nf κb p50  (Santa Cruz Biotechnology)

Journal: Cellular and Molecular Immunology

Article Title: Tofacitinib repairs inflammation and mitochondrial dysregulation in GM-CSF-reprogrammed RA macrophages

doi: 10.1038/s41423-026-01395-x

Figure Lengend Snippet: GM-CSF-differentiated RA MΦs exhibit a hyperglycolytic profile. A RA MΦs were untreated or treated with GM-CSF (100 ng/ml), and TCA enzymes (IDH, OGDH, SDHA) and HIF1α (6 h) transcription or TCA metabolite levels (citrate, succinate, 0-18 h) were quantified by qRT-PCR ( n = 10) or colorimetric assay (representative of n = 3–5). B RA MΦs were untreated or treated with GM-CSF (100 ng/ml) for 6 h, and transcript levels of MFN2 and DRP1 were determined by qRT-PCR, n = 5. C Myeloid cells were treated with GM-CSF (100 ng/ml) for 0-24 h before detecting MFN2 and DRP1 by western blotting, n = 3 (Raw WBs: Suppl- - ). D In RA synovial tissue CD68 + MΦs, expansion of the DRP1-MFN2 ratio suggests mitochondrial fragmentation as displayed by IF immunostaining ( n = 3, magx100 and magx400). E , F RA MΦs were untreated or treated with GM-CSF (1μg/ml). MitoATP and glycoATP production rates were measured by Seahorse XF Real-time ATP rate assay ( E ), and GLUT1, HK2, and LDHA transcription levels were quantified by qRT-PCR, n = 5 ( F ). G Myeloid cells were treated with GM-CSF (100 ng/ml) for 0–60 min before detection of GLUT1 and HK2 by western blot analysis, n = 3 (Raw WBs: Suppl- - ). H Representative lactate levels are shown in RA MΦ conditioned media, untreated or treated with GM-CSF (100 ng/ml, 0-18 h), n = 7. I Diagram summarizing GM-CSF-driven glycolytic reprogramming. Data are presented as mean ± SEM; statistical analysis was performed using the Mann–Whitney test: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: Blots were subsequently probed for pERK, NF-κBp65, NF-κBp50, p-p38, pSTAT1/3/5, JAK1, JAK3, GLUT1, HK2, MFN2, and DRP1 (Cell Signaling), actin (Santa Cruz), and anti-rabbit IgG HRP-linked or anti-mouse IgG HRP-linked secondary antibodies (Cell Signaling, Table ).

Techniques: Quantitative RT-PCR, Colorimetric Assay, Western Blot, Immunostaining, MANN-WHITNEY

Journal: Cellular and Molecular Immunology

Article Title: Tofacitinib repairs inflammation and mitochondrial dysregulation in GM-CSF-reprogrammed RA macrophages

doi: 10.1038/s41423-026-01395-x

Figure Lengend Snippet: Tofacitinib therapy mitigates GM-CSF-induced arthritis by resolving the inflammatory and glycolytic imprints. Schematic visualization (A) and Δ ankle circumference (B) of mice that received i.a. injection of Ad-Control or Ad-GM-CSF (days 0 and 7), and the arthritic group received TOFA (10 mg/kg) daily over 10 days, n = 10 ankles in 5 mice. C Ankles from Ad-Ctrl and Ad-GM-CSF ± TOFA were stained for H&E, F4/80, HBEGF, and HIF1α. Joint lining thickness, inflammation, and blood vessel formation ( D ), F4/80 and HBEGF ( C, E ), GLUT1, HK2, and HIF1α ( C, G ) staining were scored on a 0-5 scale , n = 4. F IL1β, IL6, CCL2, and CCL5 transcriptional regulation was determined by qRT-PCR, n = 6. H , I Ankles were stained for colocalization of F4/80 and pSTAT5 (magx100) (H) or DRP1 and TOM20 ( I ) (magx400), n = 3. J The schematic figure summarizes the mechanism by which tofacitinib counteracts shared RA blood and ST IL1β⁺S100A⁺HIF1⁺IL10 lo NFIL3/6 lo MΦs and experimental arthritis through STAT5 deactivation. Data are presented as mean ± SEM, and statistical differences were determined by a 2-way ANOVA test with Tukey’s method for adjusting for multiple comparisons: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: Blots were subsequently probed for pERK, NF-κBp65, NF-κBp50, p-p38, pSTAT1/3/5, JAK1, JAK3, GLUT1, HK2, MFN2, and DRP1 (Cell Signaling), actin (Santa Cruz), and anti-rabbit IgG HRP-linked or anti-mouse IgG HRP-linked secondary antibodies (Cell Signaling, Table ).

Techniques: Injection, Control, Staining, Quantitative RT-PCR