Journal: Clinical Cancer Research

Article Title: Fluorescence Lifetime-Based Tumor Contrast Enhancement Using an EGFR Antibody–Labeled Near-Infrared Fluorophore

doi: 10.1158/1078-0432.ccr-19-1686

Figure Lengend Snippet: Figure 1. TD fluorescence decay signals of tissue autofluorescence (solid line), anti- EGFR-800 (abbreviated as aEGFR-800) in vitro (solid line—triangles), anti- EGFR-800 in vivo in tumor (solid line—diamonds), muscle (dashed line), and liver (dotted line) 48 hours after injection. The decay curves are represented as the average of 3 3 pixels in TD reflectance fluorescence images. FLTs obtained from single exponential fits to the decay portion of the curves are shown in the inset.

Article Snippet: Tissue sections were incubated in 1:50 dilution of anti-EGFR antibody (Cat# 4267, Cell Signaling Tech.) overnight at 4 C. Secondary antibody was applied for 30minutes at 37 C and slides were developed with DAKO HRP-compatible DAB (Cat# SF-4100, Vector Laboratories) and counterstained with Harris Hematoxylin.

Techniques: In Vitro, In Vivo, Injection

Journal: Clinical Cancer Research

Article Title: Fluorescence Lifetime-Based Tumor Contrast Enhancement Using an EGFR Antibody–Labeled Near-Infrared Fluorophore

doi: 10.1158/1078-0432.ccr-19-1686

Figure Lengend Snippet: Figure 2. In vivo TD imaging, 48 hours after intravenous administration of anti-EGFR-800 (A, C, E) or IgG-800 (B, D, F) in MDA-MB-231 tumor-bearing mice. A and B, White light images with tumor boundaries indicated by red-dotted outline. C and D, CW intensity from tumors, surrounding liver and muscle. E and F, FLT maps obtained from single exponential fits to the decay of TD data. Distributions of CW intensities (G and H) and FLT distributions (I and J) in the anti-EGFR-800– (G and I) and IgG-800– (H and J) injected animals, within tumor (red) and normal tissue (green). K, ROC curves calculated on the basis of CW intensity (blue) and FLT (red) thresholding for the anti-EGFR-800– (K) and IgG-800– (L) injected animals. The arrow in (I) represents the optimal threshold FLT for tumors used for surgical guidance study shown in Fig. 5.

Article Snippet: Tissue sections were incubated in 1:50 dilution of anti-EGFR antibody (Cat# 4267, Cell Signaling Tech.) overnight at 4 C. Secondary antibody was applied for 30minutes at 37 C and slides were developed with DAKO HRP-compatible DAB (Cat# SF-4100, Vector Laboratories) and counterstained with Harris Hematoxylin.

Techniques: In Vivo, Imaging, Injection

Journal: Clinical Cancer Research

Article Title: Fluorescence Lifetime-Based Tumor Contrast Enhancement Using an EGFR Antibody–Labeled Near-Infrared Fluorophore

doi: 10.1158/1078-0432.ccr-19-1686

Figure Lengend Snippet: Figure 4. Fluorescence microscopy of ex vivo tissue sections of a tumor (A), liver (B), and muscle (C) labeled with anti-EGFR-800; D, fluorescence microscopy of tumor from IgG-800–injected mouse; H&E staining of tumor (E), liver (F), and muscle (G); IHC staining of tumor (H), liver (I), and muscle (J). Magnification: 20. Inset in (H) shows a magnified region in the tumor with predominantly membrane staining for EGFR. All ex vivo measurements were performed after sacrifice 48 hours after injection.

Article Snippet: Tissue sections were incubated in 1:50 dilution of anti-EGFR antibody (Cat# 4267, Cell Signaling Tech.) overnight at 4 C. Secondary antibody was applied for 30minutes at 37 C and slides were developed with DAKO HRP-compatible DAB (Cat# SF-4100, Vector Laboratories) and counterstained with Harris Hematoxylin.

Techniques: Fluorescence, Microscopy, Ex Vivo, Labeling, Injection, Staining, Immunohistochemistry, Membrane

Journal: Clinical Cancer Research

Article Title: Fluorescence Lifetime-Based Tumor Contrast Enhancement Using an EGFR Antibody–Labeled Near-Infrared Fluorophore

doi: 10.1158/1078-0432.ccr-19-1686

Figure Lengend Snippet: Figure 5. In situ CW intensity and FLT maps of an MDA-MB-231 tumor before and after surgical removal of tumor mass. The animal was sacrificed 48 hours after intravenous injection of anti-EGFR-800, and TD imaging was performed after the skin was removed from the top of the tumor and surrounding muscle. CW intensity image, histogram of CW intensity, FLT map, and histogram of FLT map are shown correspondingly from left to right for the whole tumor (A), residual tumor (B), and surgical bed (C); (D) IHC for EGFR in the residual tumor obtained from (C; left) and surgical bed (right) is shown. Scale bar: 5-mm unless otherwise mentioned.

Article Snippet: Tissue sections were incubated in 1:50 dilution of anti-EGFR antibody (Cat# 4267, Cell Signaling Tech.) overnight at 4 C. Secondary antibody was applied for 30minutes at 37 C and slides were developed with DAKO HRP-compatible DAB (Cat# SF-4100, Vector Laboratories) and counterstained with Harris Hematoxylin.

Techniques: In Situ, Injection, Imaging

Journal: Clinical Cancer Research

Article Title: Protein Expression of Platelet-Derived Growth Factor Receptor Correlates with Malignant Histology and PTEN with Survival in Childhood Gliomas

doi: 10.1158/1078-0432.ccr-07-1616

Figure Lengend Snippet: Fig. 2. Representative immunohistochemicalTMA protein analysis of the EGFR and PDGFR pathway in pediatric gliomas. Left, A to D, representativeTMA of pediatric glial tumors stained for PDGFR markers. p-PDGFRa ^ positive (A), p-PDGFRa ^ negative (B), PDGFRh-positive (C), and PDGFRh-negative (D) glial tumors are shown. Osteosarcoma positive control (E) and cerebral cortical gray matter negative control (F) were used for both PDGFR markers. Right, A to L, representativeTMA of pediatric glial tumors stained for EGFR and PDGFR pathway markers. Positive (A, C, E, G, I, and K) and negative (B, D, F, H, J, and L) glial tumors staining for EGFR, p-AKT, p-EGFR, p-MAPK, p-MTOR, and PTEN, respectively, are shown. Breast carcinoma positive control (M) and cerebral cortical gray matter negative control (N) for EGFR staining are shown and the same tissues were used as controls for all markers analyzed. Magnification is 200 for allTMA images, except the p-AKT ^ negative glial tumor (right, D) shown is 400 to show that no tumor was completely negative for this marker. Controls are 400 magnification, except the EGFR-negative control (right, N) shown is 100.

Article Snippet: Sections were incubated for 1 h using recommended serum block to eliminate nonspecific staining and subsequently incubated for 18 to 22 h overnight at 4jC using one of eight primary antibodies: EGFR (1:25; Cell Signaling Technology, Inc.), p-EGFR (Tyr) mouse monoclonal antibodies (1:200; Cell Signaling Technology), PDGFRh (1:50; Santa Cruz Biotechnology), p-AKT (Ser; 1:25; Cell Signaling Technology), p-mTOR (Ser; 1:50; Cell Signaling Technology), p-MAPK (44/42; 1:100; Cell Signaling Technology), PTEN mouse monoclonal antibodies (1:400; clone 6H2.1; Cascade Bioscience), and p-PDGFRa (Tyr; 1:50; Santa Cruz Biotechnology).

Techniques: Staining, Positive Control, Negative Control, Marker

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: Endothelial PP2A deficiency induces hippocampal dysfunction in mice. (A–C) Bioinformation analysis. Gene Ontology (GO) biological process analysis of genes related to VCD (A, B). Protein interaction network of genes enriched in “positive regulation of protein phosphorylation”, “response to hydrogen sulfide” and PP2ACA via STRING (C). (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of different genes in RNA sequencing ( n = 3). (E) Immunoblots of PP2AC α proteins level in PBMECs isolated from WT sham or WT LCA mice ( n = 4). (F, G) Representative confocal image (F) and quantification (G) of PP2AC α in brain endothelium of wild type mice after LCA, PP2Ac α (Green), lectin + endothelium (red) ( n = 4). Scale bar, 20 μm. (H) Survival curves of PP2A EC-cKO versus PP2AC α f/f mice after partial ligation on left carotid artery (LCA). (I–L) Cognition of male mice was evaluated by Y-maze test (I) and Morris water maze test 28 days after LCA. (J) The typical swim path of mice in the Morris water maze test. (K) Latency to reach the hidden platform during the acquisition trial after LCA. (L) Percentage occupancy in the goal quadrant in the probe trial ( n = 10–13). (M) Nissl staining for neurodegeneration in the hippocampus after LCA ( n = 5). (N, O) Representative confocal image (N) and quantification (O) of NeuN + neurons (red) in the CA3 and DG hippocampal subfield of PP2AC α f/f and PP2A EC-cKO mice ( n = 6). Scale bar, 20 or 50 μm. (P, Q) Reduced hippocampal fEPSP slope (P) and averages of the last 10 min of fEPSP recording(Q) in the CA1 region of PP2A EC-cKO mice compared to PP2AC α f/f mice. Representative traces before (1) and after (2) tetanic stimulation was shown above fEPSP recording. Scale bars depict 10 ms or 0.25 mV. PP2AC α f/f : n = 10 from 4 mice; PP2A EC-cKO : n = 10 from 3 mice. Data are expressed mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Phospho-proteomics, RNA Sequencing, Western Blot, Isolation, Ligation, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: PP2A deficiency in brain endothelium via AAV-Tie-Cre also induces cognitive dysfunction. (A) Experimental schematic of AAV-Tie-Cre intrahippocampal injection. (B, C) Representative confocal image (B) and transduction ratio (C) of Ppp2Cα deletion in hippocampal endothelium at 3 weeks after AAV-Tie-CRE injection. PP2Ac α f/f mice injected with AAV-Tie-CRE or AAV-zsGreen as control. PP2AC α (Green), lectin + endothelium (red) ( n = 6–7). Scale bar, 50 μm. (D–G) Cognition was evaluated by Y-maze test (D) and Morris water maze test (E–G) at 3 weeks after AAV-Tie-CRE injection. (E) The typical swim path of PP2Ac α f/f -zsGreen and PP2Ac α f/f -AAV-Tie-cre mice in the Morris water maze test. (F) Latency to reach the hidden platform during the acquisition trial. (G) Percentage occupancy in the goal quadrant in the probe trial, ( n = 10–11). (H) Nissl staining for neurodegeneration in the hippocampus of PP2Ac α f/f -zsGreen and PP2Ac α f/f -AAV-Tie-cre mice at 3 weeks after AAV-Tie-CRE injection, ( n = 5). (I, J) Representative confocal image (I) and quantification (J) of NeuN + neurons (red) in the CA3 and DG hippocampal subfield of PP2Ac α f/f -zsGreen and PP2Ac α f/f -AAV-Tie-cre mice at 3 weeks after AAV-Tie-CRE injection ( n = 6–8). Scale bar, 50 μm. Data are expressed mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Injection, Transduction, Control, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: Restoration of endothelial PP2A in the brain impedes cognitive dysfunction in PP2A EC-cKO mice. (A, B) Representative confocal image (A) and ratio of Ppp2Cα restoration in hippocampal endothelium (B) at 3 weeks after AAV virus injection. PP2Ac α (Green), lectin + (red) is the marker of endothelium ( n = 6–7). Scale bar, 50 μm. (C)–(F) Cognition was evaluated by Y-maze test (C) and Morris water maze test (D)–(F) at 3 weeks after AAV virus injection. (D) The typical swim path of PP2AC α f/f -zsGreen, PP2AC α f/f -AAV-Tie- Ppp2cα , PP2A EC-cKO -zsGreen, PP2A EC-cKO -AAV-Tie- Ppp2cα mice in the Morris water maze test. (E) Latency to reach the hidden platform during the acquisition trial. (F) Percentage occupancy in the goal quadrant in the probe trial ( n = 11–14). (G) Nissl staining for neurodegeneration in the hippocampus of PP2AC α f/f -zsGreen, PP2AC α f/f -AAV-Tie- Ppp2cα , PP2A EC-cKO -zsGreen, PP2A EC-cKO -AAV-Tie- Ppp2cα mice at 3 weeks after AAV virus injection ( n = 5). Scale bar, 100 μm. (H, I) Representative confocal image (H) and quantification (I) of NeuN + neurons (red) in the CA3 and DG hippocampal subfield of PP2AC α f/f -zsGreen, PP2AC α f/f -AAV-Tie- Ppp2cα , PP2A EC-cKO -zsGreen, PP2A EC-cKO -AAV-Tie- Ppp2cα mice at 3 weeks after AAV virus injection ( n = 6–8). Scale bar, 20 or 50 μm. Data are expressed mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Virus, Injection, Marker, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: Endothelial PP2A deficiency resulted in a reduction of H 2 S production in mice. (A, B) Bioinformation analysis for the interaction between PP2A and hydrogen sulfide. (C–E) Measurement of H 2 S concentration. (C) PP2AC α f/f and PP2A EC-cKO PBMECs, ( n = 5), (D) PP2AC α f/f PBMECs infected with AAV-Tie-cre ( n = 4), (E) bEnd.3 cells were transfected with Ppp2ca shRNA, ( n = 5). (F–I) Cognition was evaluated by Y-maze test (F) and Morris water maze test (G–I) at 2 days after termination of NaHS administration. (G) The typical swim path of PP2AC α f/f -saline, PP2AC α f/f -NaHS, PP2A EC-cKO -saline, PP2A EC-cKO -NaHS mice. (H) Latency to reach the hidden platform during the acquisition trial. (I) Percentage occupancy in the goal quadrant in the probe trial, ( n = 9–14). (J) Nissl staining for neurodegeneration in the hippocampus of PP2AC α f/f -saline, PP2AC α f/f -NaHS, PP2A EC-cKO -saline, PP2A EC-cKO -NaHS mice ( n = 5). Scale bar, 100 μm. (K, L) Representative confocal image (K) and quantification (L) of NeuN + neurons (red) in the CA3 and DG hippocampal subfield of PP2AC α f/f -saline, PP2AC α f/f -NaHS, PP2A EC-cKO -saline, PP2A EC-cKO -NaHS mice at 7 days after termination of NaHS administration ( n = 5∼7). Scale bar, 20 or 50 μm. Data are expressed mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Concentration Assay, Infection, Transfection, shRNA, Saline, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: Endothelial PP2A deficiency suppressed MPST expression in mice. (A, B) Representative confocal images (A) and quantification (B) of MPST in hippocampal endothelium of PP2AC α f/f and PP2A EC-cKO mice. MPST (green), Lectin (red) ( n = 7–8). Scale bar, 50 μm. (C, D) Representative confocal images (C) and quantification (D) of MPST in hippocampal endothelium of PP2AC α f/f -zsGreen and PP2AC α f/f -AAV-Tie-cre mice ( n = 6). MPST (green), Lectin (red). Scale bar = 50 μm. (E) Immunoblots of MPST proteins level in PBMECs and bEnd.3 cells ( n = 4 for PBMECs; n = 5 for bEnd.3 cells). (F) Immunoblots of MPST proteins level in bEnd.3 cells. bEnd.3 cells were treated with 100 nmol/L okadaic acid (OA) to inhibit PP2A activity ( n = 6). (G) Immunoblots of MPST proteins level in PBMECs lysates isolated from PP2AC α f/f mice infected with AAV-zsGreen or AAV-Tie-cre ( n = 7). (H) Immunoblots of MPST proteins level in PBMECs lysates isolated from PP2AC α f/f and PP2A EC-cKO mice infected with AAV-zsGreen or AAV-Tie- Mpst ( n = 4). (I) H 2 S concentration in PBMECs lysates isolated from PP2AC α f/f and PP2A EC-cKO mice infected with AAV-zsGreen or AAV-Tie- Mpst ( n = 5–6). (J–M) Cognition was evaluated by Y-maze test (J) and Morris water maze test (K–M) at 3 weeks after AAV-Tie- Mpst injection. (K) The typical swim path of PP2AC α f/f -zsGreen, PP2AC α f/f - Mpst , PP2A EC-cKO -zsGreen, PP2A EC-cKO - Mpst mice. (L) Latency to reach the hidden platform during the acquisition trial. (M) Percentage occupancy in the goal quadrant in the probe trial ( n = 10–13). (N, O) Representative confocal image (N) and quantification (O) of NeuN + neurons (red) in the CA3 and DG hippocampal subfield of PP2AC α f/f -zsGreen, PP2AC α f/f - Mpst , PP2A EC-cKO -zsGreen, PP2A EC-cKO - Mpst ( n = 5–7). Scale bar, 50 μm. Data are expressed as mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Expressing, Western Blot, Activity Assay, Isolation, Infection, Concentration Assay, Injection

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: Endothelial PP2A deficiency promotes calpain2-mediated degradation of MPST. (A, B) Relative mRNA levels of MPST in PBMECs isolated from PP2AC α f/f or PP2A EC-cKO mice ( n = 4) (A) and bEnd.3 cells ( n = 6) (B). (C, D) Relative mRNA levels of Capn1 (C) and Capn2 (D) in PBMECs lysates isolated from PP2AC α f/f or PP2A EC-cKO mice ( n = 4–5). (E, F) Immunoblots of calpain 2 (E) and calpain1 (F) proteins level in PBMECs lysates isolated from PP2AC α f/f or PP2A EC-cKO mice ( n = 4–5). (G) Representative line-scan images of Ca 2+ sparks and time-dependent profiles of global Ca 2+ influx induced by application of 0.3 mol/L CaCl 2 for PP2AC α f/f and PP2A EC-cKO PBMECs. (H) Maximum amplitude of CaCl 2 induced Ca 2+ transients. (I) Immunoblot analysis of calpain2 phosphorylation level in PP2AC α f/f and PP2A EC-cKO PBMECs using phospho-serine antibody. (J) Immunoblot analysis of calpain2 and MPST proteins in PBMECs lysates immunoprecipitated with control IgG or anti-calpain2 antibody. (K) Immunoblot analysis of calpain2 and MPST proteins in PBMECs lysates immunoprecipitated with control IgG or anti-MPST antibody. (L–N) Immunoblots of calpain2 and MPST in PP2AC α f/f and PP2A EC-cKO PBMECs. PBMECs were treated with vehicle or 40 μmol/L PD150606 for 12 h. Data are expressed as mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Isolation, Western Blot, Phospho-proteomics, Immunoprecipitation, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: PP2A activator FTY720 restores ischemic neuron dysfunction and endothelial MPST expression. (A, B) Representative confocal image (A) and quantification (B) of MPST expression in brain endothelium. MPST (Green), lectin + (red). Scale bar, 20 μm. (C) Measurement of H 2 S concentration after FTY720 treatment. (D–G) Cognition was evaluated by Morris water maze test (D–F) and Y-maze test (G) at 2 weeks after FTY720 treatment. (D) The typical swim path of WT or LCA mice treated with either vehicle (0.4% dimethyl sulfoxide in PBS) or FTY720 (2 mg/kg) by daily gavage. (E) Latency to reach the hidden platform during the acquisition trial. (F) Percentage occupancy in the goal quadrant in the probe trial. (G) Percentage time spent in the novel arm ( n = 10–11). (H, I) Representative confocal image (H) and quantification (I) of NeuN + neurons in the brain ( n = 6). Data are expressed as mean ± SEM, ∗ P < 0.05.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Expressing, Concentration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: 3MP sodium restores ischemic neuron dysfunction and endothelial MPST expression. (A) Measurement of H 2 S concentration after 3MP sodium treatment. (B, C) Representative confocal image (B) and quantification (C) of MPST expression in brain endothelium. MPST (Green), lectin + (red). Scale bar, 20 μm. (D, E) Representative confocal image (D) and quantification (E) of PP2AC α expression in brain endothelium. PP2AC α (Green), lectin + (red). Scale bar, 20 μm. (F–H) Cognition was evaluated by Novel object recognition (F–G) and Y-maze test (H) at 12 days after 3MP sodium treatment. (F) The typical path of WT or LCA mice treated with either vehicle (0.4% dimethyl sulfoxide in PBS) or 3MP sodium (1 mg/kg) by daily i.p. (G) Discrimination index toward a novel object. (H) Percentage time spent in the novel arm ( n = 9). (I, J) Representative confocal image (I) and quantification (J) of NeuN + neurons in the brain ( n = 6). Data are expressed mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Expressing, Concentration Assay

Journal: Cancer Research

Article Title: Cross-talk between LPA1 and Epidermal Growth Factor Receptors Mediates Up-regulation of Sphingosine Kinase 1 to Promote Gastric Cancer Cell Motility and Invasion

doi: 10.1158/0008-5472.can-08-0411

Figure Lengend Snippet: Figure 3. Activation of EGFR is involved in LPA-induced SphK1 expression. A, MKN1 cells were stimulated with 10 Amol/L LPA for the indicated times and lysed, and equal amounts of protein were analyzed by immunoblotting with antibodies against phosphotyrosine (PY) or phospho-EGFR (Tyr1068). Blots were stripped and reprobed with EGFR antibody to confirm equal loading. B, MKN1 cells were stimulated with the indicated concentrations of EGF for 6 h or 10 ng/mL EGF for the indicated times. RNA was isolated and reverse transcribed, and SphK1 and GAPDH mRNA levels were measured by real-time PCR. Data are expressed as fold changes after normalization to GAPDH. C, MKN1 cells were pretreated with vehicle (DMSO; white columns) or 0.5 Amol/L AG1478 (black columns) for 30 min and then stimulated with 10 Amol/L LPA or 10 ng/mL EGF for 6 h. RNA was isolated and reverse transcribed, and SphK1, S1P3, and GAPDH mRNA levels were measured by real-time PCR. SphK1 and S1P3 mRNA levels are expressed as fold changes after normalization to GAPDH. *, P V 0.01.

Article Snippet: Antibodies against phospho-EGFR (Tyr1068) and h-tubulin were from Cell Signaling.

Techniques: Activation Assay, Expressing, Western Blot, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction