Journal: Journal of molecular and cellular cardiology

Article Title: Endothelial SHP-1 regulates diabetes-induced abnormal collateral vessel formation and endothelial cell senescence.

doi: 10.1016/j.yjmcc.2025.03.005

Figure Lengend Snippet: Fig. 7. SHP-1 regulates endothelial senescence under HG level exposure. (A) Representative images of senescence-associated β-galactosidase assay and (B) quantification of the percent of positive cells (blue staining). Immunoblot representative image and quantification of (C) p21, (D) Nrf2, and (E-F) phospho-pp2aC (Y307). EC were infected with adenoviral GFP, SHP-1 dominant-negative and native form and exposed to normal glucose (NG; 5.6 mmol/L; white bars) or high glucose (HG; 25 mmol/L; black bars) under hypoxia (1 % O2) for the last 16 h of treatment as well as in (F) nondiabetic (white bars) and diabetic (black bars) mice without (ec-SHP-1+/+) or with endothelial-specific SHP-1 deletion (ec-SHP-1−/−). Results are shown as mean ± SD of N = 7 independent cell replicates or mice per group. One-way ANOVA with Tukey's post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies for immunoblotting and immunohistochemistry; Actin (HRP; Sc-1616), GAPDH (HRP; sc-20,357), p21 (Sc-397), p53 (sc6243) and p-PP2a Cα/β Y307 (sc-271,903) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); p-Akt (S473), Akt (9272), pVEGFR2 Y1175 (2478), VEGFR2 (2479), p-eNOS Ser1171 (9571), eNOS (32027), NRF2 (12721) and secondary antibody of anti-rabbit and antimouse peroxidase-conjugated from Cell Signaling (Beverly, MA); PP2a Cα (610555) from BD bioscience, SHP-1 (ab32559), α-smooth muscle actin (Ab5694) from Abcam (Cambrige, UK); CD31 (HS-351117) from HistoSure (Goettingen, Germany); and Alexa 594–conjugated anti-rat and 488-conjugated anti-rabbit from Jackson ImmunoResearch Laboratories (West Grove, PA).

Techniques: Staining, Western Blot, Infection, Dominant Negative Mutation

Journal: mBio

Article Title: LincR-PPP2R5C deficiency enhancing the fungicidal activity of neutrophils in pulmonary cryptococcosis is linked to the upregulation of IL-4

doi: 10.1128/mbio.02130-24

Figure Lengend Snippet: IL-4 activated LincR-PPP2R5C KO neutrophils to kill C. neoformans . ( A ) Fungicidal activity of WT and LincR-PPP2R5C KO neutrophils against C. neoformans stimulated with or without IL-4 (15 ng/mL) ( n = 6 per group). ( B ) Western blot (WB) for elastase in WT and LincR-PPP2R5C KO neutrophils stimulated as described in panel A for 4 h. The data are representative of three independent experiments, and the statistical analysis is shown on the right. ( C ) ELISA detection of elastase levels in WT and LincR-PPP2R5C KO neutrophils cocultured with C. neoformans for 4 h under IL-4 stimulation. ( D ) Fungicidal activity of LincR-PPP2R5C KO neutrophils against C. neoformans after treatment with alvelestat or dimethyl sulfoxide. ( E ) MFI of ROS in WT and LincR-PPP2R5C KO neutrophils stimulated as described in panel A for 4 h, as detected by flow cytometry. The statistical diagram is shown on the right. ( F ) WB was used to detect the expression levels of total PP2A, phosphorylated PP2A, and methylated PP2A in WT and LincR-PPP2R5C KO neutrophils after coculture with C. neoformans for 4 h, with or without IL-4 stimulation. The statistical graph is shown on the right. Under the stimulation conditions depicted in panel A , the elastase content ( G ) and the MFI of ROS ( H ) in LincR-PPP2R5C KO neutrophils were detected by ELISA and flow cytometry, respectively, after treatment with okadaic acid (OA, 2 nM) or ethanol. The data are shown as mean ± SEM. * P < 0.05, ** P < 0.01; unpaired Student’s t -test. ns, not significant.

Article Snippet: The blots were then blocked with 5% non-fat dried milk in Tris-buffered saline supplemented with 0.05% Tween 20 (TBST) and incubated with primary antibodies against β-actin (1:2,000, GB15003; Servicebio), total protein phosphatase 2A (PP2A) (1:5,000, ab32104; Abcam), phosphorylated PP2A (1:1,000, sc-271903; Santa Cruz Biotechnology), methylated PP2A (1:1,000, sc-81603; Santa Cruz Biotechnology), and neutrophil elastase (NE) (1:1,000, ab68672; Abcam) overnight.

Techniques: Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Methylation

Journal: Cell communication and signaling : CCS

Article Title: Concomitant targeting of FLT3 and SPHK1 exerts synergistic cytotoxicity in FLT3-ITD + acute myeloid leukemia by inhibiting β-catenin activity via the PP2A-GSK3β axis.

doi: 10.1186/s12964-024-01774-9

Figure Lengend Snippet: Fig. 9 Co-targeting SPHK1 and FLT3 stimulates β-catenin degradation via PP2A-GSK3β axis. (a–b) Western blot analysis of Molm13 and MV4-11 cells after treatment with (a) sorafenib (15 or 30 nM) or (b) quizartinib (1.2 or 2.4 nM) either alone or in combination with SKI-II (20 µM) for 24 and 48 h. (c) Western blot analysis of Molm13 and MV4-11 cells expressing shCtrl or shSPHK1 upon sorafenib (15 or 30 nM) treatment for 24 h. (d–e) Western blot analysis of Molm13 and MV4-11 cells after treatment with SKI-II (20 µM) in the absence/presence of (d) okadaic acid (OA, PP2A inhibitor; 10 nM) or (e) TWS119 (GSK3β inhibitor; 10 µM) for 24 h

Article Snippet: Antibodies against FLT3 (#3462), phospho (p)-FLT3 (Tyr589/591) (#3464), SPHK1 (#12071), SPHK2 (#32346), GSK3β (#9315), p-GSK3β (Ser9) (#9336), β-catenin (#8480), c-Myc (#5605), Survivin (#2808), CD44 (#5640), caspase-3 (#9662), and PARP (#9532) were obtained from Cell Signaling Technology (CST, Beverly, MA, USA). p-PP2A-Cα/β (Tyr307) (sc271903) was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA), and PP2A-C subunit (#05-421) was obtained from Millipore (Billerica, MA, USA).

Techniques: Western Blot, Expressing

Journal: Cell communication and signaling : CCS

Article Title: Concomitant targeting of FLT3 and SPHK1 exerts synergistic cytotoxicity in FLT3-ITD + acute myeloid leukemia by inhibiting β-catenin activity via the PP2A-GSK3β axis.

doi: 10.1186/s12964-024-01774-9

Figure Lengend Snippet: Fig. 10 Schematic model illustrating the mechanism of combined inhibition of FLT3 and SPHK1. When FLT3-ITD+ AML cells are exposed to long-term FLT3 inhibitors, the SPHK1/S1P/S1P2 signaling is upregulated, leading to the inactivation of the PP2A-GSK3β axis. Subsequently, activated β-catenin translocates to the nucleus, facilitating the transcription of its target genes, thereby promoting the maintenance of FLT3-ITD+ AML upon TKI treatment (left panel). However, concomitant targeting of SPHK1 and FLT3 signaling disrupts the inhibitory effect of SPHK1 on the PP2A-GSK3β axis and facilitates β-catenin degradation, thus significantly enhances FLT3 inhibitor-induced killing of leukemic cells (right panel)

Article Snippet: Antibodies against FLT3 (#3462), phospho (p)-FLT3 (Tyr589/591) (#3464), SPHK1 (#12071), SPHK2 (#32346), GSK3β (#9315), p-GSK3β (Ser9) (#9336), β-catenin (#8480), c-Myc (#5605), Survivin (#2808), CD44 (#5640), caspase-3 (#9662), and PARP (#9532) were obtained from Cell Signaling Technology (CST, Beverly, MA, USA). p-PP2A-Cα/β (Tyr307) (sc271903) was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA), and PP2A-C subunit (#05-421) was obtained from Millipore (Billerica, MA, USA).

Techniques: Inhibition