*4.7. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Analysis*

The complementary DNAs (cDNAs) were obtained from RNA samples (100 ng per run) using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc, Carlsbad, CA, USA) according to the manufacturer's protocol. Expressions of miR-941 (Assay ID. 002183) and miR-146b-5p (Assay ID. 001097) were examined using TaqMan microRNA assays (Applied Biosystems; Thermo Fisher Scientific, Inc, Carlsbad, CA, USA). The following primers of miRNAs were used: hsa-miR-941, UGAGAACUGAAUUCCAUGGGUU and hsa-miR-146b-5p, UGAGAACUGAAUUCCAUAGGCU. Quantitative RT-PCR was performed on an Applied Biosystems QuantStudio 7 Flex system. Target gene expression levels were normalized to U6 (Assay ID. 4427975). We quantified the PCR values using a 2-ΔΔCt method with U6 as an internal control by adopting the method from a previous study [31].

#### *4.8. Western Blot Analysis*

Total proteins from monocytes and monocytes-derived osteoclasts from patients with PsA were separated on a 10% SDS polyacrylamide gel and the proteins transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany). Non-specific binding sites were blocked with 5% BSA for one hour at room temperature. Membranes were subsequently incubated overnight at 4 ◦C with rabbit anti-WNT16 (Invitrogen, Carlsbad, CA, USA), rabbit anti-mTOR (Invitrogen, Carlsbad, CA, USA), and mouse anti-GAPDH (Merck Millipore, Darmstadt, Germany) antibodies conjugated with horseradish peroxidase. Band densities were quantified using ImageJ software (NIH, Bethesda, MD, USA).
