Fig. 5

Overexpression of GACAT2 (ov-GACAT2) reverses the inflammation-compromised cementoblastic differentiation and mitochondrial function of PDLSCs. The cells were incubated in medium with both inflammatory cytokines and EMD (inflammatory environment). a Overexpression efficiency of GACAT2 in PDLSCs validated by qRT-PCR. b Relative cementoblastic differentiation-related gene expression levels of BSP, CAP and CEMP-1 determined by qRT-PCR. c Relative cementoblastic differentiation-related protein expression of BSP, CAP and CEMP-1 determined by Western blots. d Semiquantitative analysis of protein expression levels (normalized to β-actin) in terms of relative gray density. e mtROS levels in PDLSCs determined with the aid of a MitoSOX probe (flow cytometric analysis). f Quantification of mtROS levels (reflected by the relative fluorescence intensity of MitoSOX). g Intracellular ATP contents (ATP assay). h The MMP was determined with a JC-1 probe (immunofluorescence staining; scale bar: 50 μm). i Quantification of MMP levels (reflected by the relative ratio of red/green fluorescence intensity of JC-1). j mtDNA content determined by qRT-PCR. k Relative mitochondrial complex-related protein expression of NDUFB8 (subunit of complex I), SDHA (subunit of complex II), UQCRC1 (subunit of complex III), COXIV (subunit of complex IV) and ATP5A (subunit of complex V) determined by Western blots. l Semiquantitative analysis of protein expression levels (normalized to β-actin) in terms of relative gray density. m OCR of PDLSCs determined by a Seahorse Bioscience XF Analyzer; arrows indicate the sequential injection of 1 μmol·L⁻¹ Oligo, 1 μmol·L⁻¹ FCCP and 2 μmol·L⁻¹ R/A. n Quantification of cellular respiration (basic OCR value prior to Oligo injection). o Quantification of maximal respiration (differences between the maximum rate measurement after FCCP injection and the minimum rate measurement after R/A injection). p Quantification of ATP production (difference between the final rate measurement prior to Oligo injection and the minimum rate measurement after Oligo injection). The data are shown as the mean ± SD for n from 3 to 8; *P < 0.05, **P < 0.01 and ***P < 0.001 indicate significant differences between the indicated columns