miR-125b and mitochondrial metabolism in human CD14⁺ monocytes under inflammatory conditions. CD14⁺ monocytes were negatively selected from peripheral blood mononuclear cells by magnetic separation isolated from either healthy donors (A-D) or patients with RA (E-G). (A) Kinetics of the OCR measured in CD14⁺ after LPS stimulation (0.1 μg/mL), in real time under basal conditions: oligomycin, ATP-synthetase-inhibited rate; FCCP, uncoupled rate; and rotenone + antimycin A, inhibited rate. Data are representative of 3 independent experiments. (B) Quantification of respiratory profile parameters presents 6 technical replicates. (C) Monitoring of mitochondrial fusion in CD14⁺ stained with anti-TOM20 antibodies (n = 3). Representative images of mitochondria stained with TOM20 (left; bar represents 5 μm) and quantification of fluorescence using Cellomics ArrayScan VTi platform (right; n = 20 per experimental replicate) are shown. (D) Kinetics of expression levels of miR-125b, MTP18, BIK, TNF, and CXCL11 mRNA in CD14⁺ monocytes after LPS stimulation using real-time polymerase chain reaction. RNA quantification presents fold change to time 0 (T0), and error bars represent the SD of 3 independent experiments. (E) miR-125b expression was quantified in CD14⁺ peripheral blood monocytes isolated from healthy controls (HC; n = 5) or patients with RA (n = 8). (F) Correlation study of miR-125b and BIK or MTP18 expression levels in CD14⁺ monocytes from healthy controls (n = 5) or patients with RA (n = 8) was performed using a Spearman correlation analysis. All results are expressed as mean ± SD. *P < .05; **P < .01; ***P < .001. SRC, spare respiratory capacity.