Journal: Journal of Human Immunity
Article Title: Oncostatin M receptor deficiency as a novel candidate genetic cause of autosomal recessive hyper-IgE syndrome
doi: 10.70962/jhi.20250119
Figure Lengend Snippet: Constitutive and induced expression and signaling of OSMR in patient and control PBMC populations. (A–D) Serum-starved PBMCs from P1 and three healthy sex- and age-matched controls were stimulated with IL-6 (100 ng/ml), OSM (100 ng/ml), or IL-31 (100 ng/ml) for 30 min. Whole-cell lysates were harvested for WB for the visualization of pSTAT3, STAT3, and GAPDH (A and C), and band intensities were quantitated (B and D). (E) PBMCs from P1 and three healthy sex- and age-matched controls left untreated (UT) or stimulated with 50 ng/ml OSM, 50 ng/ml IL-6, or 50 ng/ml IL-21 for 6 h. Expression of IL-6 was measured from mRNA with RT-PCR. Data were analyzed by the double δ Ct method, and statistical differences were determined in GraphPad Prism by unpaired, nonparametric t test. (F) Gating strategies of control PBMCs stained for T cells (CD3), B cells (CD19), NK cells (CD56), monocytes (CD14), and OSMR and analyzed by FC. (G) Representative flow histograms of subpopulations of PBMCs from P1 and three HCs, which were left untreated or treated with LPS (1 µg/ml) alone or LPS (1 µg/ml) + IFNγ (50 ng/ml) for 24 h prior to OSMR surface expression analysis. The grey area (fluorescence minus one, FMO) shows the negative staining control, which represents PBMCs stained with the same staining mixture but without the anti-OSMR Ab as an indicative measure of the background signal. (H) Fold change of OSMR surface expression on monocytes (CD14) from LPS- or LPS+IFNγ-treated PBMCs. (I–L) PBMCs were pretreated with LPS (1 µg/ml) + 50 ng/ml IFNγ (50 ng/ml) for 24 h prior to stimulation with OSM (100 ng/ml) (I) or IL-31 (100 ng/ml) (K) for 30 min. Whole-cell lysates were harvested for WB for the visualization of pSTAT3, STAT3, and GAPDH, and band intensities were quantitated (J and L). HC, healthy control. Statistics were calculated using the unpaired t test. * = P < 0.05; *** = P < 0.001. Source data are available for this figure: .
Article Snippet: For RT-PCR, NHDFs were stimulated with 10 ng/ml OSM for 2 or 6 h mRNA was extracted according to manufacturer (#740466.4; MACHEREY-NAGEL).
Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Staining, Fluorescence, Negative Staining
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