plasmid encoding calfluxvtn  (Addgene inc)

Journal: bioRxiv

Article Title: Mechanical Signaling Drives Tunneling Nanotubes to Preserve Cytoskeleton Tension and Lamin Integrity Against α-Synuclein-Induced Senescence in Astroglia

doi: 10.64898/2026.03.13.711517

Figure Lengend Snippet: U87MG cells were treated with α-SYN (1μM) at intervals of 3, 6, 12, and 24 hours. A) Confocal images of phalloidin (red) stained TNTs, B) Nuclear localization of senescence marker p21 and, C) Images of β-galactosidase-stained cells in the α-SYN-treated cells, compared to control cells. The graph’s datapoints represent the number of images used for analysis, pooling data from three biological repeats. Quantification of D) percentage of TNTs by counting the numbers normalized with the number of cells, E) nuclear localization of p21, and F) β-Galactosidase activity analyzing the average intensities of positively stained cells. Western blots of G) Lamin B1, I) p53, and K) p16. Quantifications of H) Lamin B1, J) p53, and L) p16 western blots. Full blots are represented in Supplementary Material 2. Scale bars are denoted on the images. Data are expressed as mean ± SD, ∗∗∗p ≤ 0.001. Statistics were analyzed using a two-way ANOVA. N=3.

Article Snippet: The human α-SYN wild-type (Addgene ID #36046) construct was purchased and overexpressed in E. coli.

Techniques: Staining, Marker, Control, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Mechanical Signaling Drives Tunneling Nanotubes to Preserve Cytoskeleton Tension and Lamin Integrity Against α-Synuclein-Induced Senescence in Astroglia

doi: 10.64898/2026.03.13.711517

Figure Lengend Snippet: A) Fluorescence images of maximum intensity projected confocal z-stacks in U87MG cells stained with Lamin A/C (Red) and DAPI (Blue) upon α-SYN treatment (1μM) for 3, 6, 12, and 24 hours. Represented 3D volume view and 3D surface plots showing Lamin A/C distributions in the nucleus of the α-SYN-treated cell. B) Quantification of Lamin A/C intensity in the nucleus. Data are expressed as mean ± SD, ∗∗∗p ≤ 0.001. Statistics were analyzed using a two-way ANOVA. C) Orthogonal Z-projection of DAPI-stained nuclei in U87MG cells upon α-SYN treatment. D) Quantification of Flattering index (τ) or the actin tension of U87MG cells upon α-SYN treatment. Scale bars are denoted on the images. The graph shows the number of cells used for analysis, pooling data from three biological repeats. Data are expressed as mean ± SD, ∗∗∗p ≤ 0.001. Statistics were analyzed using a paired ANOVA on the means of three independent sets, since data points differ over time points. N=3.

Article Snippet: The human α-SYN wild-type (Addgene ID #36046) construct was purchased and overexpressed in E. coli.

Techniques: Fluorescence, Staining

Journal: bioRxiv

Article Title: Mechanical Signaling Drives Tunneling Nanotubes to Preserve Cytoskeleton Tension and Lamin Integrity Against α-Synuclein-Induced Senescence in Astroglia

doi: 10.64898/2026.03.13.711517

Figure Lengend Snippet: A) Phalloidin (Red), Lamin A/C (Green) and DAPI (Blue)-stained U87 MG cells treated with α-SYN and α-SYN-treated cells with respective inhibitors Jasp, Noco, Cyto D, and Nac at 3 hours. Phalloidin (yellow) stained 3D volume view of actin cytoskeleton structures obtained from super-resolution microscopy is represented. B) Quantification of Lamin A/C intensities at the nucleus, and C) TNT numbers in percentage, after treatment with the inhibitors in the presence of α-SYN at 3 hours. The graph shows the number of images used for analysis, pooling data from three biological repeats. Quantification of Flattering index (τ) of the cells treated with D) Jasp, E) Noco, and F) Cyto D after treatment of the cells with the inhibitors in the presence of α-SYN for 3,6,12 and 24 hours. The graph shows the number of cells used for analysis, pooling data from three biological repeats. Data are expressed as mean ± SD, ∗∗∗p ≤ 0.001. Statistics were analyzed using a two-way ANOVA. N=3.

Article Snippet: The human α-SYN wild-type (Addgene ID #36046) construct was purchased and overexpressed in E. coli.

Techniques: Staining, Super-Resolution Microscopy

Journal: bioRxiv

Article Title: Mechanical Signaling Drives Tunneling Nanotubes to Preserve Cytoskeleton Tension and Lamin Integrity Against α-Synuclein-Induced Senescence in Astroglia

doi: 10.64898/2026.03.13.711517

Figure Lengend Snippet: A) Log2 fold changes of the selected genes from pathway analysis with significant p-values were depicted in the heatmap. This represents the DEGs in U87MG cells after treatment with α-SYN (1 μM) for 3 hours compared to the control, as well as the same genes after 24 hours of treatment compared to the 3-hour mark. The categories include: A) oxidative stress-related senescence genes, B) genes associated with DNA repair pathways, C) genes involved in the FAK-Rho-ROCK signaling pathway, and D) Intrigin-mediated cell adhesion pathway. E) STRING analysis of DEGs maintained in the pathways (A-D). P-value adjusted <0.05. F) Schematic of α-SYN-mediated oxidative stress-induced senescence, highlighting DNA damage, nuclear lamin integrity, and the ROCK-inhibitory pathway that facilitates TNT formation and the reversal of senescent cells. G) Images of TUNNEL assay represent DNA damage, H) quantification of the cells with damaged DNA, I) Paxillin represents FAs, J) cytosolic and nuclear translocation of YAP, and K) quantification of cells with nuclear YAP, in α-SYN (1 μM) treated U87 MG cells for 3,6,12, and 24hours. The graph’s data points represent the number of images used for analysis, pooling data from three biological repeats. Data are expressed as mean ± SD, ∗∗∗p ≤ 0.001. Statistics were analyzed using a two-way ANOVA. N=3.

Article Snippet: The human α-SYN wild-type (Addgene ID #36046) construct was purchased and overexpressed in E. coli.

Techniques: Control, Translocation Assay

Journal: bioRxiv

Article Title: Mechanical Signaling Drives Tunneling Nanotubes to Preserve Cytoskeleton Tension and Lamin Integrity Against α-Synuclein-Induced Senescence in Astroglia

doi: 10.64898/2026.03.13.711517

Figure Lengend Snippet: A) Images depict the levels of p21 and p53 expression in the nucleus of α-SYN and α-SYN treated with respective inhibitors Jasp, Noco, Cyto D, and Nac in U87 MG cells at 3 and 24 hours. Quantification of B) p21 and C)p53 levels in the nucleus of the images that are represented in panel (A). The graph’s data points represent the number of images used for analysis, pooling data across three biological repeats. Data are expressed as mean ± SD, ∗∗∗p ≤ 0.001. Statistics were analyzed using a two-way ANOVA. N=3.

Article Snippet: The human α-SYN wild-type (Addgene ID #36046) construct was purchased and overexpressed in E. coli.

Techniques: Expressing

Journal: bioRxiv

Article Title: Mechanical Signaling Drives Tunneling Nanotubes to Preserve Cytoskeleton Tension and Lamin Integrity Against α-Synuclein-Induced Senescence in Astroglia

doi: 10.64898/2026.03.13.711517

Figure Lengend Snippet: A) Phalloidin (Red), and YAP (Green)-co-stained and B)YAP (green)-stained astrocytes treated with α-SYN for 3 ,6 ,12 and 24 hours. C)quantification of YAP localization in the nucleus. D) YAP (Green) and Phalloidin (Red) co-localization in the TNT. E) Percentage of TNTs between cells: with either cytosolic, and/or nuclear YAP localization. F) Lamin A/C-stained astrocytes treated with α-SYN for 3 and 24 hours. Quantification of nuclear G) Lamin A/C. The graph shows the number of images used for analysis, pooling data across three biological repeats. Data are expressed as mean ± SD, ∗∗∗p ≤ 0.001. Statistics were analyzed using a two-way ANOVA. N=3.

Article Snippet: The human α-SYN wild-type (Addgene ID #36046) construct was purchased and overexpressed in E. coli.

Techniques: Staining