant dexamethasone exposure
Creators: name: Jiang, Tao
name: Xiao, Hao
name: Li, Bin
name: He, Hangyuan
name: Wang, Hui
name: Chen, Liaobin
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Genetics
subject: FOS: Biological sciences
subject: Physiology
subject: Ecology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Fig. S1. The timetable and schematic procedure of animal treatment from gestational day (GD) 0 to postnatal week (PW) 12. Fig. S2. The experiments of the knockdown efficiency of LOX-siRNA on BMMs. (A, D): LOX mRNA expression in osteoclasts. (B, E): Representative images of LOX immunohistochemical-paraffin staining with DAPI in osteoclasts. (C, F): Quantification analyses of LOX protein mean optical density. Mean ± S.E.M., n=6 per group for mRNA expression, n=3 per group for LOX immunohistochemical-paraffin. **P&lt;1.01 vs. NC-siRNA in vitro or CON+NC in vivo. ##P&lt;0.01 vs. DEX+NC-siRNA in vitro or PDE+NC in vivo. DEX: dexamethasone; LOX: lysyl oxidase; NC: negative control; CON: control; PDE: prenatal dexamethasone exposure. Fig. S3. Cell viability experiments on BMMs. (A) The cytotoxic effect of DEX on BMMs at different concentrations was measured by the MTS assay. (B) The cytotoxic effect of TEM on BMMs at different concentrations was measured by the MTS assay. (C) The cytotoxic effect of H2O2 on BMMs at different concentrations was measured by the MTS assay. Mean ± S.E.M., n=6 per group. **P&lt;0.01 vs. 0 group. DEX: dexamethasone; TEM: tempol; BMMs: bone marrow-derived macrophages; MTS: 3-(4,5-dimethyltiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium. Fig. S4. PDE induced no significant change of osteoclast function in male offspring rats. (A, E): Representative images of TRAP staining in decalcified bone sections of fetal rats and adult offspring rats. (B, F): Quantification analyses of N.Oc/B.Pm. (C, G): Quantification analyses of Oc.S/BS. (D): NFATc1, c-Fos, Acp5, CtsK, Oscar and DC-stamp mRNA expression in bone tissue of fetal rats. (H-M): NFATc1, c-Fos, Acp5, CtsK, Oscar and DC-stamp mRNA expression in bone tissue of adult offspring rats. Mean ± S.E.M., n=3 per group for TRAP staining, n=8 per group for mRNA expression. **P&lt;0.01 vs. Control. ##P&lt;0.01 vs. PDE. PDE: prenatal dexamethasone exposure; TRAP: tartrate resistant acid phosphatase; N.Oc/B.Pm: osteoclast number per bone perimeter; Oc.S/BS: osteoclast surface per bone surface; NFATc1: nuclear factor of active T cells 1; c-Fos: protooncogene c-Fos; Acp5: acid phosphatase 5; CtsK: cathepsin K; Oscar: osteoclast-associated receptor; DCstamp: dendritic cell specific transmembrane protein; SDHA: succinate dehydrogenase; TBP: TATA box binding protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; YWHAZ: tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, zeta. Fig. S5. Transcriptome sequencing results of female fetal rat long bone tissue and related experiments. (A, B): Transcriptome sequencing assays were performed in bone tissue of fetal rats. (C): ROS synthesis-related genes mRNA expression in bone tissue of fetal rats. (D): LOX mRNA expression in bone tissue of offspring rats. (E, F): NOX2 and COX1 mRNA expression in bone tissue of offspring rats. (G, H): NOX2 and COX1 mRNA expression in osteoclasts treated with different concentrations of DEX. (I, J): Predicted binding site of GR to LOX promoter region. Mean ± S.E.M., n=8 per group for mRNA expression in vivo, n=6 per group for mRNA expression in vitro. *P&lt;0.05, **P&lt;0.01 vs. Control in vivo or 0 nM DEX in vitro. LOX: lysyl oxidase; PDE: prenatal dexamethasone exposure; NOX: NADPH oxidases; NOS: NO synthase; COX: cyclooxygenase; SDHA: succinate dehydrogenase; TBP: TATA box binding protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; YWHAZ: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta; DEX: dexamethasone. Table S1. The LOX siRNA sequences used in BMMs transfection experiment. Table S2. Rat oligonucleotide primers and reaction conditions used in RT-qPCR. Table S3. Rat oligonucleotide primers and reaction conditions used in ChIP-PCR.