Journal: Virulence

Article Title: The tyrosine phosphatase SHP-2 dephosphorylated by ALV-J via its Env efficiently promotes ALV-J replication

doi: 10.1080/21505594.2021.1939952

Figure Lengend Snippet: The effect of ALV-J on SHP-2 phosphorylation in vitro and in vivo . (a) DF-1 cells infected with ALV-J GY03 at an MOI of 1 for 3 or 4d, (b) HD11 cell infected with ALV-J GY03 at an MOI of 1 for 3 or 4 d, (c) DF-1 cells infected with ALV-J different strains at an MOI of 0.1 for 6 d, (d) PBL from SPF chickens infected with ALV-J J1 at 6-week post-infection, and the control PBL were lysed and analyzed by Western blot using the indicated antibodies

Article Snippet: Then, the cells were cultured with 1% FBS DMEM with SHP-2 allosteric inhibitor SHP099 (MedChemExpress) in the indicated concentration or with DMSO solvent in the same volume.

Techniques: Phospho-proteomics, In Vitro, In Vivo, Infection, Control, Western Blot

Journal: Virulence

Article Title: The tyrosine phosphatase SHP-2 dephosphorylated by ALV-J via its Env efficiently promotes ALV-J replication

doi: 10.1080/21505594.2021.1939952

Figure Lengend Snippet: The effect of ALV-J Env on SHP-2 phosphorylation. DF-1 cells (a) and HD11 cells (b) transfected with 4 μg of different types of ALV-J Env plasmids were respectively lysed at 48 hours post-transfection (hpt) and analyzed by Western blot using the indicated antibodies

Article Snippet: Then, the cells were cultured with 1% FBS DMEM with SHP-2 allosteric inhibitor SHP099 (MedChemExpress) in the indicated concentration or with DMSO solvent in the same volume.

Techniques: Phospho-proteomics, Transfection, Western Blot

Journal: Virulence

Article Title: The tyrosine phosphatase SHP-2 dephosphorylated by ALV-J via its Env efficiently promotes ALV-J replication

doi: 10.1080/21505594.2021.1939952

Figure Lengend Snippet: The key domain within ALV-J Env in regulation of SHP-2 phosphorylation. (a) DF-1 cell transfected with 4 μg of EAV-HP-env, Gp85, Gp37 and pc3.1, respectively, (b) DF-1 cell transfected with ALV-J Gp37 and its truncations, respectively, were lysed at 48 hpt and analyzed by Western blot using the indicated antibodies. (c) ALV-J Gp37 sequence structure model diagram. The amino sequence of three types of ALV-J Env were aligned and the sequences of ALV-J Gp37 with blue, green and red line blow them were Ecto, MSD and CTD of ALV-J Gp37, respectively

Article Snippet: Then, the cells were cultured with 1% FBS DMEM with SHP-2 allosteric inhibitor SHP099 (MedChemExpress) in the indicated concentration or with DMSO solvent in the same volume.

Techniques: Phospho-proteomics, Transfection, Western Blot, Sequencing

Journal: Virulence

Article Title: The tyrosine phosphatase SHP-2 dephosphorylated by ALV-J via its Env efficiently promotes ALV-J replication

doi: 10.1080/21505594.2021.1939952

Figure Lengend Snippet: The effect of SHP-2 on ALV-J replication. (a) LMH cells respectively transfected with 4 μg of pc3.1-SHP-2 and pc3.1 were infected with ALV-J GY03 at an MOI of 0.01 at 24 hpt, and then cells were lysed at 3 dpi. (b) LMH cells and SHP-2-KO LMH cells were infected with ALV-J GY03 at an MOI of 0.01 and then cells were lysed at 1dpi or 3 dpi. (c) DF-1 cells and SHP-2-KO DF-1 cells were infected with ALV-J GY03 at an MOI of 0.01 and then cells were lysed at 3 dpi. All the cells were lysed and analyzed by Western blot using the indicated antibodies. (d) Growth curves of ALV-J J1 in DF-1 cells and SHP-2-KO DF-1 cells. ALV-J J1 was inoculated into DF-1 cells and SHP-2-KO DF-1 cells at an MOI of 0.01, respectively, and the supernatants from the infected cells were collected at the indicated time-points for virus titration using TCID 50.

Article Snippet: Then, the cells were cultured with 1% FBS DMEM with SHP-2 allosteric inhibitor SHP099 (MedChemExpress) in the indicated concentration or with DMSO solvent in the same volume.

Techniques: Transfection, Infection, Western Blot, Virus, Titration

Journal: Virulence

Article Title: The tyrosine phosphatase SHP-2 dephosphorylated by ALV-J via its Env efficiently promotes ALV-J replication

doi: 10.1080/21505594.2021.1939952

Figure Lengend Snippet: Allosteric inhibition of SHP-2 efficiently inhibited ALV-J replication. DF-1 cells were infected with ALV-J GY03 at an MOI of 0.01, 2 h post infection cell culture medium was replaced with fresh medium with SHP099 or with DMSO. (a) At 3 dpi, the cells were lysed and analyzed by Western blot using the indicated antibodies; (b) At 5 dpi, the supernatants from the infected cells were titrated in DF-1 cells by IFA for TCID 50 ; (c) At 3 dpi, the cells cultured with different concentration of SHP099 or DMSO were lysed and analyzed by Western blot using the indicated antibodies

Article Snippet: Then, the cells were cultured with 1% FBS DMEM with SHP-2 allosteric inhibitor SHP099 (MedChemExpress) in the indicated concentration or with DMSO solvent in the same volume.

Techniques: Inhibition, Infection, Cell Culture, Western Blot, Concentration Assay

Journal: Virulence

Article Title: The tyrosine phosphatase SHP-2 dephosphorylated by ALV-J via its Env efficiently promotes ALV-J replication

doi: 10.1080/21505594.2021.1939952

Figure Lengend Snippet: Knock-out of SHP-2 significantly increased the proliferation of DF-1 cells. (a) Sequence structure model diagram and alignment for chicken SHP-2 and human SHP-2. The sequence structure model diagram of chicken SHP-2 showed the important functional domains and tyrosine sites, and the SH2 domains were in blue squares, the PTP domain was in red square, and the tyrosine sites of Y542 and Y580 were indicated with arrows. The amino sequence of chicken SHP-2 and human SHP-2 were compared and aligned. The tyrosine sites of Y542 and Y580 within chicken SHP-2 and human SHP-2 were in the red and blue frames, respectively. (b) 10 4 cells of DF-1 and SHP-2-KO DF-1 cells were respectively inoculated into 96 well-plate. After cultured for 24 h and 48 h, the cells were respectively analyzed by CCK8 assay as described in the method. (c) Two hundred cells of DF-1 and SHP-2-KO DF-1 cells were respectively inoculated into 6 well-plate and further cultured for 13 d for clonal formation assay. The cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet solution

Article Snippet: Then, the cells were cultured with 1% FBS DMEM with SHP-2 allosteric inhibitor SHP099 (MedChemExpress) in the indicated concentration or with DMSO solvent in the same volume.

Techniques: Knock-Out, Sequencing, Functional Assay, Cell Culture, CCK-8 Assay, Tube Formation Assay, Staining

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Chemical structure of RMC-4550 and X-ray crystal structure of SHP2 in complex with RMC-4550 (PDB code 7RCT). Surface representation of SHP2 in complex with RMC-4550 bound in the central tunnel formed at the interface of N-SH2 (green), C-SH2 (blue) and PTP (wheat) domains. (B) Chemical structures of RMC-4550-based PROTAC candidates, R1–1C, R1–3C and R1–5C.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques:

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Inhibition of SHP2-F285S- or PTP-mediated DIFMUP dephosphorylation by R1–1C, R1–3C, R1–5C and RMC-4550. MV4;11 cells were treated with increasing doses of R1–3C (B), R1–1C or R1–5C (C) for 24 h and subjected to Western blotting using SHP2, GAPDH and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Inhibition, De-Phosphorylation Assay, Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Time course of SHP2 degradation by R1–5C (100 nM) in MV4;11 cells. Immunoblotting with SHP2 and β-actin antibodies. (B) CRBN−/− and parental MOLT4 cells were treated with increasing doses of R1–5C for 24 h and subjected to Western blotting using SHP2, CRBN and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A-D) Scatterplots displaying relative fold-change in SHP2 abundance following treatment of MV4;11 cells with 100 nM R1–5C for 4 h (A), 8 h (B), 16 h (C) or 100 nM RMC-4550 (D). SHP2/PTPN11 is highlighted in red. Hits highlighted in blue in (C) and (D) indicate changes in abundance of proteins at 16 h time point due to secondary effects (such as transcriptional responses) of SHP2 degradation or inhibition. (E) Heatmap of the protein abundance changes in MV4;11 cells comparing treatment with 100 nM R1–1C (4 h and 16 h), 100 nM R1–3C (4 h and 16 h), 100 nM R1–5C (2 h, 4 h, 8 h and 16 h), 100 nM RMC-4550 (16 h) and 1 μM pomalidomide (5 h). The heatmap colors are scaled with red indicating a decrease in protein abundance (−2 log2 FC) and blue indicating an increase (2 log2 FC) in protein abundance.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Inhibition

Journal: Acta physiologica (Oxford, England)

Article Title: Short-term fasting of mice elevates circulating fibroblast growth factor 23 (FGF23).

doi: 10.1111/apha.14049

Figure Lengend Snippet: FIGURE 5 Altered NaPiIIa (Slc34a1), 24-hydroxylase (Cyp24a1) expression and αKlotho formation in fasted mice. Arithmetic means ± SEM (n = 5) of relative Slc34a1 (A), Cyp24a1 (C), 1α-hydroxylase (Cyp27b1) (D), and αKlotho (Kl) (E) mRNA abundance normalized to hypoxanthine guanine phosphoribosyl transferase (Hprt) expression in kidneys from fed mice and mice fasted for 16 h. Representative Western Blots and arithmetic means ± SEM of renal NaPiIIa (⁓69 kDa, isoform X1: ⁓59 kDa, isoform X2: ⁓48 kDa; B: n = 7) and Klotho (⁓130 to 135 kDa; F: n = 5) protein abundance relative to loading control β-tubulin (⁓55 kDa) in fed and fasted mice. For all panels, n denotes the number of specimens included. There were no dropouts in any analysis. *p < 0.05, †p < 0.01, ‡p < 0.001 indicate significant differences compared to fed mice. ns, non-significant. (A: Mann–Whitney U test, B–F: unpaired t-test).

Article Snippet: Proteins from UMR106 cells, NRVM, kidney, heart, thymus, bone marrow, pancreas (each 30 μg), or bone (10 μg) were subjected to standard Western Blot procedure using 10% or 12% SDS- PAGE gels or 4%– 20% precast SDS- PAGE gels (for FGF23 detection in NRVM), respectively, and the following primary antibodies: anti- NFκB- p65 (#8242, Cell Signaling Technology), anti- phospho- NFκB- p65 (#3033, Cell Signaling Technology), anti- FGF23 (MAB26291, R&D Systems), anti- FGF23 (#6320, Immutopics), anti- NaPiIIa (NBP2- 13328, Novus Biologicals, Bio- Techne), anti- Klotho (AF1819, R&D Systems), anti- β- actin ((#3700 (for lysates from UMR106 cells) and #8457 (for mouse tissue lysates), Cell Signaling Technology)), anti- GAPDH (#2118, Cell Signaling Technology), or anti- β- tubulin (#2146, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Quantitative Proteomics, Control, MANN-WHITNEY

Journal: Oncotarget

Article Title: An increase in galectin-3 causes cellular unresponsiveness to IFN-γ-induced signal transduction and growth inhibition in gastric cancer cells

doi: 10.18632/oncotarget.7750

Figure Lengend Snippet: A PIP3 MASS ELISA Kit assay and Western blot analysis showing PI3K activity A. and expressions of the indicated proteins B. in AGS cells (IFN-γ insensitive) transfected with shRNA targeting luciferase ( shLuc ) and shRNA targeting galectin-3 ( shGal3 ; clones 1-4) and MKN45 cells (IFN-γ sensitive) transfected with pcDNA3- and pcDNA3-Gal3. For kinase activity, the data are presented as the mean ± SD from three independent experiments. ns, not significant. C. Representative Western blot of the indicated proteins in pcDNA3-, pcDNA3-AKT-, GFP-, GFP-tagged GSK-3β R96A -, pBABE-, pBABE-SHP2 wild-type-, and pBABE-SHP2 D61A -tranfected MKN45 cells (IFN-γ sensitive). For Western blotting, β-actin was used as an internal control. pAKT T, phospho-AKT at Thr308; pAKT S, phospho-AKT at Ser473. A representative dataset from triplicate experiments is shown. D. In cells transfected with or without treatment with IFN-γ or the SHP2 inhibitor, NSC87877, for 6 h, the luciferase reporter assay showed the ratio of IRF1 to control Renilla. The data are presented as the mean ± SD from three independent experiments. ** p < 0.01 and *** p < 0.001 compared to the untreated sample; ## p < 0.01 and ### p < 0.001 compared to the relative control. †† p < 0.01 and ††† p < 0.001 compared to IFN-γ.

Article Snippet: The plasmid expressing GFP-PTEN (ID NM_000314; Plasmid 13039) and its control pcDNA3-GFP (Plasmid 13031); pcDNA3 flag HA AKT1 (Plasmid 9021) and its control pcDNA3 flag HA (Plasmid 1436); and pBABE-puro SHP2 (Plasmid 8329) and pBABE-puro SHP2 D61A (Plasmid 8330) and their control vector pBABE-puro (Plasmid 1764) were purchased from Addgene (Cambridge, MA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay, Transfection, shRNA, Luciferase, Clone Assay, Control, Reporter Assay

Journal: Oncotarget

Article Title: An increase in galectin-3 causes cellular unresponsiveness to IFN-γ-induced signal transduction and growth inhibition in gastric cancer cells

doi: 10.18632/oncotarget.7750

Figure Lengend Snippet: A proliferation assay A. and propidium iodide (PI) staining-based flow cytometric analysis B. showing cell growth and apoptosis, respectively, in IFN-γ-treated AGS cells transfected with shRNA targeting luciferase ( shLuc ) and shRNA targeting galectin-3 ( shGal3 ). The data (mean ± SD from three independent experiments) are shown as multiples of change compared to the normalized value of the control sample. ** p < 0.01 and *** p < 0.001. ns, not significant. C. A hypothetical model for aberrant galectin-3 involved in facilitating AKT/GSK-3β/SHP2 signaling to inhibit IFN-γ STAT1/IRF1 signaling and IFN-γ-induced cell growth inhibition and apoptosis.

Article Snippet: The plasmid expressing GFP-PTEN (ID NM_000314; Plasmid 13039) and its control pcDNA3-GFP (Plasmid 13031); pcDNA3 flag HA AKT1 (Plasmid 9021) and its control pcDNA3 flag HA (Plasmid 1436); and pBABE-puro SHP2 (Plasmid 8329) and pBABE-puro SHP2 D61A (Plasmid 8330) and their control vector pBABE-puro (Plasmid 1764) were purchased from Addgene (Cambridge, MA).

Techniques: Proliferation Assay, Staining, Transfection, shRNA, Luciferase, Control, Inhibition

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: ( A ) Recombinant human Shp2 activity after 30-min incubation with SFX-01. Data represent mean activity ( ± SEM; n = 3 biological replicates) and were fitted to a one-phase exponential decay curve (gray line; r 2 = 0.987). ( B ) Shp2 activity after incubation with SFX-01 for the indicated times and concentrations. Data shown are mean activity ( ± SEM; n = 3 biological replicates). ( C ) Shp2 activity after 30-minute incubation with or without bisphosphorylated IRS1 and SFX-01. Bar represents mean activity (± SEM; n = 4 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. ( D ) Representative immunoblots showing SFN-modification of recombinant Shp2 (1.6 nM) following incubation with 1.75 or 0.109 µM SFX-01 for 30 min. ( E ) Precursor isotopic envelop spectrum of 0.1 µM recombinant human Shp2 protein incubated with equimolar SFN for 6 h at 37 °C corresponding to a dithiolethione modification adducted between Cys 333 and Cys 367 . ( F ) Schematic representing the proposed mechanism of Shp2-dithiolethione formation by SFN. The isothiocyanate group reacts with a cysteine residue to form a dithiocarbamate intermediate, which further reacts with a second cysteine residue to yield the dithiolethione modification. ( G ) Immunoblot of immunoprecipitated WT or active site mutant Shp2 and SFN-modification from HEK cells treated with SFX-01. “E” represents non-transfected cells, and 0 h represents untreated cells. The graph represents densitometric analysis of Shp2-SFN adduct formation in WT or mutant Shp2 exposed to SFX-01 for 2 or 4 h. Bars represent mean values (± SEM; n = 3 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. .

Article Snippet: C459S Shp2 , Addgene , #8382.

Techniques: Recombinant, Activity Assay, Incubation, Western Blot, Modification, Residue, Immunoprecipitation, Mutagenesis, Transfection

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: Shp2 phosphatase activity in ( A ) cardiac tissue ( n = 8 mice) and ( B ) spleen of WT ( n = 4 mice) or Ptpn11 D61G (−/+) mice ( n = 5 mice) that received SFX-01 in the drinking water for 10 days. ( A , B ) Bars represent mean activity ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. ( C ) Representative immunoblots showing the input and immunoprecipitated Shp2 protein and SFN-labeled Shp2 from cardiac tissue isolated from mice with or without oral SFX-01 administration as in ( A ). ( D ) Representative immunoblot of Shp2 immunoprecipitated from cardiac tissue of WT mice following treatment with SFX-01 for 4 days after incubation with biotin-iodoacetamide. ( E ) Representative far-western immunoblot of Shp2 immunoprecipitated from cardiac tissue of WT mice following treatment with SFX-01 for 4 days. The immunoblot was then exposed to biotin-phenylarsenic acid (PAA). .

Article Snippet: C459S Shp2 , Addgene , #8382.

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Labeling, Isolation, Incubation

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: ( A ) Changes in leukocyte cell populations in blood isolated from individual WT or Ptpn11 D61G (−/+) mice before and after 10 weeks of SFX-01 in drinking water. P values were calculated by paired t tests. For WT mice: n = 14 before and after vehicle treatment, n = 14 before SFX-01 treatment, and n = 15 after SFX-01 treatment. For Ptpn11 D61G (−/+) mice: n = 14 before vehicle treatment, n = 16 after vehicle treatment, n = 11 before SFX-01 treatment, and n = 14 after SFX-01 treatment. ( B ) Blood leukocyte cell population after 10 weeks SFX-01 treatment. ( left ) Bars represent mean leukocyte population ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 14 vehicle treatment, and n = 15 SFX-01 treatment. For Ptpn11 D61G (−/+) mice: n = 16 vehicle treatment, and n = 14 SFX-01 treatment. ( right ) Representative microphotographs of Wright-Giemsa-stained blood isolated from mice after 10 weeks SFX-01 treatment or controls. Scale bars show 200 µm. ( C ) Violin plots of blood myeloid-derived (CD11b + /Ly6G + and CD11b + /Ly6C + ) cell populations from WT or Ptpn11 D61G (−/+) mice after 10 weeks of SFX-01 in drinking water. Horizontal lines represent quartiles and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 13 vehicle or SFX-01 treatment. For Ptpn11 D61G (−/+) mice: n = 9 vehicle treatment, and n = 13 SFX-01 treatment. ( D ) Changes in spleen size from individual WT or Ptpn11 D61G (−/+) mice before and after 10 weeks of SFX-01 in drinking water. P values were calculated by paired t tests. For WT mice: n = 14 before and after vehicle treatment ( P = 0.0102), n = 14 before and after SFX-01 treatment ( P = 0.0890). For Ptpn11 D61G (−/+) mice: n = 13 before and after vehicle treatment ( P = 0.0253), n = 15 before and after SFX-01 treatment ( P = 0.1694) for each group. ( E ) Mean changes in spleen size in WT or Ptpn11 D61G (−/+) mice over 10 weeks of SFX-01 treatment compared to before treatment. Bars represent mean spleen size ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 14 vehicle or SFX-01 treatment. For Ptpn11 D61G (−/+) mice: n = 13 vehicle treatment, and n = 15 SFX-01 treatment. ( F ) Violin plots of spleen myeloid-derived (CD11b + /Ly6G + and CD11b + /Ly6C + ) cell populations from WT or Ptpn11 D61G (−/+) mice after 10 weeks of SFX-01 in drinking water. Horizontal lines represent quartiles and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 14 vehicle treatment, and n = 15 SFX-01 treatment ( P = 0.6138). For Ptpn11 D61G (−/+) mice: n = 16 vehicle treatment, and n = 12 SFX-01 treatment ( P = 0.0251). .

Article Snippet: C459S Shp2 , Addgene , #8382.

Techniques: Isolation, Staining, Derivative Assay

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: ( A ) Representative immunoblot of relative STAT1 phosphorylation and cyclin D1 expression in leukocytes isolated from WT or Ptpn11 D61G (−/+) mice after 10-week SFX-01 treatment. Graphs represent densitometric analyses of relative STAT1 (Ser 727) phosphorylation and cyclin D1 expression. Bars represent mean expression level ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. n = 5 mice for STAT1 (Ser 727) phosphorylation and n = 3 mice biological replicates for cyclin D1 expression. ( B ) Representative immunoblots of relative STAT1 phosphorylation and cyclin D1 expression in a Shp2-activating mutant expressing human GDM-1 cell line treated with SFX-01 or vehicle. Graph bars represent mean expression levels ( ± SEM; n = 4 biological replicates) and P values calculated by unpaired t test. ( C ) Cell cycle analysis of GDM-1 cells cultured with SFX-01. Bars represent mean percentage ( ± SEM), and significance compared to controls was calculated by one-way ANOVA with Dunnett’s test. n = 4 for vehicle or 250 µM SFX-01 treatment, n = 3 for 1, 5, 25, or 100 µM SFX-01 treatment, biological replicates. ( D ) Colony-forming ability of GDM-1 cells treated with SFX-01. Bars represent mean normalized values (± SEM), and because of the low sample size and not assuming a normal Gaussian distribution, significance was calculated using a Kruskal–Wallis test with an uncorrected Dunn’s test for comparison to the control group. n = 2 for vehicle treatment, n = 3 for 0.1 µM SFX-01 treatment ( P = 0.3957), n = 3 for 1 µM SFX-01 treatment ( P = 0.2167), n = 5 for 10 µM SFX-01 ( P = 0.0245) and n = 3 for 100 µM SFX-01 ( P = 0.0018) treatment, biological replicates. ( E ) Cell cycle analysis and ( F ) colony-forming ability of control (CB) or JMML patient-derived HSCs cultured with or without SFX-01. Clonogenicity of HSC was normalized to control cells, and bars represent mean ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. For ( E ): CB: n = 3 for vehicle, 5, or 25 µM SFX-01 treatment, and n = 2 for 100 µM SFX-01 treatment. JMML: n = 7 for vehicle or 5 µM SFX-01 treatment, and n = 8 for 25 or 100 µM SFX-01 treatment, biological replicates. For ( F ): CB: n = 14 for vehicle treatment, n = 4 for 10 µM SFX-01 treatment, n = 17 for 50 µM SFX-01 treatment, and n = 19 for 100 µM SFX-01 treatment. JMML: n = 6 for vehicle or 10 µM SFX-01 treatment, n = 11 for 50 µM SFX-01 treatment, and n = 12 for 100 µM SFX-01 treatment, biological replicates. ( G ) A heatmap displaying the significantly altered Shp2 substrates in CD11b+ myeloid bone marrow cells isolated from WT or Ptpn11 D61G (−/+) mice, under conditions with or without SFX-01 treatment based on data from the phosphoproteomics analysis ( n = 3 biological replicates). .

Article Snippet: C459S Shp2 , Addgene , #8382.

Techniques: Western Blot, Phospho-proteomics, Expressing, Isolation, Mutagenesis, Cell Cycle Assay, Cell Culture, Comparison, Control, Derivative Assay

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: CD11b+ bone marrow cells isolated from WT and Ptpn11 D61G (−/+) mice treated with SFX-01, SHP099 or both together. SFX-01 or SHP099 attenuated cell proliferation and this was not accentuated when these interventions were combined. Data are presented as means ( ± SEM; n = 5–6) with P values calculated by two-way ANOVA with Tukey’s multiple comparison test. * P < 0.00001 versus untreated WT or # P < 0.00001 versus untreated Ptpn11 D61G (−/+) .

Article Snippet: C459S Shp2 , Addgene , #8382.

Techniques: Isolation, Comparison