Fig 2: Generation of macrophage-specific RGS12-KO mice. (A) Organization of the wild-type RGS12 allele, the floxed RGS12 targeting construct, the LysM-Cre+ transgene, and the recombinant RGS12 allele. Exons are shown as black boxes and are numbered. Cre: Cre recombinase coding sequence; neo: neomycin resistance gene; and loxP: loxP sites. (B) Immunoblot analysis of bone marrow macrophage (BMM) lysates isolated from the long bone showing the absence of RGS12 protein in RGS12-cKO mice. (C) Quantitative analysis of the results in (B). Note that an immunoreactive band for RGS12 (150 kD) is detectable in LysM-Cre+ mice, whereas LysM-Cre+;RGS12fl/fl mice almost completely lack the RGS12 protein. ***, P<0.001, n=5.

Fig 3: RGS12 can strengthen the transcriptional regulation of COX2 via NF-κB. (A) Western blot analysis of RGS12 expression. Primary BMMs were transfected with pCMV-empty (control) or pCMV-RGS12 (RGS12 OE) plasmids by Lipofectamine 3000 for 24 hours, and the expression levels of RGS12 were analyzed. **, P<0.01 vs. the control group, n=3. (B) Relative mRNA expression of COX2 was analyzed by real-time PCR. Primary BMMs were treated as described in (A), and the expression levels of COX2 were analyzed. **, P<0.01, ***, P<0.001 compared with the control group. The values are the mean ± SEM (n=5). (C) COX2 expression levels in BMMs were analyzed by ELISA. Primary BMMs were treated as described in (A), and COX2 expression levels were analyzed. Note that RGS12 OE enhanced the expression level of COX2. **, P<0.01, ***, P<0.001 compared with the control group. (D) Relative protein levels of NF-κBp65. RAW264.7 cells were transfected with shControl (pSicoR-empty) or shNF-κB plasmids (pSicoR-shNFκBp65-1 and pSicoR-shNFκBp65-2) for 24 hours. β-Actin was used as an internal reference. The data are presented as the means ± SEM. ***, P<0.001 vs. shControl group, n=3. (E) Western blot analysis of NF-κBp65. RAW264.7 cells were transfected with pcDNA3.1-empty (control) or pcDNA3.1-NF-κBp65 (NF-κBp65 OE) plasmids for 24 hours. β-Actin was used as an internal reference. The data are presented as the means ± SEM. ***, P<0.001 vs. the control group, n=3. (F) RAW 264.7 cells were transfected with pUC-shNF-κBp65 and/or pCMV-RGS12 for 24 hours and then induced with TNFα for 24 hours. Relative mRNA expression was measured by real-time PCR. Note that RGS12 could not increase COX2 expression when NF-κB expression was decreased. The values are the mean ± SEM. ***, P<0.001, n=5. (G) Analysis of the interactions between RGS12-PTB and NF-κBp65 by pulldown assay. RAW264.7 cells were transfected with pCMV-Flag or pCMV-PTB-Flag plasmids, and cell lysates were precipitated with anti-Flag affinity gel and immunoblotted (IB) with anti-NF-κB p65 and anti-Flag antibodies. (H) RGS12 promotes the transcriptional activity of NF-κB and COX2 expression (luciferase assay). Stable knockdown of NF-κB in RAW264.7 cells was achieved via the transfection of pSicoR-shNFκBp65-1 and pSicoR-shNFκBp65-2 vectors. The stably transfected RAW264.7 cells were treated with COX2-Luc, pcDNA3.1-NF-κBp65, pCMV-RGS12 and/or pCMV-PTB for 48 hours. *, P<0.05, ***, P<0.001 versus the control. The data are presented as the means ± SEM. Note that RGS12 or PTB could increase the transcriptional activity of NF-κB.

Fig 4: COX2/PGE2 enhances RGS12 expression through EP2 and EP4. (A) Western blot analysis of COX2 overexpression (OE) in BMMs. Primary BMMs were isolated from WT mice and transfected with pCMV-empty vector (control) and pCMV-COX2 (COX2 OE) plasmids by Lipofectamine 3000 for 24 hours. Immunoblot quantification shows significantly increased expression of COX2. ***, P<0.001 versus the control. n=5. (B) Analysis of RGS12 levels after COX2 overexpression (OE) and PGE2 induction. RGS12 levels were determined by strand-specific RT-qPCR analysis of total RNA. The data are presented as the mean ± SEM, n=5. **, P<0.01; ***, P<0.001. (C,D) Western blot analysis of RGS12 expression after COX2 overexpression (OE) and PGE2 induction in BMMs (C). RGS12 levels were measured by semiquantitative Western blotting (D). The data are presented as the mean ± SEM, n=5. **, P<0.01 and ***, P<0.001. (E) RGS12 mRNA levels were determined by real-time PCR. Primary BMMs were treated with PGE2 (Cayman) or PGE2 plus individual PGE2 receptor antagonists (EP1 (ONO-8711, Cayman) 10 µM; EP2 (TG11-77, Cayman) 10 µM; EP3 (Sulprostone, Cayman) 10 µM; or EP4 (GW 627368X, Cayman) 10 µM). The data were normalized to GAPDH levels and are presented as the mean ± SEM, n=5. *, P<0.05 and ***, P<0.001. (F,G) RGS12 protein levels were quantitatively analyzed. Primary BMMs were treated as described in (E). **, P<0.01 and ***, P<0.001, n=5. The data are presented as the mean ± SEM.

Fig 5: The loss of RGS12 in macrophages attenuates RA pain. (A) Effect of RGS12 deletion in macrophages on pain thresholds as measured by the von Frey pain test in arthritic mice. A significant decrease in the pain thresholds of LysM-Cre+;RGS12fl/fl mice compared to LysM-Cre+ mice was observed in arthritic animals beginning on day 5 after induction with mAbs. The data are expressed as the means ± SEM. n=5, **, P<0.01. (B) Effect of RGS12 deficiency in macrophages on the latency of paw withdrawal from the thermal stimulus in arthritic mice. Each value in the line graph represents the mean ± SEM. A significant decrease in the latency time of LysM-Cre+;RGS12fl/fl mice compared to LysM-Cre+ mice was observed in arthritic animals beginning on day 5 after induction with mAbs. (C,D) Behavioral responses of LysM-Cre+ and LysM-Cre+;RGS12fl/fl mice with the same clinical score (score level 2) to mechanical and heat stimulation in the context of RA. (C) Mechanical paw withdrawal thresholds of naive LysM-Cre+ and LysM-Cre+;RGS12fl/fl mice obtained by von Frey monofilament stimulation. (D) Heat-induced paw withdrawal thresholds of LysM-Cre+ and LysM-Cre+;RGS12fl/fl mice obtained by the plantar heat test. **, P<0.01; ***, P<0.001.