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Global Deletion of Protein Tyrosine Phosphatase 1B (PTP1B-/-) Results In

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Global Deletion of Protein Tyrosine Phosphatase 1B (PTP1B-/-) Results In Supplemental Figures and Supplemental Methods Section: Supplemental Figure 1: Global deletion of protein tyrosine phosphatase 1B (PTP1B-/-) results in shortened lifespan and splenomegaly. (a) Survival curve of male global PTP1B knockout mice (PTP1B-/-) and wild type controls (PTP1B+/+). PTP1B-/- mice have a significantly shorter life span (p<0.0001) with a median survival of 76 weeks compared to PTP1B+/+ mice, whose median survival could not be calculated as more than 50% of mice survived the predetermined census period (n=40 PTP1B-/- male mice and 22 PTP1B+/+ male mice). (b) PTP1B-/- female mice have a significantly shorter lifespan (p<0.0001) when compared to PTP1B+/+ controls with a median survival time of 64 weeks in comparison to 90 weeks in control mice (n=23 PTP1B-/- female mice versus 17 PTP1B+/+ female mice). (c) Global deletion of PTP1B in results in splenomegaly in PTP1B-/- mice (n=8/genotype) (p<0.01), with a two fold increase in spleen weight at the time of death. (d) Representative images of splenomegaly and lymph node enlargement in PTP1B-/- mice in comparison to control wild type and PTP1B heterozygous mice (PTP1B+/-) littermates. (e) Survival curve of male global PTP1B knockout mice (PTP1B-/-) and wild type controls (PTP1B+/+) expressed as percent healthy, sick or dead at 9-14 months of age versus 15-20 months of age. (f) Survival curve of female global PTP1B knockout mice (PTP1B-/-) and wild type controls (PTP1B+/+) expressed as percent healthy, sick or dead at 9-14 months of age versus 15-20 months of age. Supplemental Figure 2: Development of leukaemia is associated with protein tyrosine phosphatase 1b (PTP1B) deletion efficiency and time. (a) PTP1B and GAPDH bone marrow derived macrophages (BMDM) protein levels from PTP1Bfl/fl, LysM PTP1B+/- and LysM PTP1B-/- mice; LysM PTP1B-/- mice have the highest deletion efficiency (100%) with no detectable protein, and LysM PTP1B+/- mice have a varying deletion efficiency from 50-80% (n=10/genotype). (b) Quantification of PTP1B protein levels relative to GAPDH in BMDM in PTP1Bfl/fl mice (black), LysM PTP1B+/- (grey), protein was not detectable in LysM PTP1B-/- mice. Both genotypes, LysM PTP1B+/- and LysM PTP1B-/- had a significant decrease in PTP1B expression in comparison to PTP1B+/+ mice (p<0.001) (c) 100X magnification of blast cell population in blood, representative images are shown, cells have a high nuclear to cytoplasm ratio and display some nuclear membrane folding. (d) The number of blast cells present in PTP1Bfl/fl mice (black) and LysM PTP1B+/- mice peripheral circulation as determined by blood film differentials, there was no statistical difference between PTP1B+/+ and LysM PTP1B+/- mice (n=12/genotype). (e) White blood cell populations in the peripheral circulation of 9 month old PTP1Bfl/fl mice (black) and LysM PTP1B-/- mice (grey) as determined by blood film differential counts. LysM PTP1B-/- have a substantial blast cell population in the circulation of around 10% which is absent in PTP1B+/+ mice (p<0.001) (n=12/genotype). The increase in blast cell population is time dependent, with an (f) increase to 40% at 18 months of age in peripheral circulation of 18 month old PTP1Bfl/fl mice (black) and PTP1B-/- mice (grey), (n=12 PTP1B+/+ mice and 4 LysM PTP1B-/- mice, this is due to early death of LysM PTP1B-/- mice on the study). Supplemental Figure 3: LysM PTP1B-/- mice develop splenomegaly, hepatomegaly and tumours. (a) Representative post-mortem photograph of LysM PTP1B-/- mouse with large extranodal tumour. (b) 40x histology using haemotoxylin and eosin (H&E) staining of solid tumours in LysM PTP1B-/- mice from various locations, clockwise; top left - attached to the small intestine, top right – back of the neck, bottom right – hip joint of the right hind leg, bottom left – lower back. Tumours consist of pleomorphic cells with positive evidence of fibrosis. (c) 100x magnification of tumour cells from LysM PTP1B-/- solid tumours stained with H&E. Large pleomorphic cells with prominent nucleoli are pictured, many with wrinkled nuclear membranes. Supplemental figure 4: Hepatic microarray analysis from LysM PTP1B-/- and PTP1Bfl/fl mice A: Principal Component Analysis plot of hepatic microarray gene expression data from Ptp1b fl/fl mice (red circles; n = 5) or LysM-Ptp1b-/- mice (blue circles; n = 4), aged 6 months, injected with LPS for 3 hours. PCA plot was generated using Partek Genomics Suite. B: Heatmap of significantly differentially expressed genes in LysM-Ptp1b-/- vs Ptp1b fl/fl mice (red is upregulated and blue is downregulated). Top KEGG terms (C) and top GO terms (D) for genes (involved in cellular or molecular functions) significantly upregulated or downregulated in LysM-Ptp1b-/- vs Ptp1b fl/fl mice. Supplemental Figure 5: LysM PTP1B-/- have increased STAT5 expression. Representative immunoblots of (a) liver STAT5 protein and (b) Spleen STAT5 protein immunoblots with GAPDH loading control. STAT5 is increased in LysM PTP1B-/- when compared to control PTP1Bfl/fl mice in the c) liver (n=10/genotype) (p<0.05) and (d) spleen (n=10/genotype) (p<0.01). This is confirmed with qPCR gene expression analysis of (e) liver stat5 and (f) spleen stat5. (n=12/genotype)(p values - p*<0.05. **p<0.01, ***p<0.001). Supplemental figure 6: STAT3 ChIP-seq in LysM PTP1B-/- and PTP1Bfl/fl mice STAT3 binding peaks at selected genes were visualised in the UCSC genome browser. Chromosomal locations are shown at the top of each panel and gene positions are indicated at the bottom. Abbreviations: Bcl2l14, BCL2-like 14 (apoptosis facilitator), also known as Bclg; Capn1, calpain 1; Casp1, Caspase 1; Ddx5, DEAD box helicase 5, Fasl, Fas ligand; Mctp1, multiple C2 and transmembrane domain containing 1; Pdia4, protein disulfide isomerase family A member 4; Slc38a9, solute carrier family 38 member 9. Supplemental Methods: ChIP-seq analysis: BMDM extracted from PTP1Bfl/fl and LysM-PTP1B-/- mice aged 6 months were treated ± IL10 20ng/ml (Peprotech) for 4 hours(n=4/group). ChIP was performed using the SimpleChIP Enzymatic Chromatin IP kit (Cell Signalling) according to manufacturer’s instructions, with chromatin sheered to 200 bp and 10µl of STAT3 Mouse mAb #9139 per IP (Cell Signalling). Purified DNA was used to generate sequencing libraries with the TruSeq ChIP Library Preparation Kit (Illumina, San Diego, CA) to manufacturer’s instructions (50 ng of DNA per sample). Libraries were sequenced on the NextSeq- 500 Desktop sequencer platform (Illumina) with a 1x75 paired-end read length. Reads were processed within the Galaxy bioinformatics environment and filtered using TrimGalore to remove adapter sequences and reads with pHred score <20 or length <20bp. Filtered reads were checked using FastQC before aligning with HiSAT to the Mus musculus UCSC mm10 genome. BAM files were exported to the Cistrome bioinformatics environment and peaks called using MACS v1.4 using an input control and p-value cut-off 10-5. All peaks, including those in distal regions were included in the downstream analysis. Peaks were analysed using CEAS, GREAT was used to annotate the regions nearest to peaks (within 3 kb upstream and 1 kb downstream) and MEME suite tools were used to analyse motifs. Gene ontology and KEGG analysis on annotated genes nearest peaks was performed using DAVID v6.8. Microarray bioinformatics analysis: PTP1Bfl/fl and LysM-PTP1B-/- mice (n=5 and 4, respectively) were injected with 0.5 mg/kg LPS i.p. for 3 hours. Livers were snap frozen in liquid nitrogen and homogenised in TriZol (Sigma). Microarray was performed using Gene 2.0ST array (Affymetrix), according to manufacturer’s instructions. Assay quality control, differential expression analysis, hierarchical clustering and SOM clustering were performed using Partek Genomics Suite. Gene ontology and KEGG analysis of differentially expressed genes was performed using DAVID v6.8. a b 100 100 l l a a v v 80 i i 80 v v r r u u s 60 s 60 t t n n e e 40 40 c c r r e e P P 20 20 0 0 0 20 40 60 80 100 0 20 40 60 80 100 Weeks Weeks +/+ +/- -/- c d 0.15 ** ) g ( 0.10 t h g i e 0.05 W 0.00 - + / / - + B B 1 1 P P T T P P e Longevity - Males Longevity - females 100% 100% 90% 90% 80% 80% 70% 70% 60% 60% Dead 50% 50% Sick 40% 40% 30% Healthy 30% 20% 20% 10% 10% 0% 0% s s s s s s s s h h h h h h t h t h t t t t t t n n n n n n n n o o o o o o o o m m m m m m m m 4 0 0 4 0 4 0 4 1 2 2 1 2 1 2 - 1 - - - - - - - 9 8 8 2 5 2 5 9 1 - 1 1 1 1 1 / - T - - - T / T / / T - B - - W W 1 W B B B W 1 P 1 1 T P P P T P T T P P P Supplemental Figure 1 a PTP1B fl/fl LysM PTP1B+/- LysM PTP1B-/- PTP1B GAPDH Blast cells b PTP1B protein c LysM PTP1B-/- d 2.5 0.15 N.S 2.0 0.10 1.5 ve to GAPDH ve to Ɵ 1.0 0.05 ve percent of total RBC 0.5 N.D Ɵ 0.00 0.0 fl fl Rela / / expression rela fl fl PTP1B PTP1B PTP1B LysM PTP1B -/- LysM PTP1B +/- LysM PTP1B +-/- e * f 60 *** *** *** 60 * 40 40 20 20 Percent of Leukocytes Percent Percent of Leukocytes Percent 0 0 Basophils Basophils Blast Cells Blast Cells Basophils Basophils Monocytes Monocytes Eosinophils Eosinophils Blast Cells Blast Cells Neutrophils Neutrophils Monocytes Monocytes Eosinophils Eosinophils Lymphocytes Lymphocytes Neutrophils Neutrophils Lymphocytes Lymphocytes PTP1B fl/fl LysM PTP1B -/- PTP1B fl/fl LysM PTP1B -/- Supplemental Figure 2 a LysM PTP1B-/- Post Mortem b LysM PTP1B-/- tumours c LysM PTP1B-/- tumours small intesƟne neck small intesƟne neck on – staining H&E Ɵ on – staining H&E ca Ɵ fi ca fi 100 x 100 x magni 40 x magni right hip joint lower back right hip joint lower back Supplemental Figure 3 Supplemental Figure
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