Mice
C57BL/6 J (JAX:000664) mice were obtained from The Jackson Laboratory. B6;129-Osmrtm1.1Nat/J (JAX: 011081) were crossed to B6.Cg-Tg(Mx1-cre)1Cgn/J mice (JAX:003556). To induce Mx1-Cre, mice were intraperitoneally injected once every other day for five total injections with 15 mg/kg high molecular weight polyinosinic-polycytidylic acid (polyI:C) (InvivoGen). Mice used for experiments were young female adults (2–4 months of age) and were used >10 weeks after polyI:C administration. The Jackson Laboratory’s Institutional Animal Care and Use Committee approved all experiments.
Genotyping and recombination PCR
Genomic DNA was extracted from lysed peripheral blood samples using the DNeasy Blood & Tissue Kit (Qiagen) for genotyping. Genotyping primers were used as suggested on the B6;129-Osmrtm1.1Nat/J (JAX: 011081) mice webpage (protocol ID 29086 and 23692). Images were taken using the InGenuis LHR2 Gel imaging system (Syngene). Recombination of the Osmrfl locus was evaluated by PCR using the following primers: Forward: 5’-GGAAATACCTTGGCAGTGGTG and Reverse: 5’-GCTACCAAACCTCGGTAATCC.
Western blotting
Total protein was extracted from snap frozen tissues with a Tissue Lyser II (Qiagen) using 5 mm Bead Beater balls (Qiagen) after the addition of 50 mM HEPES pH 7.5 (Gibco) and 6 M urea (Sigma Aldrich) at 1.5 mL per 200 mg tissue. Insoluble material was spun out at 21,000 × g for 20 m. Total protein concentration of each sample was determined by microBCA (ThermoFisher). SDS-PAGE gel samples were prepared. Two 4–15% Mini-PROTEAN TGX Precast, 1.0 mm, 15-well, SDS-PAGE Gel (BioRad) were loaded with 25 ug total protein per well. The gels were run at 150 V for 1 h at room temperature in Tris·Glycine·SDS buffer (BioRad) then transferred overnight at 4 °C at 40 mAmp to 0.22 mm PVDF (BioRad) in Tris·Glycine buffer (BioRad) with 10% methanol. The blots were placed in Block (2% BSA + 4% Dry Milk) at room temperature for 1 h. The blots were then placed in primary antibody Polyclonal Goat IgG anti-mouse OSMRβ (R&D systems AF662) diluted in fresh block at 1:2500 and incubated on an orbital shaker at 4 °C overnight. Then the blots were washed with TBS with 0.03% Tween 20 (TBS-T) for 15 min and three more washes for 5 minutes each. Then the blots were placed in Peroxidase AffiniPure Bovine Anti-Goat IgG (H + L) (Jackson Immuno Research) diluted 1:5,000 in block. The blots rotated for 1 h at room temperature and then were washed with TBS-T for 15 min and three more washes for 5 minutes each. The blot was then developed with (ThermoFisher) SuperSignal West Pico Plus reagents (ThermoFisher) and visualized with a Syngene G-Box. The blots were then stripped with Restore™ PLUS Western Blot Stripping Buffer (ThermoFisher) at room temperature for 5 min. The blots were re-blocked and then incubated with anti-β-actin (Cell Signaling Technology clone D6A8) at 1:5000 for 1 h at room temperature. Then the blots were washed with TBS-T for 15 min and three more washes for 5 minutes each. Then the blots were placed in Goat Anti-Rabbit IgG (H + L)-HRP (Bio-Rad) diluted 1:5,000 in block. The blots rotated for 1 h at room temperature and then were washed with TBS-T for 15 min and three more washes for 5 minutes each. The blot was then developed with SuperSignal West Femto Maximum Sensitivity reagents (ThermoFisher) and visualized with a Syngene G-Box. Images were quantified by ImageJ by relative intensity, adjusted for background and normalized to β-Actin expression per sample. Significance was calculated using paired t test with correction for multiple comparisons using the Holm-Sidak method. Calculation of significance and graphing was performed using Prism 10 (GraphPad).
RNA sequencing
Bone marrow (BM) cells were isolated from OsmrΔ/Δ Mx1-Cre and Mx1-Cre from pooled and crushed femurs, tibiae, iliac crests, sternums, forepaws, and spinal columns of individual mice. BM mononuclear cells (MNCs) were isolated by 1X Red Blood Cell lysis Buffer (eBioscience) and stained with a panel of fluorochrome-conjugated antibodies against c-Kit (BD Biosciences, BioLegend clone 2B8), Sca-1 (BioLegend clone D7), CD150 (BioLegend clone TC15-12F12.2), CD48 (BioLegend clone HM48-1), FLT3 (BioLegend clone A2F10), (Lin) marker mix (B220 (BD Biosciences, BioLegend clone RA3-6B2), and 4′,6-diamidino-2-phenylindole (DAPI). 2,000 hematopoietic stem cells (HSCs) were prospectively isolated based on the cell surface marker combination: Lin- Sca-1+ c-Kit+ Flt3- CD150+ CD48- on a FACSAria with >95% purity (Fig. 2). HSCs were directly sorted into StemSpan SFEM II media with SCF (100 ng/ml, BioLegend), TPO (50 ng/ml, Peprotech), with or without OSM (500 ng/ml, BioLegend) and incubated at 37 °C for 60 min. Cells were then washed, pelleted and flash frozen. Total RNA was isolated from flash-frozen pellets using the RNeasy Micro Kit (Qiagen) including the optional DNase digest step. RNA concentration and quality were assessed using the RNA 6000 Pico Assay (Agilent Technologies). Libraries were constructed using the SMARTer Stranded Total RNA-Seq Kit v2-Pico (Takara) according to the manufacturer’s protocol. Library concentration and quality were assessed using the D5000 ScreenTape (Agilent Technologies) and Qubit dsDNA HS Assay (ThermoFisher). Libraries were subject to 75 bp paired-end sequencing on an Illumina NextSeq 500 using the High Output Reagent Kit v2.5 or 150 bp paired-end on an Illumina NovaSeq 6000 using the S4 Reagent Kit v1.5. Trimmed alignment files were processed using RSEM (v1.3.3 or v1.3.1). Alignment to the mm10 reference genome was completed using Bowtie 2 (v2.4.1 or v2.4.5). Expected read counts per gene produced by RSEM were rounded to integer values. Filtered total read counts had an average of 151 million counts (±32 million standard deviation), Q30 rate of 93% (±1% standard deviation), 52% GC rate (±2% standard deviation), and 28 million (±9 million standard deviation) read pairs mapped to genes (Table S1, see supplementary xlsx file). Counts were filtered to include only genes that had at least two samples within a sample group having a counts per million reads >1 in R (v4.1.3), and edgeR (v3.36.0) was used for differential expression analysis. A negative binomial generalized log-linear model was fit to the read counts for each gene. The dispersion trend was estimated by Cox-Reid approximate profile likelihood followed by empirical Bayes estimate of the negative binomial dispersion parameter for each tag, with expression levels specified by a log-linear model. Likelihood ratio tests for coefficient contrasts in the linear model were evaluated producing a p value per contrast. The Benjamini and Hochberg’s algorithm was used to control the false discovery rate. Features with fold change (FC) >2 or <−2 and p < 0.05 were declared significantly differentially expressed. Predicted genes and pseudogenes were removed from the list of significantly differentially expressed genes before downstream comparisons were performed.
Cell sorting plots and purity for control Mx1-Cre and OsmrΔ/Δ Mx1-Cre HSCs. (A) Representative flow cytometric gating used for isolation of HSCs as viable Lin- Sca-1+ c-Kit+Flt3- CD150+ CD48- cells. (B) Representative purity of isolated Lin- Sca-1+ c-Kit+ HSPCs after cell sorting, which was >95%. (C) Representative purity of isolated Lin- Sca-1+ c-Kit + Flt3- CD150+ CD48- HSCs after cell sorting, which was >99%.
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- Source: https://www.nature.com/articles/s41597-024-03839-3