Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: ODN 2088 modulates the release of chemokines by SC astrocytes, in vitro. a Representative chemokine arrays used to detect chemokines in CM of vehicle- and ODN 2088-treated astrocytes. The chemokine arrays were independently repeated twice, showing similar results. Results from a representative experiment are shown. The dots enclosed in rectangular boxes show chemokines whose levels were decreased (> 5% difference) in the CM of ODN 2088-treated astrocytes compared to the CM of vehicle-treated astrocytes. The dots enclosed in the oval box show the chemokine whose levels were increased (> 5% difference) in the CM of ODN 2088-treated astrocytes compared to CM of vehicle-treated astrocytes. 1: CCL1; 2: CCL9/MIP-1γ; 3: CCL2/MCP-1; 4: CCL20/MIP-3α; 5: CX3CL1. b Densitometric quantification of the signal obtained in the chemokine array using the Image Lab software (Bio-Rad). c Quantification of CCL9 levels in CM obtained from ODN 2088- or vehicle-treated astrocytes [** p < 0.01, independent-sample t -test, two-tailed]. The experiment was independently repeated four times, and the mean of 4 experiments ( n = 4) is shown. d Quantification of CCL2 levels in CM obtained from ODN 2088- or vehicle-treated astrocytes [* p < 0.05, independent-sample t -test, two-tailed]. The experiment was independently repeated three times, and the mean of 3 experiments ( n = 3) is shown

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: In Vitro, Software, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: CCL1 released by ODN 2088-treated astrocytes mediates the chemotaxis of peritoneal macrophages. Quantification of F4/80 + cells that crossed to the lower surface of the membrane in response to CM derived from vehicle- or ODN 2088-treated TLR9 astrocytes in the absence or presence of CCL1 neutralizing antibody or IgG 2A isotype control [ F (5, 48) = 81.03, p < 0.0001 by one-way ANOVA, **** p < 0.0001 by Tukey’s post hoc test]. The results of three independent experiments ( n = 3) are shown. Data are presented as mean ± SEM

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Chemotaxis Assay, Membrane, Derivative Assay, Control

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: Astrocyte-derived CCL2 and CCL9 but not CCL1 regulate macrophage polarization, in vitro. a Macrophage cultures were exposed to ODN 2088-treated astrocyte CM (ODN 2088-CM), in the absence or presence of CCL1 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [ p = 0.7228, independent-sample t -test, two-tailed]. b Macrophage cultures were exposed to vehicle-treated astrocyte CM, in the absence or presence of CCL2 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [** p < 0.01, independent-sample t -test, two-tailed]. c Macrophage cultures were exposed to vehicle-treated astrocyte CM, in the absence or presence of CCL9 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [*** p < 0.001, independent-sample t -test, two-tailed]. d Macrophage cultures were exposed to vehicle-treated astrocyte CM (Veh-CM) or ODN 2088-treated astrocyte CM (ODN 2088-CM) for 24 h, with or without (control) addition of rmCCL9 (20 pg/ml). The graph shows the quantification of the F4/80 + /Arg-1 + double-labeled cells expressed as percent of total F4/80 + cells in macrophage cultures [ F (2, 6) = 53.68, p < 0.0001 by one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 by Tukey’s post hoc test]. The experiments were independently repeated twice, yielding similar results. Results from a representative experiment are shown. Results obtained from additional biological repeats of these experiments can be found in Additional file D-G. Data are presented as mean ± SEM

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Derivative Assay, In Vitro, Two Tailed Test, Control, Labeling

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: A scheme summarizing the effects of ODN 2088-treated astrocytes on macrophages. TLR9 antagonism increases the release of CCL1 by astrocytes, which enhances macrophage chemotaxis. In contrast, CCL2 and CCL9 release are decreased in response to ODN 2088. This reduces the negative regulatory effect of CCL2 and CCL9 on M2 macrophage polarization and fosters the M2 phenotype

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Chemotaxis Assay

Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: ( a, b ) DOCK7 (TMW = 239 kDa, a ) and DNMT1 (TMW = 183 kDa, b ) protein interaction validated by DNMT1 co-immunoprecipitation and Western blot analysis by using a specific antibody against DOCK7 and DNMT1 in E14.5 cortical lysates. N = 3 experiments. + = DNMT1-antibody pulldown, - = IgG-antibody pulldown. ( c ) Representative images of N2a cells (grown for 48 h) co-stained with conjugated antibodies directed against DNMT1 (red) and DOCK7 (green), additionally stained with DAPI (blue), and captured by high-resolution STED microscopy. Scale bars: 5 µm. The white squares depict the magnification of the merge. Scale bars: 2 µm. Overlapping volumes are highlighted by white arrowheads. ( d, e ) Analysis of cytosolic colocalization between DNMT1 and DOCK7 by Pearson’s correlation coefficient (PCC) ( d ) and the percentage of colocalized volumes ( e ) in comparison to the corresponding rotated and randomized controls. n (ROIs) = 133; N (cells) = 45. ( f ) Predicted interaction between DNMT1 and DOCK7 using MD simulations and docking methods (left figure). Specific hot spot residues for the interaction between DNMT1 and DOCK7 are represented in the right figure. Hydrogen bonding and salt bridge interactions are shown as black dashes. ( g, h ) Inverted microphotographs of exemplary βIII-tubulin immunocytochemically stained cortical neurons (E14.5 + 2 DIV) transfected with control ( g ) or Dock7 ( h ) siRNA at 1 DIV for 24 h. Scale bars: 20 µm. ( i-l ) Analysis of morphological parameters, such as the length of the longest process ( i ), the number of processes ( j ), the branches per length summed across all processes likely representing dendrites ( k ), and the branches normalized to the longest process length likely representing axons ( l ). n (Ctrl siR) = 207 cells; n ( Dock7 siR) = 198 cells. N = 3 experiments. Two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Data are presented as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Immunoprecipitation, Western Blot, Staining, Microscopy, Comparison, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: (a) Representative STED micrographs of a fixed N2a cell cultured for 48 h, labeled with MitoTracker™ Deep Red FM (Mito., false color green), DAPI (blue), and an antibody against DOCK7 (red). Scale bar: 5 µm. The white box indicates the magnified region shown in the merge (Scale bar: 2 µm). Nuclear signals were computationally removed to improve visualization of the cytosolic compartment. (b) Quantification of cytosolic colocalization between DOCK7 and mitochondria by Pearson’s correlation coefficient (PCC) compared to a rotated randomized control. n (ROIs) = 45; N (cells) = 15. Two-tailed Student’s t -test, p < 0.0001. (c) Representative images of cortical neurons (P0 + 4 DIV) co-transfected at DIV 3 with Alexa Fluor™ 555–labeled control siRNA (red) and MT-GFP plasmid (green) and imaged 24 h post-transfection. Scale bar: 10 µm. (d) Exemplary tracking of MT-GFP–labeled mitochondria in cortical neurons following control, Dnmt1 , or Dock7 siRNA treatment. Non-motile mitochondria are marked by white arrowheads, motile mitochondria by colored stars (each color indicates one mitochondrion). White boxes in overview images (first panel) mark the magnified regions shown over time. The last frame (sixth panel) depicts temporally color-coded mitochondrial trajectories. Scale bars: 5 µm. (e) Representative kymographs illustrating mitochondrial motility under the respective conditions. (f) Quantification of the ratio of non-motile to motile mitochondria in cortical neurons (P0 + 4 DIV) following control, Dnmt1 , or Dock7 siRNA-mediated knockdown for 24 h at DIV 3. N (Ctrl siR) = 35 ROIs; n ( Dnmt1 siR) = 39 ROIs; n ( Dock7 siR) = 33 ROIs. N = 3 independent experiments. Kruskal–Wallis test followed by Dunn’s post hoc multiple comparison test, p < 0.01, p < 0.001. Data are shown as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Cell Culture, Labeling, Control, Two Tailed Test, Transfection, Plasmid Preparation, Knockdown, Comparison

Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: ( a, b ) Live cell imaging analysis capturing mitochondria accumulation using the MitoTracker TM Deep Red FM prior to branch formation in N2a cells 24 h after control, Dnmt1 , or Dock7 siRNA transfection. The white squares in the first panel indicate the magnified regions shown for each time frame. (a) Inverted grayscale images of mitochondria accumulation at branch initiation sites, Scale bars: 10 µm. The MitoTracker TM integrated density, normalized to the corresponding integrated density of the same position in the first frame, at prospective branchpoints is shown in ( b ). n (Ctrl siR) = 22 cells with 54 events; n ( Dnmt1 siR) = 13 cells with 24 events; n ( Dock7 siR) = 21 cells with 43 events. N = 4 experiments. Two-way ANOVA followed by Tukey’s post-hoc multiple comparison test, ** p < 0.01. ( c, d ) Analysis of the branch formation time ( c ) and exemplary tracking of a branching event (from the first mitochondria accumulation puncta to the formation of the branch) ( d ). The white squares in the first panel indicate the magnified regions shown for each time frame. Scale bars: 20 µm. n (Ctrl siR) = 22 cells with 54 events; n ( Dnmt1 siR) = 13 cells with 24 events; n ( Dock7 siR) = 21 cells with 43 events. N = 4 experiments. One-way ANOVA followed by Dunnett’s post-hoc multiple comparison test, ** p < 0.01, **** p < 0.0001. Data are presented as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Live Cell Imaging, Control, Transfection, Comparison

Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: ( a, b ) Microphotographs of cortical neurons ( a, Scale bars: 20 µm) and N2a cells ( b, Scale bars: 40 µm), which were transfected with control, Dnmt1 , or Dock7 siRNA for 24 h after growing for one day, co-stained for DAPI (blue), ßIII-tubulin (TUBB3, green), and acetylated tubulin (AcTUB, magenta). ( c, d ) Analysis of the integrated density for the acetylated tubulin (AcTUB) normalized to the βIII-tubulin (TUBB3) integrated density after the respective knockdown in cortical neurons ( c ) and N2a cells ( d ). N = 3 experiments. One-way ANOVA followed by Dunnett’s post-hoc multiple comparison test, * p < 0.05, **** p < 0.0001. ( e, f ) Analysis of the acetylated tubulin (AcTUB) integrated density normalized to the βIII-tubulin (TUBB3) integrated density after the RG108 inhibitor treatment in cortical neurons ( e ) and N2a cells ( f ). N = 3 experiments. Two-tailed Student’s t-test, * p < 0.05. For ( c, e ): n (Ctrl siR) = 314 cells; n ( Dnmt1 siR) = 263 cells; n ( Dock7 siR) = 277 cells; n (DMSO) = 365 cells; n (RG108) = 358 cells. For ( d, f ): n (Ctrl siR) = 279 cells; n ( Dnmt1 siR) = 117 cells; n ( Dock7 siR) = 175 cells; n (DMSO) = 506 cells; n (RG108) = 525 cells. ( g, h ) Western blots revealing protein bands of STMN1 ( g , TMW = 17 kDa) and the phosphorylated version of STMN1 ( h , S16-P) (TMW = 17 kDa) in cortical single cell lysates (E14.5 + 2 DIV) treated previously with control, Dnmt1 , or Dock7 siRNA at 1 DIV for 24 h. γ-tubulin ( g , h , TUBG1, TMW = 51 kDa) was used as a housekeeper. ( i, j ) Analysis of the mean grey value of STMN1 normalized against TUBG1 ( i ) and the mean grey value of the phosphorylated version of STMN1 (S16-P) ( j ) normalized against STMN1. N = 3 experiments. One-way ANOVA followed by Dunnett’s post-hoc multiple comparison test, * p < 0.05, ** p < 0.01. Data are presented as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Transfection, Control, Staining, Knockdown, Comparison, Two Tailed Test, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Serine/Threonine‐Protein Kinase 3 Facilitates Myocardial Repair After Cardiac Injury Possibly Through the Glycogen Synthase Kinase‐3β/β‐Catenin Pathway

doi: 10.1161/JAHA.121.022802

Figure Lengend Snippet: A , SGK3, phosphorylated (p‐) proteins of SGK3 Thr320 in neonatal cardiomyocytes transfected with Ad5:cTNT‐CON or Ad5:cTNT‐SGK3 were detected by Western blot. B through E , cardiomyocytes isolated from 100 P1 mice were transfected with Ad5:cTNT‐CON or Ad5:cTNT‐SGK3, and immunofluorescence staining was then used to evaluate the cardiomyocyte proliferation for EDU + (5639 cardiomyocytes in the Ad5:cTNT‐SGK3 group and 5996 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( B ); Ki67 + (6511 cardiomyocytes in the Ad5:cTNT‐SGK3 group and 6852 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( C ); pH3 + (8391 cardiomyocytes in the Ad5:SGK3 group and 7121 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( D ); and Aurora B + (8503 cardiomyocytes in the Ad5:SGK3 group and 14 162 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( E ). F , SGK3 expression in neonatal cardiomyocytes transfected with Ad5:cTNT‐SGK3i or Ad5:cTNT‐CONi were analyzed by Western blot analysis. G through I , Immunofluorescence staining of cardiomyocytes isolated from 100 P1 mice transfected with Ad5:cTNT‐SGK3i or Ad5:cTNT‐CONi and quantification of EDU + (4716 cardiomyocytes in the Ad5:SGK3i group and 4736 cardiomyocytes in the Ad5:cTNT‐CONi group, n=6 ( G ); Ki67 + (6388 cardiomyocytes in the Ad5:cTNT‐SGK3i group and 9583 cardiomyocytes in the Ad5:cTNT‐CONi group, n=6 ( H ); and pH3 + (5642 cardiomyocytes in the Ad5:cTNT‐SGK3i group and 6576 cardiomyocytes in the Ad5:cTNT‐CONi group, n=6 ( I ). J and K , Cell flow cytometry was performed to detect the cell cycle of cardiomyocytes after SGK3 overexpression or inhibition. L , Terminal deoxynucleotidyl transferase‐mediated dUTP in situ nick end labeling (TUNEL) staining was performed to evaluate the effect of Ad5:cTNT‐SGK3 on oxygen glucose deprivation/reoxygenation (OGD/R)‐induced neonatal mouse cardiomyocyte apoptosis (3657 cardiomyocytes in the Ad5:cTNT‐CON group, 3017 cardiomyocytes in the Ad5:cTNT‐SGK3 group, 2049 cardiomyocytes in the OGD/R+Ad5:cTNT‐CONi group, and 1303 cardiomyocytes in the OGD/R+Ad5:cTNT‐SGK3 group, n=6). The cells indicated by the arrows are immunofluorescence‐positive cardiomyocytes. Data are presented as mean±SEM. * P ≤0.05; ** P ≤0.01; *** P ≤0.001. Ad5:cTNT‐CON indicates control adenovirus serotype 5; Ad5:cTNT‐CONi, knockdown control adenovirus serotype 5; Ad5:cTNT‐SGK3, cardiomyocyte‐specific SGK3 overexpression adenovirus serotype 5; Ad5:cTNT‐SGK3i, cardiomyocyte‐specific SGK3 knockdown adenovirus serotype 5; CON, control; CONi, knockdown control; cTNT, cardiac troponin T; EDU, 5‐ethynyl‐2′‐deoxyuridine; G1, G1 phase; G2/M, G2/M phase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; ki67+, ki67 positive; NS, no significance; P1, postpartum day 1; ph3+:phospho‐histone H3 positive; S, S phase; and SGK3i, serine/threonine‐protein kinase 3 knockdown.

Article Snippet: To identify cell‐cycle activities and cytokinesis, the Click‐iT EdU Imaging Kits (Thermo Fisher), anti‐Ki67 antibody (Abcam; ab16667), anti‐Aurora B antibody (Abcam; ab2254), and anti‐phosphorylated‐histone 3 (pH3) antibodies (CST; 9701) were used to culture sections and formaldehyde‐fixed cardiomyocytes.

Techniques: Transfection, Western Blot, Isolation, Immunofluorescence, Staining, Expressing, Flow Cytometry, Over Expression, Inhibition, In Situ, End Labeling, TUNEL Assay, Control, Knockdown

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Serine/Threonine‐Protein Kinase 3 Facilitates Myocardial Repair After Cardiac Injury Possibly Through the Glycogen Synthase Kinase‐3β/β‐Catenin Pathway

doi: 10.1161/JAHA.121.022802

Figure Lengend Snippet: A , Experimental pattern: myocardium targeting SGK3 overexpression of adeno‐associated virus serotype 9 (AAV9:cTNT‐SGK3) or control (AAV9:cTNT‐CON) was intraperitoneally injected into P1 mice, and intraperitoneally injected EDU solution at P8, P10, and P12, and the hearts were then collected at P14 for relevant experiments. B through E , After 14 days treatment of AAV9:cTNT‐SGK3 or AAV9:cTNT‐CON in P1 mice, immunofluorescence staining was then performed at P14 to evaluate the cardiomyocyte proliferation for EDU + (12 565 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 12 678 cardiomyocytes in the AAV9:cTNT‐CON group, n=6 ( B ); pH3 + (10 067 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 5788 cardiomyocytes in the AAV9:cTNT‐CON group, n=6 ( C ); Ki67 + (9908 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 10 241 cardiomyocytes in the AAV9:cTNT‐CON group, n=6 ( D ); and Aurora B + (18 132 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 17 707 cardiomyocytes in the AAV9:cTNT‐CON group, n=6 ( E ). F , Terminal deoxynucleotidyl transferase‐mediated dUTP in situ nick end labeling (TUNEL) staining was used to evaluate the effect of AAV9:cTNT‐SGK3 or AAV9:CTNT‐CON on cardiomyocyte apoptosis in mice at P14 (5723 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 5457 cardiomyocytes in the AAV9:cTNT‐CON group, n=6). G , Heart weight/body weight (HW/BW) ratio and cardiac morphology between AAV9:cTNT‐SGK3 (n=5) and AAV9:cTNT‐CON mice (n=5) at P14. H , Survival rate was analyzed by between AAV9:cTNT‐CON (n=10) and AAV9:cTNT‐SGK3 (n=10). I , Wheat germ agglutinin (WGA) immunofluorescence was used to detect the cardiomyocyte size between AAV9:cTNT‐SGK3 and AAV9:cTNT‐CON mice at P14 (14 687 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 14 582 cardiomyocytes in the AAV9:cTNT‐CON group, n=6). Data are presented as mean±SEM. *** P ≤0.001. CON indicates control; cTNT, cardiac troponin T; EDU, 5‐ethynyl‐2′‐deoxyuridine; i.p., intraperitoneal; ki67+, ki67 positive; NS, no significance; P1, postpartum day 1; P14, postnatal day 14; and pH3+, phospho‐histone H3 positive.

Article Snippet: To identify cell‐cycle activities and cytokinesis, the Click‐iT EdU Imaging Kits (Thermo Fisher), anti‐Ki67 antibody (Abcam; ab16667), anti‐Aurora B antibody (Abcam; ab2254), and anti‐phosphorylated‐histone 3 (pH3) antibodies (CST; 9701) were used to culture sections and formaldehyde‐fixed cardiomyocytes.

Techniques: Over Expression, Virus, Control, Injection, Immunofluorescence, Staining, In Situ, End Labeling, TUNEL Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Serine/Threonine‐Protein Kinase 3 Facilitates Myocardial Repair After Cardiac Injury Possibly Through the Glycogen Synthase Kinase‐3β/β‐Catenin Pathway

doi: 10.1161/JAHA.121.022802

Figure Lengend Snippet: A , Experimental pattern: adenovirus serotype 5 SGK3i (Ad5:cTNT‐SGK3i) or control (Ad5:cTNT‐CONi) was injected into border myocardium after apical resection (AR) in P1 mice using microinjector. EDU solution was then intraperitoneally injected at 4 days post resection (dpr). Finally, the cardiomyocyte proliferation was detected at 6 dpr, and cardiac function was evaluated at 22 dpr. B and C , Cardiac function of ejection fraction and fractional shortening between Ad5:cTNT‐SGK3i or Ad5:cTNT‐CONi treated mice at 1 and 22 days after AR (Ad5:cTNT‐SGK3i group, n=13; Ad5:cTNT‐CONi, n=13). D through F , Immunofluorescence staining was used to evaluate the cardiomyocyte proliferation between Ad5:cTNT‐SGK3i– or Ad5:cTNT‐CONi–treated mice at P6 for EDU + (11 873 cardiomyocytes in the Ad5:cTNT‐SGK3i group and 11 755 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( B ); pH3 + (15 210 cardiomyocytes in the Ad5:cTNT‐SGK3i group and 15 084 cardiomyocytes in the Ad5:cTNT‐CON group, n=6( C ); and Ki67 + (12 290 cardiomyocytes in the Ad5:cTNT‐SGK3i group and 13 119 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( D ). G , Masson staining of mouse ventricular cross‐sections between Ad5:cTNT‐SGK3i– or Ad5:cTNT‐CONi–treated mice at 22 days after AR (n=7 in each group). Data are presented as mean±SEM. * P ≤0.05; ** P ≤0.01; *** P ≤0.001. CON indicates control; CONi, knockdown control; cTNT, cardiac troponin T; EDU, 5‐ethynyl‐2′‐deoxyuridine; ki67+, ki67 positive; NS, no significance; P1, postpartum day 1; and pH3+, phospho‐histone H3 positive.

Article Snippet: To identify cell‐cycle activities and cytokinesis, the Click‐iT EdU Imaging Kits (Thermo Fisher), anti‐Ki67 antibody (Abcam; ab16667), anti‐Aurora B antibody (Abcam; ab2254), and anti‐phosphorylated‐histone 3 (pH3) antibodies (CST; 9701) were used to culture sections and formaldehyde‐fixed cardiomyocytes.

Techniques: Control, Injection, Immunofluorescence, Staining, Knockdown

Journal: Neuron

Article Title: Zika Virus Protease Cleavage of Host Protein Septin-2 Mediates Mitotic Defects in Neural Progenitors

doi: 10.1016/j.neuron.2019.01.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Flavivirus envelope protein, 4G2 clone 1 EMD Millipore MAB10216; RRID:AB_827205 Phopho-H3-Ser10 EMD Millipore 06–570; RRID:AB_310177 α-tubulin Santa Cruz sc-53030; RRID:AB_2272440 CEP63 EMD Millipore 06–1292; RRID:AB_10918481 active-Caspase-3 EMD Millipore AB3623; RRID:AB_303959 SEPT2 Proteintech 60075; RRID:AB_2187016 SEPT2 (N-terminal) Santa Cruz 20408; RRID:AB_2187133 SEPT2 (C-terminal) Abcam ab185998 SEPT7 Abcam ab158073 SEPT9 Thermo-Fisher PA5–13200; RRID:AB_10987482 GAPDH EMD Millipore MAB374; RRID:AB_2107445 NS2B GeneTex GTX124246; RRID:AB_11170698 NeuN Abcam ab104224; RRID:AB_10711040 Bacterial and Virus Strains Zika virus Strain FSS13025 WRCEVA FSS13025 Zika virus Strain MR-766 WRCEVA MR-766 Chemicals, Peptides, and Recombinant Proteins human SEPT2 Abcam ab99296 Dorsomorphin Tocris 3093 A8301 Tocris 2939 bFGF Thermo-Fisher PHG0261 Matrigel BD Biosciences 356234 Accutase EMD Millipore SCR005 TRIzol Thermo-Fisher 15596026 laminin Sigma L2020 puromycin Sigma P8833 Critical Commercial Assays Gibson Assembly® Master Mix New England Biolabs E2611S Q5® Site-Directed Mutagenesis Kit New England Biolabs E0554S Pierce GST Protein Interaction Pull-Down Kit Thermo-Fisher 21516 RNeasy Mini Kit QIAGEN 74104 SuperScript III First-Strand Synthesis System Thermo-Fisher 18080051 iTaq Universal SYBR® Green Supermix Bio-rad 1725120 Experimental Models: Cell Lines HEK293T ATCC CRL-3216; RRID:CVCL_0063 HeLa ATCC CCL-2; RRID:CVCL_0030 C6/36 ATCC CRL-1660; RRID:CVCL_Z230 H9 WiCell WA09; RRID:CVCL_9773 Recombinant DNA pCDH-ZIKV-NS2B3 This paper N/A pCDH-ZIKV-NS2B3-S135A This paper N/A pGEX-6P-ZIKV-NS2B3 ( Shiryaev et al., 2017 ) N/A pGEX-6P-ZIKV-NS2B3-S135A ( Shiryaev et al., 2017 ) N/A REAGENT or RESOURCE SOURCE IDENTIFIER pSG5-FLAG-ZIKV-NS3 ( Yoon et al., 2017 ) N/A pcDNA3.1(+)-SEPT2 This paper N/A pcDNA3.1(+)-SEPT2-R306A This paper N/A MISSION® shRNA-SEPT2 Sigma 004404 and 010891 MISSION® shRNA-SEPT7 Sigma 001788 and 009859 pEGFP-SEPT2 ( DeMay et al., 2011 ) N/A pEGFP-SEPT2-R306A This paper N/A pGEX-6P-SEPT2 This paper N/A pGEX-6P-SEPT2-R306A This paper N/A Software and Algorithms ImageJ NIH https://imagej.net/Fiji GraphPad Prism GraphPad https://www.graphpad.com String String https://string-db.org/cgi/input.pl Open in a separate window KEY RESOURCES TABLE Zika protease NS2B3 is a heterodimer that cleaves host proteins in hNPCs NS2B3 expression causes protease-dependent cytokinesis defects and apoptosis NS2B3 associates with the septin cytoskeleton and cleaves Septin-2 Non-cleavable Septin-2 partially restores cytokinesis in NS2B3-expressing cells

Techniques: Recombinant, Mutagenesis, SYBR Green Assay, shRNA, Software