Journal: Frontiers in immunology

Article Title: SIRPα Suppresses Response to Therapeutic Antibodies by Nurse Like Cells From Chronic Lymphocytic Leukemia Patients.

doi: 10.3389/fimmu.2020.610523

Figure Lengend Snippet: FIGURE 5 | SHP1 but not SHP2 is downstream of SIRPa. (A) Total and phosphorylated SHP1 and SHP2 expression were examined in phenotypically sensitive and resistant NLCs by western blotting. Representative blots and quantified data are shown. Each point represents a single patient. (B) NLCs were transfected with SIRPa siRNA C or scrambled control siRNA. SIRPa protein expression, as well as (C) total and phosphorylated SHP1 (pSHP1) or SHP2 (pSHP2) expression was estimated in transfected NLCs. Representative blots are shown and quantified data shown graphically. Data presented as mean value ± SEM and normalized to scrambled siRNA control. Each point represents an individual patient. Statistical differences were calculated by a Mann Whitney U Test. ***p < 001 and ****p < 0.0001. ns, not significant.

Article Snippet: Where protein expression has been quantitated, results represent relative protein levels normalized to corresponding total protein levels or b-actin or GAPDH using Image J software. siRNA Knockdown Predesigned stealth small interfering RNAs (siRNAs) were used to silence protein expression of SIRPa (27mer Human siRNA duplexes; SR315411; OriGene) and Shp1 (27mer Human siRNA duplexes; SR321517; OriGene).

Techniques: Expressing, Western Blot, Transfection, Control, MANN-WHITNEY

Journal: Frontiers in immunology

Article Title: SIRPα Suppresses Response to Therapeutic Antibodies by Nurse Like Cells From Chronic Lymphocytic Leukemia Patients.

doi: 10.3389/fimmu.2020.610523

Figure Lengend Snippet: FIGURE 7 | SHP1-dependent ADP repression is SYK-dependent. NLCs were transfected with 50 nM scrambled siRNA control or three different SHP1-siRNAs (siRNA A–C). (A) Protein was harvested after 48 h and SHP1 and GAPDH expression visualized and quantitated by western blot to examine knockdown efficiency. (B) NLCs were co- cultured with autologous CLL cells for 48 h after SHP1 siRNA C or scrambled siRNA transfection. Cultures were then treated with or without obinutuzumab (10 µg/ml) for 2 h before ADP responses examined. Data presented as mean value ± SEM from five CLL patients. (C) Western blot data analysis of SYK (pSYKY525/526) and AKT phosphorylation (pAKTS473) in NLCs following SHP1 siRNA transfection. Representative blots and quantified data are shown. Data presented as mean value +/−SEM. Each point represents an individual patient. Statistical analysis used a Mann Whitney U Test. *p < 0.05, ***p, 0.001, and ****p < 0.0001.

Article Snippet: Where protein expression has been quantitated, results represent relative protein levels normalized to corresponding total protein levels or b-actin or GAPDH using Image J software. siRNA Knockdown Predesigned stealth small interfering RNAs (siRNAs) were used to silence protein expression of SIRPa (27mer Human siRNA duplexes; SR315411; OriGene) and Shp1 (27mer Human siRNA duplexes; SR321517; OriGene).

Techniques: Transfection, Control, Expressing, Western Blot, Knockdown, Cell Culture, Phospho-proteomics, MANN-WHITNEY

Journal: Frontiers in immunology

Article Title: SIRPα Suppresses Response to Therapeutic Antibodies by Nurse Like Cells From Chronic Lymphocytic Leukemia Patients.

doi: 10.3389/fimmu.2020.610523

Figure Lengend Snippet: FIGURE 8 | Overview of FcgR signaling pathway in antibody resistant NLC. Therapeutic antibody binding to cell-surface receptor, FcgR independently activates SYK/BTK and p110d/AKT pathways. Activation of SIRPa by tumor cell CD47 (not shown) activates SHP1 phosphatase that suppresses SYK activity.

Article Snippet: Where protein expression has been quantitated, results represent relative protein levels normalized to corresponding total protein levels or b-actin or GAPDH using Image J software. siRNA Knockdown Predesigned stealth small interfering RNAs (siRNAs) were used to silence protein expression of SIRPa (27mer Human siRNA duplexes; SR315411; OriGene) and Shp1 (27mer Human siRNA duplexes; SR321517; OriGene).

Techniques: Binding Assay, Cell Surface Receptor Assay, Activation Assay, Activity Assay

Journal: Cancers

Article Title: Evidence for a Tumor-Suppressive Role of SHP-1 in EMT Regulation in Bladder Cancer Cells

doi: 10.3390/cancers18091401

Figure Lengend Snippet: ( A ) Relative SHP-1 protein expression in the 19 BCa cell lines examined in this study. ( B ) Expression levels were significantly higher in epithelial-like cells (n = 11) than in intermediate and mesenchymal-like lines (n = 8). Error bars represent the standard error of the mean (SEM).

Article Snippet: Lentiviral shRNA constructs targeting SHP-1 were obtained from Origene (Rockville, MD, USA), and the manufacturer’s protocol was used to establish stable cell lines for each construct.

Techniques: Expressing

Journal: Cancers

Article Title: Evidence for a Tumor-Suppressive Role of SHP-1 in EMT Regulation in Bladder Cancer Cells

doi: 10.3390/cancers18091401

Figure Lengend Snippet: ( A ) SHP-1 protein expression in bladder tissue was significantly lower in muscle-invasive (MI, n = 8) tumor than in non-muscle-invasive (NMI, n = 8) and urothelial tissue with no residual disease (NRD, n = 10). ( B ) Examples of DAB and corresponding H&E staining in the bladder tissues examined in this study. The error bars represent the interquartile range.

Article Snippet: Lentiviral shRNA constructs targeting SHP-1 were obtained from Origene (Rockville, MD, USA), and the manufacturer’s protocol was used to establish stable cell lines for each construct.

Techniques: Expressing, Staining

Journal: Cancers

Article Title: Evidence for a Tumor-Suppressive Role of SHP-1 in EMT Regulation in Bladder Cancer Cells

doi: 10.3390/cancers18091401

Figure Lengend Snippet: Correlation plots (n = 19) of SHP-1 protein expression with the EMT markers E-cadherin, N-cadherin, and Vimentin. Gray areas represent the 95%CI. Representative Western blot images: from the BCa cell lines EJ and HT-1376, the first lane of each pair corresponds to the protein indicated above each lane, and the second corresponds to the total protein detected for the same lane.

Article Snippet: Lentiviral shRNA constructs targeting SHP-1 were obtained from Origene (Rockville, MD, USA), and the manufacturer’s protocol was used to establish stable cell lines for each construct.

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: Evidence for a Tumor-Suppressive Role of SHP-1 in EMT Regulation in Bladder Cancer Cells

doi: 10.3390/cancers18091401

Figure Lengend Snippet: ( A ) SHP-1 protein expression following transduction with SHP-1 shRNA (shSHP-1) or SHP-1 cDNA (+SHP-1) and corresponding negative controls (NC): CUBIII n = 6, RT-112 n = 7, TCCSUP n = 4, UM-UC-3 n = 6. ( B ) Representative Western blot images: the first lane of each pair corresponds to SHP-1, and the second corresponds to the total protein detected for the same lane. Error bars represent the SEM. ( C ) Relative proliferation rates of BCa cells transduced with SHP-1 shRNA (shSHP-1) or SHP-1 cDNA (+SHP-1) and corresponding negative controls (NC): CUBIII n = 5, RT-112 n = 5, TCCSUP n = 9, UM-UC-3 n = 5. Error bars represent the SEM.

Article Snippet: Lentiviral shRNA constructs targeting SHP-1 were obtained from Origene (Rockville, MD, USA), and the manufacturer’s protocol was used to establish stable cell lines for each construct.

Techniques: Expressing, Transduction, shRNA, Western Blot

Journal: Cancers

Article Title: Evidence for a Tumor-Suppressive Role of SHP-1 in EMT Regulation in Bladder Cancer Cells

doi: 10.3390/cancers18091401

Figure Lengend Snippet: ( A ) Relative migration rates of BCa cells transduced with SHP-1 shRNA (shSHP-1) or SHP-1 cDNA (+SHP-1) and corresponding negative controls (NC): CUBIII n = 5, RT-112 n = 4, TCCSUP n = 3, UM-UC-3 n = 3. ( B ) Representative images from in vitro migration assays (scale bar = 200 μm). ( C ) Relative invasion rates of BCa cells transduced with SHP-1 shRNA (shSHP-1) or SHP-1 cDNA (+SHP-1) and corresponding negative controls (NC): CUBIII n = 3, RT-112 n = 5, TCCSUP n = 3, UM-UC-3 n = 3. ( D ) Representative images from in vitro invasion assays (scale bar = 200 μm). Error bars represent the SEM.

Article Snippet: Lentiviral shRNA constructs targeting SHP-1 were obtained from Origene (Rockville, MD, USA), and the manufacturer’s protocol was used to establish stable cell lines for each construct.

Techniques: Migration, Transduction, shRNA, In Vitro

Journal: Cancers

Article Title: Evidence for a Tumor-Suppressive Role of SHP-1 in EMT Regulation in Bladder Cancer Cells

doi: 10.3390/cancers18091401

Figure Lengend Snippet: Significant gene sets resulting from GSEA of RNA sequence data comparing the transduced BCa lines with high-SHP-1-expressing lines versus low-SHP-1-expressing lines (q-value < 0.1).

Article Snippet: Lentiviral shRNA constructs targeting SHP-1 were obtained from Origene (Rockville, MD, USA), and the manufacturer’s protocol was used to establish stable cell lines for each construct.

Techniques: Sequencing, Expressing

Journal: Cancers

Article Title: Evidence for a Tumor-Suppressive Role of SHP-1 in EMT Regulation in Bladder Cancer Cells

doi: 10.3390/cancers18091401

Figure Lengend Snippet: Relative expression of pAkt/Akt in BCa cells transduced with SHP-1 shRNA (shSHP-1) or SHP-1 cDNA (+SHP-1) and corresponding negative controls (NC): RT-112 n = 7 and TCCSUP n = 8. Representative Western blot images: the first lane of each pair corresponds to Akt or pAkt (ser473), and the second corresponds to the total protein detected for the same lane. Error bars represent the SEM.

Article Snippet: Lentiviral shRNA constructs targeting SHP-1 were obtained from Origene (Rockville, MD, USA), and the manufacturer’s protocol was used to establish stable cell lines for each construct.

Techniques: Expressing, Transduction, shRNA, Western Blot

Journal: Cells

Article Title: Regulation of Microtubule Nucleation in Mouse Bone Marrow-Derived Mast Cells by Protein Tyrosine Phosphatase SHP-1

doi: 10.3390/cells8040345

Figure Lengend Snippet: The SHP-1 and SHP-2 in complexes containing γ-tubulin. ( I ) The expression profile of SHP-1 and SHP-2 in selected mouse tissues and BMMCs: A gel-based RT-PCR analysis of mouse SHP-1 ( Ptpn6 ) and SHP-2 ( Ptpn11 ) is shown. Mouse spleen and heart served as positive controls for SHP-1 and SHP-2, respectively. ( II ) The extracts from BMMCs precipitated with immobilized Abs specific to SHP-1 ( A , D , G ), SHP-2 ( B , E , H ), or γ-tubulin sequence 434–449 ( C , F , I ): The blots were probed with Abs to γ-tubulin (γ-Tb), SHP-1, and SHP-2. The load ( lane 1 ), the immobilized Abs not incubated with cell extracts ( lane 2 ), the precipitated proteins ( lane 3 ), and the carriers without Abs and incubated with cell extracts ( lane 4 ).

Article Snippet: To prepare C-terminally enhanced green fluorescent protein (EGFP)-tagged mouse SHP-1 (gene Ptpn6 ; RefSeq ID: NM_013545.3), the coding sequence without a stop codon was amplified from the C-terminally Myc-DDK-tagged Ptpn6 (tv1) (OriGene Technologies, Rockville, MD, USA; MR209258) by PCR using the forward 5′-GCTC GAATTC ATGGTGAGGTGGTTTC-3′ and reverse 5′-AGC GTCGAC CTTCCTCTTGAGAGAACCT-3′ primers.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Incubation

Journal: Cells

Article Title: Regulation of Microtubule Nucleation in Mouse Bone Marrow-Derived Mast Cells by Protein Tyrosine Phosphatase SHP-1

doi: 10.3390/cells8040345

Figure Lengend Snippet: The generation of SHP-1 deficient cell lines. ( A ) A schematic diagram of Ptpn6 with sites targeted by guide RNA (sgRNA) sequences: The targeted sites (blue) and protospacer-adjacent motifs (PAM; red) on the gene (18.01 kb) containing 16 exons are shown. The defined domains are indicated. ( B ) The PCR amplification of genomic DNA from the control cells (BMMC) and SHP-1-deficient cell lines (SHP-1_KO1, SHP-1_KO2, SHP-1_KO3) with primers flanking the deleted region: The template is not present in the control sample. Due to the large size of the deleted region (approx. 6 kb), no amplification was found in the control BMMCs. The amplification of short fragments (approx. 560 bp) was detected in SHP-1-deficient clones. ( C ) The SHP-1 protein levels in BMMCs and SHP-1-deficient cell lines analyzed by an immunoblotting of whole-cell lysates: GCP2 served as the loading control.

Article Snippet: To prepare C-terminally enhanced green fluorescent protein (EGFP)-tagged mouse SHP-1 (gene Ptpn6 ; RefSeq ID: NM_013545.3), the coding sequence without a stop codon was amplified from the C-terminally Myc-DDK-tagged Ptpn6 (tv1) (OriGene Technologies, Rockville, MD, USA; MR209258) by PCR using the forward 5′-GCTC GAATTC ATGGTGAGGTGGTTTC-3′ and reverse 5′-AGC GTCGAC CTTCCTCTTGAGAGAACCT-3′ primers.

Techniques: Amplification, Clone Assay, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: JAK2V617F ‐dependent down regulation of SHP ‐1 expression participates in the selection of myeloproliferative neoplasm cells in the presence of TGF ‐β

doi: 10.1111/jcmm.70138

Figure Lengend Snippet: Expression of SHP‐1 is reduced in JAK2 V617F mutated cells. (A) Volcano plot obtained by comparing RNA‐seq data from UT‐7 cells (4 replicates) with or without treatment with TGF‐β. Left panel: JAK2 WT cells; middle panel: JAK2 V617F cells; right panel: Volcano plot comparing WT and JAK2 V617F cells under TGF‐β. (B) RT‐qPCR analysis (three independent experiments) of the expression of PTPN6/SHP‐1 in UT‐7 cells expressing WT or mutated JAK2. (C) Western blot analysis of SHP‐1 in UT‐7 cells expressing WT or mutated JAK2 (one representative experiment out of 2). (D) RT‐qPCR analysis (three independent experiments) of the expression of PTPN6/SHP‐1 in total bone marrow cells from JAK2 V617F knock‐in mice or syngenic WT mice. (E) Western blot analysis of SHP‐1 in total bone marrow cells from JAK2 V617F knock‐in mice or syngenic WT mice. (one representative experiment out of 2). (F) RT‐qPCR analysis (two independent experiments) of the expression of PTPN6/SHP‐1 in CD34 positive cells isolated from MPN patients or healthy controls, normalized to the expression of the housekeeping gene TBP. (G) PTPN6/SHP‐1 expression in RNA‐seq data obtained from purified sub‐populations of HSPCs from JAK2 V617F knock‐in mice or syngenic WT mice. ¹⁷

Article Snippet: The SHP‐1 cDNA plasmid ‘SHP1 (PTPN6) Human Tagged ORF Clone’ for overexpression of SHP‐1 was purchased from Origene (clone no RC213896L4).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Knock-In, Isolation, Purification