Journal: Advanced Science

Article Title: A Bioinspired Manganese‐Organic Framework Ameliorates Ischemic Stroke through its Intrinsic Nanozyme Activity and Upregulating Endogenous Antioxidant Enzymes

doi: 10.1002/advs.202206854

Figure Lengend Snippet: The effect of pDA‐MNOF on upregulating HO1 and SOD2 via STAT3 signaling. a) Western blot analysis of p‐STAT3, STAT3, HO1, and SOD2 expression in N2a cells treated with 12.5 µg mL −1 pDA‐MNOF for various time durations. b) Quantification of the band intensity ratios of p‐STAT3/STAT3, HO1/ β ‐actin, and SOD2/ β ‐actin in (a). t‐STAT3 indicated total proteins of p‐STAT3 and STAT3. c) Quantification of fluorescent intensity of p‐STAT3, HO1, and SOD2 in PBS or 12.5 µg mL −1 pDA‐MNOF‐treated N2a cells ( n = 4). Data were presented with mean ± s.d.; *, p < 0.05; **, p < 0.01; T ‐test. d) Representative images of immunofluorescent staining for p‐STAT3, HO‐1, and SOD‐2 (red) in indicated groups. Nuclei were stained in blue, and cell body was outlined with white dashed lines. Scale bar, 20 µm. e) Western blot analysis of p‐STAT3, STAT3, HO1, and SOD2 expression in N2a cells treated with different concentrations of pDA‐MNOF for 12 h. f) Quantification of the band intensity ratios of p‐STAT3/STAT3, HO1/ β ‐actin, and SOD2/ β ‐actin in (e). g) Experimental scheme for the RNA interference and pathway inhibition assays. h) Western blot analysis of p‐STAT3 and STAT3 in N2a cells treated with PBS, siRNA Control (siRNA Ctrl), siRNA 1#, siRNA 2#, and siRNA 3#. i) Quantification of the band intensity ratios of p‐STAT3/ β ‐actin and STAT3/ β ‐actin in (h). j) N2a cells were transfected with siRNA Ctrl, siRNA 2#, and siRNA 3#, followed by treatment with pDA‐MNOF; the lysates were analyzed with indicated antibodies. k) Quantification of the band intensity ratios of p‐STAT3/ β ‐actin, HO1/ β ‐actin, and SOD2/ β ‐actin in (j). Data were presented with mean ± s.d.; *, p < 0.05; ANOVA. l) N2a cells were treated with PBS or pDA‐MNOF, followed by treatment with AG490; the lysates were analyzed with indicated antibodies. m) Quantification of the band intensity ratios of p‐STAT3/STAT3, HO1/ β ‐actin, and SOD2/ β ‐actin in (l) ( n = 3). Data were presented with mean ± s.d.; *, p < 0.05; **, p < 0.01; ANOVA.

Article Snippet: To assess the protein levels of VEGF, the supernatant of N2a cells that were treated with PBS or various concentrations of pDA‐MNOF for 24 h was collected for ELISA detection (Elabscience, China).

Techniques: Western Blot, Expressing, Staining, Inhibition, Control, Transfection

Journal: Advanced Science

Article Title: A Bioinspired Manganese‐Organic Framework Ameliorates Ischemic Stroke through its Intrinsic Nanozyme Activity and Upregulating Endogenous Antioxidant Enzymes

doi: 10.1002/advs.202206854

Figure Lengend Snippet: pDA‐MNOF protects neuronal cells against ROS‐induced injury. a) DCFH‐DA staining for detecting ROS in the PBS or pDA‐MNOF‐treated N2a cells after being treated with an 8 h OGD. N2a cells cultured in regular condition were set as the control. Scale bars, 50 µm. b) Quantification of percentages of DCFH‐DA positive (DCFDA + ) cells in indicated groups in (a) ( n = 5). Data were presented with mean ± s.d.; *, p < 0.05; ****, p < 0.0001; ANOVA. c) Flow cytometric analysis of DCFH‐DA positive cell proportions in the three groups. d) Fluorescence intensity detection in cell lysis of DCFH‐DA‐stained N2a cells in indicated groups ( n = 5). Data were presented with mean ± s.d.; *, p < 0.05; ***, p < 0.001; ANOVA. e) Schematic illustration of determining the contribution ratios of two effects of pDA‐MNOF on scavenging ROS. Briefly, N2a cells were treated with PBS, or 12.5 µg mL −1 pDA‐MNOF for 12 h, and their SOD‐like activities were determined as A 0 and A 1 , respectively. Cell lysis of pDA‐MNOF‐treated N2a cells was subject to ultracentrifugation for removing the residual pDA‐MNOF, and the SOD‐like activity was determined as A 3 . f) Quantification of contribution ratios of two effects of pDA‐MNOF on scavenging ROS. The formula was listed to the right. g) Live & Dead staining for PBS or pDA‐MNOF‐treated N2a cells after an 8 h OGD followed by a 16 h regular incubation. Scale bars, 100 µm. h) Quantification of percentages of dying cells in indicated groups in (g) ( n = 5). Data were presented with mean ± s.d.; ****, p < 0.0001; ANOVA. i) Cell viability of N2a cells in indicated groups in CCK‐8 assay ( n = 5). Data were presented with mean ± s.d.; **, p < 0.01; ****, p < 0.0001; ANOVA. j) Schematics of pDA‐MNOF utilizing nanozyme activity and upregulating endogenous HO1/SOD2, jointly scavenging ROS.

Article Snippet: To assess the protein levels of VEGF, the supernatant of N2a cells that were treated with PBS or various concentrations of pDA‐MNOF for 24 h was collected for ELISA detection (Elabscience, China).

Techniques: Staining, Cell Culture, Control, Fluorescence, Lysis, Activity Assay, Incubation, CCK-8 Assay

Journal: Advanced Science

Article Title: A Bioinspired Manganese‐Organic Framework Ameliorates Ischemic Stroke through its Intrinsic Nanozyme Activity and Upregulating Endogenous Antioxidant Enzymes

doi: 10.1002/advs.202206854

Figure Lengend Snippet: pDA‐MNOF promotes angiogenesis. a) Schematics showing VEGF, a downstream gene of STAT3 signaling, was activated by pDA‐MNOF. b) The mRNA levels of VEGF in N2a cells treated with PBS (control) or pDA‐MNOF ( n = 3). Data were presented with mean ± s.d.; **, p < 0.01; ****, p < 0.0001; ANOVA. c) The levels of VEGF protein secreted from the PBS (control) or pDA‐MNOF‐treated N2a cells ( n = 3). Data were presented with mean ± s.d.; ***, p < 0.001; ****, p < 0.0001; ANOVA. d) Capillary‐like tube formation assay performed on C166 cells (murine endothelial cell line) that were cultured with regular N2a culture medium or conditioned medium obtained from the pDA‐MNOF‐treated N2a cells. 5pDA‐MNOF, 12.5pDA‐MNOF, or 25pDA‐MNOF represent conditioned medium that was from the 5, 12.5, or 25 µg mL −1 pDA‐MNOF‐treated N2a cells, respectively. The branch points were indicated by red arrowheads, and the structure between two adjacent arrowheads was a branch. e) Quantification of branch point numbers, tube length, and tube numbers in capillary‐like tube formation assay in (d) ( n = 5 repeats). f) Schematics of cerebral ventricle injection of PBS and pDA‐MNOF for further evaluating in vivo angiogenesis. g) Relative VEGF mRNA levels in brain tissue of the MCAo mice receiving cerebral ventricle injection of PBS or pDA‐MNOF ( n = 9). The mice received sham operation were set as control ( n = 3). Data were presented with mean ± s.d.; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ANOVA. h) Representative fluorescence images of vessels (CD31, red) and astrocytes (GFAP, green) in the infarct (an asterisk) and peri‐infarct 4 weeks after the mice received cerebral ventricle injection of PBS or pDA‐MNOF. The boundary between the infarct and peri‐infarct was indicated by white dashed lines. i,j) Quantification of percentages of CD31 positive (CD31 + ) area in i) the infarct and j) peri‐infarct, respectively ( n = 9). Data were presented with mean ± s.d.; *, p < 0.05; **, p < 0.01; ANOVA.

Article Snippet: To assess the protein levels of VEGF, the supernatant of N2a cells that were treated with PBS or various concentrations of pDA‐MNOF for 24 h was collected for ELISA detection (Elabscience, China).

Techniques: Control, Capillary Tube Formation Assay, Cell Culture, Injection, In Vivo, Fluorescence

Journal: Journal of Alzheimer's Disease

Article Title: Halting of Caspase Activity Protects Tau from MC1-Conformational Change and Aggregation

doi: 10.3233/JAD-150960

Figure Lengend Snippet: Correlation between total tau and conformationally-changed MC1-tau levels. A–C) ELISA assay analyses of cell lysates from N2a cells transiently transfected with increasing amounts of 2N4R tau cDNA or mock transfected controls. Relative amounts of total tau measured in DA9/CP27 ELISA (A), conformationally changed tau measured in MC1/CP27 ELISA (B), and correlation of mean values from the two (C). Linear regression with variable slope analyses were used to calculate relative amounts of total tau and conformationally-changed tau from standard curves made of purified PHFs. A 50μg and 0.625μg of cell lysates were loaded per well for detection of MC1-tau and total tau, respectively. Amounts of total tau and conformationally-changed tau are expressed as mean values for six individual data sets. A.U., arbitrary units. D–F) Fluorescence micrographs of N2a cells immunostained for total tau with CP27 antibody (D), conformationally-changed tau with MC1 antibody (E), nuclear staining and merged MC1 and CP27-stained images (F).

Article Snippet: N2a cell medium was subjected to the CyTox 96 ® Non-Radioactive cytotoxicity assay.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Purification, Fluorescence, Staining

Journal: Journal of Alzheimer's Disease

Article Title: Halting of Caspase Activity Protects Tau from MC1-Conformational Change and Aggregation

doi: 10.3233/JAD-150960

Figure Lengend Snippet: Effects of tau expression on stress-related cellular events. A) Relative amounts of total tau in DA9/CP27 ELISA in lysates from N2a cells transiently transfected with 2N4R tau cDNA for 24 and 48 h. B) Lactate dehydrogenase (LDH) release monitored in medium from N2a cells transiently transfected with 2N4R tau cDNA for 24 and 48 h, and mock transfected controls. C) Results of AlphaScreen assays for D421-caspase cleaved tau and (D, E) phospho-tau corrected to total tau levels in N2a cells expressing 2N4R tau for 24 and 48 h. Linear regression with variable slope analyses were used to calculate relative amounts of total tau from standard curves made of purified PHFs. Relative amounts of D421-caspase cleaved tau were calculated from standard curves made of N2a cell lysates transiently expressing 2N4R tau and treated with staurosporine. Amounts of total tau, D421-tau, pS202 and pS396/pS404 are expressed as mean values of six individual datasets from three independent experiments. Statistical analyses were done using one-way ANOVA with Dunnett’s test or unpaired t test. A.U., arbitrary units.

Article Snippet: N2a cell medium was subjected to the CyTox 96 ® Non-Radioactive cytotoxicity assay.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Amplified Luminescent Proximity Homogenous Assay, Purification

Journal: Journal of Alzheimer's Disease

Article Title: Halting of Caspase Activity Protects Tau from MC1-Conformational Change and Aggregation

doi: 10.3233/JAD-150960

Figure Lengend Snippet: Induction of caspase activation and cleavage of tau at D421 by treatment of N2a cells with staurosporine. A) N2a cells transiently transfected with 2N4R tau cDNA and treated with 0.0625-1μM staurosporine. Caspase activation was detected using a fluorochrome-conjugated pan-caspase inhibitor (SR-VAD-FMK) in a GUAVA PCA96 flow cytometer. Membrane structural integrity was detected by the cell impermeable dye 7-AAD. Cells with low, moderate or high levels of caspase activation are depicted as filled circles, filled squares and filled triangles respectively. Dead cells with no active caspases present and permeable membranes are depicted as open circles. Each point is the mean value of three independent experiments. B) Induction of caspase-3 and caspase-6 was confirmed in two independent experiments in fluorescence activated cell sorting (FACS). N2a cells transiently transfected with 2N4R tau with or without staurosporine treatment and immunolabeled with active caspase-3 or active caspase-6 antibodies in a total population of 100,000 cells for each group. C) Amounts of D421-caspase cleaved tau corrected to total tau levels measured by ELISA in N2a cells transiently transfected with 2N4R tau cDNA and treated with 0.0625–1μM staurosporine. Linear regression with variable slope analyses was used to calculate relative amounts of total tau from standard curves based on purified PHFs. Relative amounts of D421-caspase cleaved tau were calculated from standard curves derived from N2a cell lysates transiently expressing 2N4R tau and treated with staurosporine. Mean values of six individual datasets from three independent experiments are presented. (D) Fluorescence micrographs of N2a cells transiently transfected with 2N4R tau with or without treatment with 0.158μM staurosporine and immunostained for D421 caspase-cleaved tau.

Article Snippet: N2a cell medium was subjected to the CyTox 96 ® Non-Radioactive cytotoxicity assay.

Techniques: Activation Assay, Transfection, Flow Cytometry, Membrane, Fluorescence, FACS, Immunolabeling, Enzyme-linked Immunosorbent Assay, Purification, Derivative Assay, Expressing

Journal: Journal of Alzheimer's Disease

Article Title: Halting of Caspase Activity Protects Tau from MC1-Conformational Change and Aggregation

doi: 10.3233/JAD-150960

Figure Lengend Snippet: Dose-dependent increase in levels of conformationally-changed tau after treatment with staurosporine. Levels of total tau and conformationally-changed tau were measured in cell lysates of N2a cells transiently transfected with 2N4R tau cDNA and treated with 0.0625–1μM staurosporine. Relative amounts of total tau in DA9/CP27 ELISA (A), relative amounts of conformationally-changed tau levels measured by MC1/CP27 ELISA (B), levels of conformationally-changed tau corrected for total tau levels C). Linear regression with variable slope analyses were used to calculate relative amounts of total tau and conformationally-changed tau from standard curves derived from purified PHFs. Amounts of total tau and conformationally-changed tau are expressed as mean values of six individual data sets relative to non-treated controls set to unity. Statistical analyses for the relative amounts of conformationally-changed tau using one-way ANOVA with Dunnett’s test confirmed significant differences between the values obtained for 0.25 ( p < 0.5), 0.5 ( p < 0.001), and 1 ( p < 0.001) μM staurosporine when compared to untreated controls. A.U., arbitrary units. D) Fluorescence micrographs of N2a cells immunostained for total tau with CP27 antibody, conformationally-changed tau with MC1 antibody, and merged with nuclear stained images.

Article Snippet: N2a cell medium was subjected to the CyTox 96 ® Non-Radioactive cytotoxicity assay.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Derivative Assay, Purification, Fluorescence, Staining

Journal: Journal of Alzheimer's Disease

Article Title: Halting of Caspase Activity Protects Tau from MC1-Conformational Change and Aggregation

doi: 10.3233/JAD-150960

Figure Lengend Snippet: Time-course of caspase activation and induction of conformationally-changed tau after treatment of N2a cells with staurosporine. Caspase activation in N2a cells transiently transfected with the 2N4R tau cDNA alone (A) and treated with 1μM staurosporine (D). Samples were collected at time zero, 6 h, 12 h, and 24 h. Caspase activation was detected using a fluorochrome-conjugated pan-caspase inhibitor (SR-VAD-FMK) in a GUAVA PCA96 flow cytometer. Membrane structural integrity was detected by the cell impermeable dye 7-AAD. Cells with low, moderate, or high levels of caspase activation correspond to filled circles, filled squares, and filled triangles, respectively. Dead cells with no active caspases present and permeable membranes are marked as open circles (A, D). Relative amounts of total tau in DA9/CP27 ELISA in control lysates (B) and after treatment with 1μM staurosporine (E). Relative amounts of conformationally-changed tau levels measured by MC1/CP27 ELISA in control lysates (C) and after treatment with 1μM staurosporine (F). Linear regression with variable slope analyses was used to calculate relative amounts of total tau and conformationally-changed tau from standard curves obtained from purified PHFs. Each point represents the mean value of six independent samples. A.U., arbitrary units.

Article Snippet: N2a cell medium was subjected to the CyTox 96 ® Non-Radioactive cytotoxicity assay.

Techniques: Activation Assay, Transfection, Flow Cytometry, Membrane, Enzyme-linked Immunosorbent Assay, Control, Purification

Journal: Journal of Alzheimer's Disease

Article Title: Halting of Caspase Activity Protects Tau from MC1-Conformational Change and Aggregation

doi: 10.3233/JAD-150960

Figure Lengend Snippet: Fractionation of tau in N2a cell lysates after treatment with staurosporine. A) Lysates from N2a cells transiently transfected with 2N4R tau cDNA and treated with 0.03125-1μM staurosporine subjected to low speed centrifugation (2600× g ) followed by a high speed centrifugation (100,000× g ) in the presence of Nonidet P40, as described in Materials and Methods. The low speed (LS), soluble (S1), and pelleted (P1) fractions after the high speed centrifugation step were resolved by SDS-PAGE. Total tau was detected by western blot with DA9 antibody. B) Absorbance signals in DA9/CP27 and MC1/CP27 ELISA formats for total tau and aggregated MC1-tau, respectively, for low-speed (LS), soluble (S1), and pelleted (P1) fractions of N2a cell lysates after treatment with 0.5μM staurosporine. C) Fluorescence micrographs of N2a cells transfected with 2N4R tau cDNA, treated with 0.25–0.5μM staurosporine and immunostained for total tau with CP27 antibody, aggresome detection reagent and merged with nuclear stained images. Accumulation of tau in aggresomes indicated by arrows.

Article Snippet: N2a cell medium was subjected to the CyTox 96 ® Non-Radioactive cytotoxicity assay.

Techniques: Fractionation, Transfection, Centrifugation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Fluorescence, Staining

Journal: Journal of Alzheimer's Disease

Article Title: Halting of Caspase Activity Protects Tau from MC1-Conformational Change and Aggregation

doi: 10.3233/JAD-150960

Figure Lengend Snippet: Timing of proteolytic processing of tau and its aggregation, and the effect of caspase inhibitors on tau integrity and D421-cleavage. A, B) Combined time-course and staurosporine dose-response in N2a cells transiently expressing 2N4R tau. The cells were treated with 0-1μM staurosporine for 6, 12, or 24 h marked in black, grey, and white, respectively. Relative levels of D421-tau (A) and MC1-tau (B) corrected to total tau were measured in ELISA assays. C) N2a cells transiently expressing 2N4R tau were treated with staurosporine (0.125, 0.25, and 0.5μM) or staurosporine and pan-caspase inhibitor Z-VAD(Ome)-FMK (50μM) for either 6 or 24 h. Controls with no treatment are shown as NT. Cell lysates were resolved in SDS-PAGE and tau protein was detected by western blot with DA9 antibody. Data represent three independent experiments. Results of AlphaScreen assays for total tau (D) and D421-caspase cleaved tau corrected to total tau levels (E) in N2a cells transiently transfected with 2N4R tau and treated with 0.158μM and 0.5μM staurosporine (STS) or staurosporine and 50μM of pan-caspase inhibitor (ApoBlock) for 24 h. Untreated controls are shown as NT. Linear regression with variable slope analyses were used to calculate relative amounts of total tau and D421-caspase cleaved tau for A, B, D, and E from standard curves based on N2a cell lysates transiently expressing 2N4R tau and treated with STS. Relative amounts of D421-caspase cleaved tau were corrected to total tau. Amounts of total tau and D421-cleaved tau are expressed as mean values for five individual data sets relative to untreated controls set to unity. Statistical analyses for the relative amounts of total tau and Asp421-tau using one-way ANOVA with multiple comparisons. A.U., arbitrary units.

Article Snippet: N2a cell medium was subjected to the CyTox 96 ® Non-Radioactive cytotoxicity assay.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Amplified Luminescent Proximity Homogenous Assay, Transfection

Journal: Journal of Alzheimer's Disease

Article Title: Halting of Caspase Activity Protects Tau from MC1-Conformational Change and Aggregation

doi: 10.3233/JAD-150960

Figure Lengend Snippet: Protective effect of pan-caspase inhibitor on aggregation of tau. N2a cells were transiently transfected with 2N4R tau and treated with 0.5μM staurosporine (STS) or staurosporine and 50μM pan-caspase inhibitor (ApoBlock) for 24 h. Untreated controls are shown as NT. Cell lysates were analyzed in ELISA for total tau (A) and conformationally-changed MC1-tau (B). Linear regression with variable slope analyses were used to calculate relative amounts of total tau and conformationally-changed tau from standard curves based on purified PHFs. Relative amounts of conformationally-changed were corrected for total tau. Amounts of total tau and conformationally-changed tau are expressed as mean values of six individual datasets relative to untreated controls set to unity. Statistical analyses for the relative amounts of total tau and D421-tau using one-way ANOVA with multiple comparisons. A.U. arbitrary units.

Article Snippet: N2a cell medium was subjected to the CyTox 96 ® Non-Radioactive cytotoxicity assay.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Purification