Journal: Cells

Article Title: Constitutive Cell Proliferation Regulating Inhibitor of Protein Phosphatase 2A (CIP2A) Mediates Drug Resistance to Erlotinib in an EGFR Activating Mutated NSCLC Cell Line

doi: 10.3390/cells10040716

Figure Lengend Snippet: Bortezomib downmodulates cell proliferation regulating inhibitor of phosphatase 2A (CIP2A), restores regulation of Akt, and arrests cells in the G 2 /M phase of the cell cycle. Cells were incubated with 1 µM erlotinib, 15 nM bortezomib ( A , B ) or 150 nM bortezomib ( A ) for 24 h. Proteins were separated on a 10% polyacrylamide gel and blotted on a nitrocellulose membrane, and specific target proteins were detected using target specific primary and species-specific secondary antibodies, coupled with horseradish peroxidase, as listed in the methods section. Signals were developed using a chemoluminescence substrate and detected with the ChemoCam System (Intas, Göttingen, Germany). A representative blot of at least three biological replicates is shown. ( C ) Distribution of the cell cycle phases was analyzed by propidium iodide staining of fixed cells and detection of the fluorescence intensities using a MACSQuant flow cytometer after incubating the cells with 1 µM erlotinib or 15 nM bortezomib for 24 h. ( D ) The relative amount of cells in each phase of the cell cycle is displayed as mean ± SD, n = 6, * p < 0.05, *** p < 0.001.

Article Snippet: Antibodies for CIP2A (2G10-3B5, mouse monoclonal IgG2b), p-PP2A (p-PP2A-Cα/β Rabbit pAb (Tyr 307)), and dem-PP2A (demethylated PP2A-C (4B7), Mouse mAb) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). p27 (Anti-p27 KIP 1 Rabbit mAb [Y236]), phospho-p27 (Recombinant Anti-p27 KIP 1 (phospho S10) antibody (EP233(2)Y)), and p21 (Anti-p21 Rabbit mAb (EPR3993)) antibodies were obtained from abcam (Cambridge, MA, USA).

Techniques: Incubation, Membrane, Staining, Fluorescence, Flow Cytometry

Journal: The Journal of pharmacy and pharmacology

Article Title: Magnesium isoglycyrrhizinate prevents cadmium-induced activation of JNK and apoptotic hepatocyte death by reversing ROS-inactivated PP2A.

doi: 10.1093/jpp/rgab125

Figure Lengend Snippet: Figure 4 MgIG suppressed JNK activation and apoptosis partially by attenuating Cd-inactivated PP2A in hepatocyte cultures. (a, b, e) Western blotting of the total cell lysates from hepatic cell cultures with indicated treatments. β-Tubulin served as a loading control. (c, f) Apoptotic cells were evaluated by using DAPI staining. (d, g) Trypan blue exclusion was performed to evaluate the percentage of live cells. The results are presented as the means ± SE, n = 3–5. aP < 0.05, difference versus the Ctrl group; bP < 0.05, difference versus the 50 μM Cd group; cP < 0.05, difference versus the Cd/OA group or Cd/MgIG group; dP < 0.05, Ad-PP2A-wt group versus the Ad-GFP group.

Article Snippet: Sources of antibodies used in this study were as follows: poly ADP-ribose polymerase (PARP) (CST); phospho-JNK (p-JNK) (Thr183/Tyr185), JNK, phospho-c-Jun (p-c-Jun) (Ser63), c-Jun and demethylated PP2A (de-PP2A) (Santa Cruz Biotechnology); phospho-PP2A (p-PP2A) (Epitomics); PP2ACɑ (BD Biosciences); PP2A-A and PP2A-B (Millipore); FLAG, β-tubulin (Sigma); and goat anti-rabbit IgG-horseradish peroxidase (HRP) and goat anti-mouse IgG-HRP (ThermoFisher).

Techniques: Activation Assay, Western Blot, Control, Staining

Journal: The Journal of pharmacy and pharmacology

Article Title: Magnesium isoglycyrrhizinate prevents cadmium-induced activation of JNK and apoptotic hepatocyte death by reversing ROS-inactivated PP2A.

doi: 10.1093/jpp/rgab125

Figure Lengend Snippet: Figure 5 MgIG blocks Cd-induced ROS generation involved in the activation of the PP2A/JNK cascade and apoptotic hepatocyte cell death. (a, c) Cell ROS im- aging (green) and ROS levels were detected by using the oxidant-sensitive probe CM-H2DCFDA. Scale bar: 20 μm. (b, d, k) Quantitation of ROS fluorescence intensity from images. (e, h) Western blotting of the total cell lysates from hepatic cell cultures with indicated treatments. β-Tubulin served as a loading control. (f, i) The percentages of apoptotic cells were quantified by DAPI staining. (g, j) Trypan blue exclusion was performed to evaluate the percentage of live cells. The results are presented as the means ± SE, n = 3–5. aP < 0.05, difference versus the Ctrl group; bP < 0.05, difference versus the 50-μM Cd group; cP < 0.05, difference versus the Cd/NAC group or Cd/MgIG group; dP < 0.05, the Ad-PP2A-wt group versus the Ad-LacZ group.

Article Snippet: Sources of antibodies used in this study were as follows: poly ADP-ribose polymerase (PARP) (CST); phospho-JNK (p-JNK) (Thr183/Tyr185), JNK, phospho-c-Jun (p-c-Jun) (Ser63), c-Jun and demethylated PP2A (de-PP2A) (Santa Cruz Biotechnology); phospho-PP2A (p-PP2A) (Epitomics); PP2ACɑ (BD Biosciences); PP2A-A and PP2A-B (Millipore); FLAG, β-tubulin (Sigma); and goat anti-rabbit IgG-horseradish peroxidase (HRP) and goat anti-mouse IgG-HRP (ThermoFisher).

Techniques: Activation Assay, Quantitation Assay, Fluorescence, Western Blot, Control, Staining

Journal: The Journal of pharmacy and pharmacology

Article Title: Magnesium isoglycyrrhizinate prevents cadmium-induced activation of JNK and apoptotic hepatocyte death by reversing ROS-inactivated PP2A.

doi: 10.1093/jpp/rgab125

Figure Lengend Snippet: Figure 6 Schematic model showcasing the hepatoprotective effect of MgIG on Cd-triggered apoptotic hepatocyte cell death. MgIG protected against Cd-triggered hepatocyte apoptosis by suppressing excess intracellular ROS inactivation of PP2A, thus inhibiting the JNK cascade activation.

Article Snippet: Sources of antibodies used in this study were as follows: poly ADP-ribose polymerase (PARP) (CST); phospho-JNK (p-JNK) (Thr183/Tyr185), JNK, phospho-c-Jun (p-c-Jun) (Ser63), c-Jun and demethylated PP2A (de-PP2A) (Santa Cruz Biotechnology); phospho-PP2A (p-PP2A) (Epitomics); PP2ACɑ (BD Biosciences); PP2A-A and PP2A-B (Millipore); FLAG, β-tubulin (Sigma); and goat anti-rabbit IgG-horseradish peroxidase (HRP) and goat anti-mouse IgG-HRP (ThermoFisher).

Techniques: Activation Assay

Journal: The Journal of pharmacy and pharmacology

Article Title: Magnesium isoglycyrrhizinate prevents cadmium-induced activation of JNK and apoptotic hepatocyte death by reversing ROS-inactivated PP2A.

doi: 10.1093/jpp/rgab125

Figure Lengend Snippet: Figure 4 MgIG suppressed JNK activation and apoptosis partially by attenuating Cd-inactivated PP2A in hepatocyte cultures. (a, b, e) Western blotting of the total cell lysates from hepatic cell cultures with indicated treatments. β-Tubulin served as a loading control. (c, f) Apoptotic cells were evaluated by using DAPI staining. (d, g) Trypan blue exclusion was performed to evaluate the percentage of live cells. The results are presented as the means ± SE, n = 3–5. aP < 0.05, difference versus the Ctrl group; bP < 0.05, difference versus the 50 μM Cd group; cP < 0.05, difference versus the Cd/OA group or Cd/MgIG group; dP < 0.05, Ad-PP2A-wt group versus the Ad-GFP group.

Article Snippet: Sources of antibodies used in this study were as follows: poly ADP-ribose polymerase (PARP) (CST); phospho-JNK (p-JNK) (Thr183/Tyr185), JNK, phospho-c-Jun (p-c-Jun) (Ser63), c-Jun and demethylated PP2A (de-PP2A) (Santa Cruz Biotechnology); phospho-PP2A (p-PP2A) (Epitomics); PP2ACɑ (BD Biosciences); PP2A-A and PP2A-B (Millipore); FLAG, β-tubulin (Sigma); and goat anti-rabbit IgG-horseradish peroxidase (HRP) and goat anti-mouse IgG-HRP (ThermoFisher).

Techniques: Activation Assay, Western Blot, Control, Staining

Journal: The Journal of pharmacy and pharmacology

Article Title: Magnesium isoglycyrrhizinate prevents cadmium-induced activation of JNK and apoptotic hepatocyte death by reversing ROS-inactivated PP2A.

doi: 10.1093/jpp/rgab125

Figure Lengend Snippet: Figure 5 MgIG blocks Cd-induced ROS generation involved in the activation of the PP2A/JNK cascade and apoptotic hepatocyte cell death. (a, c) Cell ROS im- aging (green) and ROS levels were detected by using the oxidant-sensitive probe CM-H2DCFDA. Scale bar: 20 μm. (b, d, k) Quantitation of ROS fluorescence intensity from images. (e, h) Western blotting of the total cell lysates from hepatic cell cultures with indicated treatments. β-Tubulin served as a loading control. (f, i) The percentages of apoptotic cells were quantified by DAPI staining. (g, j) Trypan blue exclusion was performed to evaluate the percentage of live cells. The results are presented as the means ± SE, n = 3–5. aP < 0.05, difference versus the Ctrl group; bP < 0.05, difference versus the 50-μM Cd group; cP < 0.05, difference versus the Cd/NAC group or Cd/MgIG group; dP < 0.05, the Ad-PP2A-wt group versus the Ad-LacZ group.

Article Snippet: Sources of antibodies used in this study were as follows: poly ADP-ribose polymerase (PARP) (CST); phospho-JNK (p-JNK) (Thr183/Tyr185), JNK, phospho-c-Jun (p-c-Jun) (Ser63), c-Jun and demethylated PP2A (de-PP2A) (Santa Cruz Biotechnology); phospho-PP2A (p-PP2A) (Epitomics); PP2ACɑ (BD Biosciences); PP2A-A and PP2A-B (Millipore); FLAG, β-tubulin (Sigma); and goat anti-rabbit IgG-horseradish peroxidase (HRP) and goat anti-mouse IgG-HRP (ThermoFisher).

Techniques: Activation Assay, Quantitation Assay, Fluorescence, Western Blot, Control, Staining

Journal: The Journal of pharmacy and pharmacology

Article Title: Magnesium isoglycyrrhizinate prevents cadmium-induced activation of JNK and apoptotic hepatocyte death by reversing ROS-inactivated PP2A.

doi: 10.1093/jpp/rgab125

Figure Lengend Snippet: Figure 6 Schematic model showcasing the hepatoprotective effect of MgIG on Cd-triggered apoptotic hepatocyte cell death. MgIG protected against Cd-triggered hepatocyte apoptosis by suppressing excess intracellular ROS inactivation of PP2A, thus inhibiting the JNK cascade activation.

Article Snippet: Sources of antibodies used in this study were as follows: poly ADP-ribose polymerase (PARP) (CST); phospho-JNK (p-JNK) (Thr183/Tyr185), JNK, phospho-c-Jun (p-c-Jun) (Ser63), c-Jun and demethylated PP2A (de-PP2A) (Santa Cruz Biotechnology); phospho-PP2A (p-PP2A) (Epitomics); PP2ACɑ (BD Biosciences); PP2A-A and PP2A-B (Millipore); FLAG, β-tubulin (Sigma); and goat anti-rabbit IgG-horseradish peroxidase (HRP) and goat anti-mouse IgG-HRP (ThermoFisher).

Techniques: Activation Assay

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