Journal: International Journal of Molecular Sciences

Article Title: Mesenchymal Stem Cells Restore Endothelial Integrity and Alleviate Emotional Impairments in a Diabetic Mouse Model via Inhibition of MMP-9 Activity

doi: 10.3390/ijms26073355

Figure Lengend Snippet: Figure 2. DM mice exhibit synaptic deficits and increased neuroinflammation in the HIP. (A) Repre- sentative WB images showing the expression levels of the presynaptic protein synapsin I (SYN1) and the postsynaptic protein postsynaptic density protein 95 (PSD95) in the HIP of CTL and DM mice. β-actin was used as an internal control. (B) Semi-quantitative analysis of PSD95 and SYN1 expression levels from immunoblot experiments. n = 6. (C) Representative images of Iba1 immunostaining in the HIP of CTL and DM mice. The area within the dashed box is magnified and displayed in the insets. Scale bars: 100 µm (main images) and 20 µm (insets). (D) Quantitative analysis of Iba1-positive microglia in the HIP of CTL and DM mice. n = 5. (E) qPCR analysis of iNOS and Arg1 mRNA levels in the HIP of CTL and DM mice. n = 6. (F) qPCR analysis of IL-6, IL-1β, and TNF-α mRNA levels in the HIP of CTL and DM mice. n = 6. (G) ELISA measuring IL-6, IL-1β, and TNF-α levels in the serum of CTL and DM mice. n = 3. Data are presented as mean ± SEM. Statistical significance was determined using a one-tailed unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were blocked and incubated overnight with the following antibodies: PSD95 (SYSN, Toronto, ON, Canada, 124002, 1:1000); SYN1 (SYSN, 106011, 1:1000); Cldn5 (Invitrogen, 35-2500, 1:1000); Ocln (Invitrogen, 71-1500, 1:1000); and MMP9 (Boster, Shanghai, China; PB9669, 1:1000).

Techniques: Expressing, Control, Western Blot, Immunostaining, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Mesenchymal Stem Cells Restore Endothelial Integrity and Alleviate Emotional Impairments in a Diabetic Mouse Model via Inhibition of MMP-9 Activity

doi: 10.3390/ijms26073355

Figure Lengend Snippet: Figure 6. MSC treatment attenuated DM-induced neuroinflammation and synaptic deficits. (A) Representative immunofluorescence images of Iba1 staining in the HIP of mice from the CTL, MSC, DM, and DM+MSC groups. The dashed box indicates the magnified region shown in the insets. Scale bars: 100 µm (main images) and 20 µm (insets). (B) Quantitative analysis of Iba1+ microglia in the HIP. n = 4. (C) qPCR analysis of iNOS and Arg1 mRNA levels in the HIP of mice from the CTL, MSC, DM, and DM+MSC groups. n = 9. (D) qPCR analysis of mRNA levels of IL-6, IL-1β, and TNF-α in the HIP of mice from the CTL, MSC, DM, and DM+MSC groups. n = 9. (E) ELISA results showing serum levels of IL-6, IL-1β, and TNF-α in CTL, MSC, DM, and DM+MSC mice. n = 4. (F) Representative WB images of PSD95 and SYN1 expression in the HIP. β-actin was used as a loading control. (G) Semi-quantitative analysis of PSD95 and SYN1 protein levels. n = 6. Data are presented as mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test. ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were blocked and incubated overnight with the following antibodies: PSD95 (SYSN, Toronto, ON, Canada, 124002, 1:1000); SYN1 (SYSN, 106011, 1:1000); Cldn5 (Invitrogen, 35-2500, 1:1000); Ocln (Invitrogen, 71-1500, 1:1000); and MMP9 (Boster, Shanghai, China; PB9669, 1:1000).

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Control

Journal: Autophagy

Article Title: Imbalanced autophagy causes synaptic deficits in a human model for neurodevelopmental disorders

doi: 10.1080/15548627.2021.1936777

Figure Lengend Snippet: Synaptic phenotype in KdVS patient derived iNeurons. (A) Schematic presentation of the protocol to colocalize LC3 and SYN1-SYN2. Image of a dendrite of a control iNeuron at DIV21 after overnight incubation without B27 and treated with 200 nM BAF for 10 min before fixation. Scale bar: 10 µm. (B) Representative images showing dendrites stained for MAP2 and SYN1-SYN2 and SYN1-SYN2 puncta quantification at DIV21 for all lines. n = 60 for C 1 ; n = 57 for KdVS 1 ; n = 24 for CRISPR 1 ; n = 15 for C 2 ; n = 20 KdVS 2 ; n = 34 for KdVS 3 . Scale bar: 20 µm. One-way ANOVA and Sidak’s multiple comparison test were used to test for statistically significant differences. (C) Representative voltage clamp recordings at V h = −60 mV showing sEPSCs at DIV21. (D) sEPSC amplitude and (E) frequency quantification. n = 9 for C 1 ; n = 11 for KdVS 1 ; n = 10 for CRISPR 1 ; n = 15 for C 2 ; n = 8 for KdVS 2 and KdVS 3 (obtained in two independent experiments). (F) Schematic representation for neuronal network measurements on MEAs (3 min of recording). Representative raster plots for C 1 , KdVS 1 and CRISPR 1 derived networks that were plated at similar high densities, measured at DIV 30. (G) Quantification of the mean firing rate and (H) network burst rate, (I) percentage of random spikes, and (J) coefficient of variation (CV) calculated on the inter network burst interval. n = 15 for C 1 ; n = 18 for KdVS 1 ; n = 16 for CRISPR 1 . If not stated differently, data presented in this figure were obtained in at least 3 independent experiments and statistically significant differences were tested through Kruskal-Wallis and Dunn’s multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.0001.

Article Snippet: Used primary antibodies were: rabbit anti-KANSL1 (1:500; Sigma-Aldrich, HPA006874); mouse anti-MAP2 (1:1000; Sigma-Aldrich, M4403); guinea pig anti-MAP2 (1:1000; Synaptic Systems, 188004); guinea pig anti-SYN1-SYN2 (1:1000; Synaptic Systems, 106004); rabbit anti-SYN1 (1:500; BioConnect, AB1543P); mouse anti-HOMER1 (1:500; Synaptic Systems, 160,011); rabbit anti-DLG4/PSD95 (1:50; Cell Signaling Technology, D27E11); mouse anti-pan axon (1:1000; Covance, SMI-312 R); rabbit anti-SQSTM1 (1:500; Sigma-Aldrich, P0067); mouse anti-LC3 (1:500; NanoTools, 0231–100/LC3-5 F10); rabbit anti-LAMP1 (1:200; Sigma-Aldrich, L1418-200ul); mouse anti-8-oxo-dG (1:100; R&D Systems, 4354-MC-050), rabbit anti-H4K16ac (1:400; Abcam, ab109463), rabbit anti-NANOG (1:100; Abcam, ab21624), mouse anti-SSEA4 (1:50; Abcam, ab16287), rabbit anti-POU5F1(1:250; Abcam, ab19857), mouse anti-TRA1-81 (1:100; Millipore, MAB4381) .

Techniques: Derivative Assay, Incubation, Staining, CRISPR

Journal: Autophagy

Article Title: Imbalanced autophagy causes synaptic deficits in a human model for neurodevelopmental disorders

doi: 10.1080/15548627.2021.1936777

Figure Lengend Snippet: Apocynin treatment rescues synaptic phenotype. (A) Representative images of 8-oxo-dG stainings and 8-oxo-dG quantification for untreated and APO treated iNeurons relative to respective untreated control cells at DIV21. n = 39 for C 1 ; n = 46 for C 1 + APO; n = 42 for KdVS 1 ; n = 51 for KdVS 1 + APO; n = 26 for CRISPR 1 ; n = 25 for CRISPR 1 + APO; n = 26 for C 2 ; n = 25 for C 2 + APO; n = 28 for KdVS 2 ; n = 27 for KdVS 2 + APO; n = 31 for KdVS 3 ; n = 23 for KdVS 3 + APO. Scale bar: 20 µm. (B) Representative images of SQSTM1 stainings of iNeurons at DIV21 and fluorescence quantification in untreated and APO treated iNeurons relative to untreated control cells at DIV21. n = 43 for C 1 ; n = 34 for C 1 + APO; n = 34 for KdVS 1 ; n = 38 for KdVS1+ APO; n = 40 for CRISPR 1 ; n = 39 for CRISPR 1 + APO; n = 16 for C 2 ; n = 19 for C 2 + APO; n = 25 for KdVS 2 ; n = 27 for KdVS 2 + APO; n = 34 for KdVS 3 ; n = 24 for KdVS 3 + APO. Scale bar: 20 µm. (C) Representative images of dendrites stained for MAP2 and SYN1-SYN2 for C 1 , KdVS 1 and CRISPR 1 at DIV21 either untreated or APO treated and SYN1-SYN2 quantification. n = 11 for C 1 ; n = 9 for C 1 + APO; n = 12 for KdVS 1 ; KdVS 1 + APO; n = 10 for CRISPR 1 ; CRISPR 1 + APO; n = 7 for C 2 ; C 2 + APO; KdVS 2 ; KdVS 2 + APO; KdVS 3 , KdVS 3 + APO. Scale bar: 20 µm. Two-way ANOVA was used to determine statistically significant changes. (D) Representative voltage clamp recordings at V h = −60 mV showing sEPSCs at DIV21 with and without APO treatment during differentiation. (E) sEPSC frequency and (F) amplitude quantifications. n = 8 for C 1 ; C 1 + APO; n = 9 for KdVS 1 ; KdVS1+ APO; n = 11 for CRISPR 1 ; n = 8 for CRISPR 1 + APO; n = 15 for C 2 ; n = 14 for C 2 + APO; n = 8 for KdVS 2 ; n = 9 for KdVS 2 + APO; n = 8 for KdVS 3 ; KdVS 3 + APO. (G) Representative raster plots for untreated C 1 network and untreated and APO treated CRISPR 1 network at DIV30 (3 min. of recording). Quantification of (H) mean firing rate, (I) network burst frequency, (J) percentage of random spikes, and (K) CV of inter-network burst interval. n = 15 for C 1 ; n = 17 for C 1 + APO; n = 16 for CRISPR 1 ; n = 15 for CRISPR 1 + APO. All data presented in this figure were generated in at least 2 independent experiments and statistically significant differences were tested through Kruskal-Wallis and Dunn’s multiple comparison test, if not mentioned differently. All samples were always compared to the respective untreated control; only significant differences were indicated. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.0001.

Article Snippet: Used primary antibodies were: rabbit anti-KANSL1 (1:500; Sigma-Aldrich, HPA006874); mouse anti-MAP2 (1:1000; Sigma-Aldrich, M4403); guinea pig anti-MAP2 (1:1000; Synaptic Systems, 188004); guinea pig anti-SYN1-SYN2 (1:1000; Synaptic Systems, 106004); rabbit anti-SYN1 (1:500; BioConnect, AB1543P); mouse anti-HOMER1 (1:500; Synaptic Systems, 160,011); rabbit anti-DLG4/PSD95 (1:50; Cell Signaling Technology, D27E11); mouse anti-pan axon (1:1000; Covance, SMI-312 R); rabbit anti-SQSTM1 (1:500; Sigma-Aldrich, P0067); mouse anti-LC3 (1:500; NanoTools, 0231–100/LC3-5 F10); rabbit anti-LAMP1 (1:200; Sigma-Aldrich, L1418-200ul); mouse anti-8-oxo-dG (1:100; R&D Systems, 4354-MC-050), rabbit anti-H4K16ac (1:400; Abcam, ab109463), rabbit anti-NANOG (1:100; Abcam, ab21624), mouse anti-SSEA4 (1:50; Abcam, ab16287), rabbit anti-POU5F1(1:250; Abcam, ab19857), mouse anti-TRA1-81 (1:100; Millipore, MAB4381) .

Techniques: CRISPR, Fluorescence, Staining, Generated