Journal: Cell Death & Disease

Article Title: Understanding the function of Pax5 in development of docetaxel-resistant neuroendocrine-like prostate cancers

doi: 10.1038/s41419-024-06916-y

Figure Lengend Snippet:

Article Snippet: Phospho-EGFR , 1:1000 , Cell Signaling, 1068S.

Techniques:

Journal: Cell Death & Disease

Article Title: Understanding the function of Pax5 in development of docetaxel-resistant neuroendocrine-like prostate cancers

doi: 10.1038/s41419-024-06916-y

Figure Lengend Snippet:

Article Snippet: Phospho-EGFR , 1:1000 , Cell Signaling, 1068S.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness

doi: 10.1074/jbc.m113.497685

Figure Lengend Snippet: FIGURE 1. Up-regulation of CD147 induces activation of EGFR-Ras-ERK signaling. A, Western blot depicting surface biotinylation of MCF-10A cells, treated with an MOI of 2 of -gal or CD147 adenovirus (Adv), compared with untreated MDA-MB-231 cells. An MOI of 2 was used in all subsequent adenovirus experiments. For a negative control, a lane was loaded with eluate from NeutrAvidin resin only (labeled Beads), which was prepared similarly to experimental groups but without cell lysate; n 3. B, evaluation of Ras activity and ERK phosphorylation in lysates of MCF-10A cells that had been pretreated in culture with -galorCD147adenovirus.ActivatedRaswasidentifiedbyprobingwithapan-Rasantibody.ParentalMCF-10AwholecelllysatestreateddirectlywithGDPand GTP were used as negative and positive controls, respectively.-Actin was used as a loading control. Left, a representative gel; right, densitometric quantitation of Ras-GTP versus total Ras. Columns are means S.E. (error bars); n 4; *, p 0.05. C, effect of inhibition of EGFR activity on ERK phosphorylation in MCF-10A cells treated with -gal or CD147 adenovirus. After treatment with adenovirus for 24 h in full medium, cells were incubated in supplement-free medium for 4 h and subsequently treated with vehicle (DMSO) or 0.5 and 5.0 M tyrphostin (AG1478) for 12 h. -Actin was used as a loading control. Left, a representative gel; right, densitometric quantitation of p-ERK versus total ERK. Columns are means S.E.; n 3; **, p 0.01; ***, p 0.001. D, EGFR and ERK phosphorylation in MCF-10Acellstreatedwith-galorCD147adenovirusfor24hinfullysupplementedmedium,followedbyincubationinmediumcontainingsupplementswith and without EGF (20 ng/ml) for 6 h; EGFR activation was evaluated by probing for phosphorylation of the tyrosine 1173 or 1068 residues. For controls, parental MCF-10A cells were incubated in medium containing supplements with and without EGF (20 ng/ml) for 6 h. -Actin was used as a loading control. Left, a representative gel; middle, densitometric quantitation of p-ERK versus total ERK; right, densitometric quantitation of p-EGFR versus total EGFR. Columns are means S.E.; n 3; *, p 0.05; **, p 0.01; ***, p 0.001.

Article Snippet: Briefly, primary antibodies targeting CD147 (mouse; BD Pharmingen), EGFR (rabbit; Cell Signaling), and CD44 (mouse (Santa Cruz Biotechnology) and rabbit (Santa Cruz Biotechnology)) were applied using standard procedures.

Techniques: Activation Assay, Western Blot, Negative Control, Labeling, Activity Assay, Phospho-proteomics, Control, Quantitation Assay, Inhibition, Incubation

Journal: Journal of Biological Chemistry

Article Title: CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness

doi: 10.1074/jbc.m113.497685

Figure Lengend Snippet: FIGURE 2. Hyaluronan-CD44 interaction mediates CD147-induced activation of EGFR signaling. A, hyaluronan secretion in MCF-10A cells treated with -gal or CD147 adenovirus. After seeding the adenovirus-treated cells into 24-well plates and incubating for 24 h, the media were replaced with fresh media. After another 24-h incubation, media were collected and analyzed for hyaluronan concentration using an ELISA-like assay. Hyaluronan (HA) in the media was normalized to cell number and depicted as mean -fold change. Columns are means S.E. (error bars); n 3; **, p 0.01. B, effects of hyaluronan oligomers (o-HA) on activation of EGFR and ERK. MCF-10A cells treated with -gal or CD147 adenovirus were subsequently treated with 100 g/ml o-HA for 1, 12, and 24 h and probed for p-EGFR (Tyr-1068) and p-ERK. -Actin was used as a loading control. Left, a representative gel; middle, densitometric quantitation of p-EGFR (Tyr-1068) versus total EGFR; right, densitometric quantitation of p-ERK versus total ERK. Columns are means S.E.; n 3; *, p 0.05; **, p 0.01; ns, not significant. C, effects of CD44 knockdown on activation of EGFR and ERK. MCF-10A cells treated with CD147 adenovirus were subsequently treated with nonspecific control siRNA or pooled CD44-specific siRNA as described under “Experimental Procedures” and probed for the indicated proteins. -Actin was used as a loading control. Left, a representative gel; middle, densitometric quantitation of p-ERK versus total ERK; right, densitometric quantitation of p-EGFR (Tyr-1068) versus total EGFR. Columns are means S.E.; n 3; *, p 0.05; **, p 0.01. D, effect of CD44 knockdown on invasion. Shown are quantitation and representative images of cell invasion through Matrigel by CD147-up-regulated MCF-10A cells after treatment with nonspecific control siRNA or pooled CD44-specific siRNA. The cell invasion index was calculated as the number of invasive cells that had been treated with CD147 adenovirus and then pooled CD44-specific siRNA, normalized to the number of invasive cells that had been treated with CD147 adenovirus then nonspecific control siRNA. Columns are means S.E.; n 3; **, p 0.01.

Article Snippet: Briefly, primary antibodies targeting CD147 (mouse; BD Pharmingen), EGFR (rabbit; Cell Signaling), and CD44 (mouse (Santa Cruz Biotechnology) and rabbit (Santa Cruz Biotechnology)) were applied using standard procedures.

Techniques: Activation Assay, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Quantitation Assay, Knockdown

Journal: Journal of Biological Chemistry

Article Title: CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness

doi: 10.1074/jbc.m113.497685

Figure Lengend Snippet: FIGURE 3. CD147 induces assembly of signaling complexes. A, representative images demonstrating protein interactions (40 nm) using PLA (see “Exper- imental Procedures” for details). CD147-treated MCF-10A cells were probed with primary antibodies in the following combinations to detect protein interac- tions in the cell population: CD44 (mouse) and EGFR (rabbit), CD147 (mouse) and EGFR (rabbit), and CD147 (mouse) and CD44 (rabbit). Protein interactions appearassmallpunctategreensignals(i.e.dots),andcellnucleiwereidentifiedbyco-stainingwithDAPI.ExamplesoftheseproteininteractionsinMCF-10Acells treated with -gal or CD147 adenovirus, in this case interactions between CD147 and CD44, are indicated by the red arrowheads. The bottom panel shows a representative negative control where only one primary antibody was employed. B, quantitation of the indicated protein interactions in the cell population over five random fields with at least 30 cells/field. Left, percentage of cells showing interaction; column values are means S.E. (error bars) over three independent experiments. Right, interactions per optical slice, estimated to be at the center of the cell based on DAPI signal; scatter plot showing each interaction with mean S.E.; n 3; **, p 0.01; ***, p 0.001. C, representative image of MCF-10A cells treated with CD147 adenovirus and evaluated for CD147 (red), cholera toxin-B (green), and colocalization (yellow; indicated by yellow arrowheads). Examples of filopodia are identified by red arrowheads. Scale bar, 10 m. The CD147 channel was pseudocolored from blue to red for easier visualization of colocalization. D, MCF-10A cells treated with -gal or CD147 adenovirus were subjected to detergent-resistant membrane isolation (see “Experimental Procedures”) and probed for the indicated proteins. Light fractions are from the gradient interface (0–20%), where detergent-resistant membrane domains, such as lipid rafts, localize; n 3. Cav-1, caveolin (lipid raft marker). E, CD44 and EGFR surface expression was analyzed by FACS in CD147-treated MCF-10A cells. Distributions with dark gray shading, light shading, and no shading represent IgG-isotype control, -gal, and CD147 treated cells, respectively; n 3.

Article Snippet: Briefly, primary antibodies targeting CD147 (mouse; BD Pharmingen), EGFR (rabbit; Cell Signaling), and CD44 (mouse (Santa Cruz Biotechnology) and rabbit (Santa Cruz Biotechnology)) were applied using standard procedures.

Techniques: Negative Control, Quantitation Assay, Membrane, Isolation, Marker, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness

doi: 10.1074/jbc.m113.497685

Figure Lengend Snippet: FIGURE 7. Identification and localization of endogenous protein interactions in MCF-10A-EV, MCF-10A-K-RasV12, and MDA-MB-231 cells. A, endoge- nousproteininteractions(40nm)wereanalyzedbyPLA(see“ExperimentalProcedures”fordetails)andquantitatedforthenumberofindicatedinteractions/ optical slice in a similar manner as in Fig. 3. Shown is a scatter plot of the indicated protein interactions in MCF-10A-EV and MCF-10A-K-RasV12 cells with mean S.E.; n 3; *, p 0.05; ***, p 0.001. B, representative image demonstrating colocalization (yellow) of CD147 (red) and cholera toxin-B (green) in 10A-K-RasV12 cells. Scale bar, 10 m; the CD147 channel was pseudocolored from blue to red for easier visualization. Filopodia are highlighted with yellow arrowheads. C, Western blot comparing protein distribution between 10A-EV and 10A-K-RasV12 cells subjected to detergent-resistant membrane isolation and probed for indicated proteins. Light fractions are from the gradient interface (0–20%), where detergent-resistant membrane domains, such as lipid rafts, localize; n 2. Cav-1,caveolin(lipidraftmarker).D,WesternblotshowingproteindistributionforMDA-MB-231cellssubjectedtolipidraftanalysisasinC;n2.Tfr,transferrin receptor (non-raft plasma membrane marker); Flot, flotillin-1 (lipid raft marker); -COP, coatomer protein (Golgi marker). E, co-immunoprecipitation of CD147, CD44, and EGFR from lysates of MDA-MB-231 cells. Whole cell lysates from MDA-MB-231 cells were co-immunoprecipitated (IP) with mouse Ig antibodies against human CD147 or CD44, as described under “Experimental Procedures”; controls (Cont) were performed in a similar manner with mouse Ig. Immuno- blotting of aliquots of test and control eluates from the antibody-conjugated resins was performed with antibodies against CD147, CD44, and EGFR; similar results were obtained in two independent experiments.

Article Snippet: Briefly, primary antibodies targeting CD147 (mouse; BD Pharmingen), EGFR (rabbit; Cell Signaling), and CD44 (mouse (Santa Cruz Biotechnology) and rabbit (Santa Cruz Biotechnology)) were applied using standard procedures.

Techniques: Western Blot, Membrane, Isolation, Clinical Proteomics, Marker, Immunoprecipitation, Control

Journal: Journal of Biological Chemistry

Article Title: CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness

doi: 10.1074/jbc.m113.497685

Figure Lengend Snippet: FIGURE 8. MCF-10A-K-RasV12 cells with high constitutive surface levels of CD147 have elevated activation of EGFR and ERK and are more invasive. A, representative micrographs demonstrating heterogeneity in CD147 and CD44 staining intensity across the 10A-K-RasV12 cell population. Scale bar, 25 m. B, FACS scatter distribution of total 10A-K-RasV12 cell population (left) and delineation of CD147Lo and CD147Hi cells (bottom and top 20%, respectively)inthedistributionofCD147-expressingcells(topright)withcorrespondingscattercharacteristics(bottomright).Notethatthiscellsortingscheme identifies two distinct cell populations. C, FACS analysis of cell surface expression of CD44 and EGFR in CD147Lo and CD147Hi cells in 10A-K-RasV12 (left; n 3), MDA-MB-231 (middle; n 3), and MCF-7 (right; n 2) cells. Distributions with dark gray shading, no shading, and light gray shading represent IgG-isotype control,CD147Lo,andCD147Hicells,respectively.D,left,immunoblotcomparingproteinexpressionofindicatedproteinsinCD147LoandCD147Hi10A-K-RasV12 cells. -Actin was used as a loading control; n 2. Right, histogram demonstrating -fold change between CD147Hi and CD147Lo cells for pEGFR (Tyr-1068), pERK1, and pERK2 protein levels; densitometric intensities were normalized to total EGFR and ERK1/2 protein; n 2. E, quantitation of the percentage of cells with actin-cortactin aggregates with or without underlying degraded gelatin matrix in CD147Lo and CD147Hi 10A-K-RasV12 cells was calculated by evaluating random fields containing at least 15 cells/field over three independent experiments. Column values are means S.E. (error bars); ***, p 0.001; ns, not significant.

Article Snippet: Briefly, primary antibodies targeting CD147 (mouse; BD Pharmingen), EGFR (rabbit; Cell Signaling), and CD44 (mouse (Santa Cruz Biotechnology) and rabbit (Santa Cruz Biotechnology)) were applied using standard procedures.

Techniques: Activation Assay, Staining, Expressing, Control, Quantitation 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: 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