axio observer z1 microscope  (Carl Zeiss)

Journal: bioRxiv

Article Title: The SV40 T-ag nuclear localization signal affects Toxoplasma gondii viability by targeting importin α

doi: 10.1101/2025.07.20.665720

Figure Lengend Snippet: (a) T. gondii tachyzoite vacuoles expressing 1xGFP construct alone and when fused with SV40-NLS. The images were captured on Zeiss observer Z1 Spinning Disc microscope with 40X objective (scale bar 50 μm); (b) Percentage of GFP-positive vacuoles per field were counted (N = 3) and the mean ± SEM was plotted; (c) The alanine mutations (bold) of the SV40-NLS motif generated in our study are shown, with their dissociation constant (K d ) values and the cNLS score; (d) Percentage of GFP-positive vacuoles per field were counted (N = 3) with the mean ± SEM plotted; (e) Tachyzoite vacuoles expressing 2xGFP construct alone and when fused with SV40-NLS (scale bar 50 μm); (f) Percentage of GFP-positive vacuoles per field were counted (N = 3) and the mean ± SEM was plotted. A significant difference of p < 0.01 and p < 0.0001 is indicated by * and ****, respectively, on performing the Mann-Whitney test for all plotted graphs.

Article Snippet: To quantify GFP-positive parasitophorous vacuoles, 15 fields were imaged from each replicate in the Zeiss Observer Z1 Spinning Disc microscope at 40X.

Techniques: Expressing, Construct, Microscopy, Generated, MANN-WHITNEY

Journal: bioRxiv

Article Title: The SV40 T-ag nuclear localization signal affects Toxoplasma gondii viability by targeting importin α

doi: 10.1101/2025.07.20.665720

Figure Lengend Snippet: (a) WT-TgImpα-HA, AAA-TgImpα-HA, and KRR-TgImpα-HA localize in the nucleus and cytoplasm of T. gondii tachyzoites. Images were captured in a Zeiss LSM 780 Confocal microscope with 100X objective (scale bar 5 μm); (b) Transient co-transfection of WT and variants of TgImpα-HA (red) with SV40-NLS-2xGFP (green) resulted in live SV40-NLS-GFP expressing vacuoles. Images were captured in Zeiss Observer Z1 Spinning Disc microscope with 40X objective (scale bar 30 μm); (c) Graph shows mean ± SEM of the percentage of vacuoles after transient co-transfection expressing only SV40-NLS-2xGFP or 2xGFP (green), only TgImpα-HA (red) or both (yellow) per DAPI counts (N = 3). Mann-Whitney test performed shows a significant difference of p < 0.003 (***) or p < 0.0001 (****) for the yellow bar in the plotted graph; (d) Graph shows mean ± SEM of the percentage of vacuoles in the stable lines of TgImpα-HA (wild-type and variants) transfected with SV40-NLS-2xGFP or 2xGFP (N = 2); (e) Graph shows mean ± SEM of the CTCF intensity estimated of TgImpα-HA in transiently transfected and stable lines for 30-40 vacuoles. The Mann-Whitney test performed shows a significant difference of p < 0.0001.

Article Snippet: To quantify GFP-positive parasitophorous vacuoles, 15 fields were imaged from each replicate in the Zeiss Observer Z1 Spinning Disc microscope at 40X.

Techniques: Microscopy, Cotransfection, Expressing, MANN-WHITNEY, Transfection

Journal: bioRxiv

Article Title: The SV40 T-ag nuclear localization signal affects Toxoplasma gondii viability by targeting importin α

doi: 10.1101/2025.07.20.665720

Figure Lengend Snippet: (a) GFP-His and SV40-NLS-GFP-His recombinant proteins (14 μg/ml) were transfected into HFF cells and incubated for 24 hours. The images were captured on Zeiss observer Z1 Spinning Disc microscope with 63X objective (scale bar 30 μm); (b) Graph shows mean ± SEM of the CTCF intensity estimated of GFP in 30 fields at varying concentrations of proteins (N = 2); (c) Cell viability MTT assay performed on HFF when treated with the proteins in the respective concentration range (N = 3).

Article Snippet: To quantify GFP-positive parasitophorous vacuoles, 15 fields were imaged from each replicate in the Zeiss Observer Z1 Spinning Disc microscope at 40X.

Techniques: Recombinant, Transfection, Incubation, Microscopy, MTT Assay, Concentration Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LRRK2 kinase modulates glucose-stimulated insulin secretion via RAB8 phosphorylation and ciliogenesis

doi: 10.1007/s00018-025-05810-w

Figure Lengend Snippet: LRRK2 controls the glucose-stimulated insulin secretion through its kinase activity. A Glucose-stimulated (20 mM) insulin secretion in βtc3 cells incubated in the absence (CTR - DMSO) or presence of the LRRK2 kinase inhibitors GSK (200 nM) or MLi-2 (10 nM) (n = at least 5 independent experiments). Data are expressed as percentage of insulin content and are reported as mean ± SD. Two-way ANOVA: * p < 0.05, ** p < 0.01. B Glucose-stimulated (16.7 mM) insulin secretion in isolated human islets incubated in the absence (CTR - DMSO) or presence of the LRRK2 kinase inhibitors GSK (200 nM) and MLi-2 (10 nM) ( n = 5 independent experiments). Data are expressed as percentage of insulin content and are reported as mean ± SD. Two-way ANOVA: *p<0.05, **p < 0.01, ***p < 0.005. C Representative images of insulin granules density in the TIRF zone (100 nm) under basal (1 mM glucose) and stimulated (20 mM glucose) conditions in βtc3 cells incubated with 10 nM MLi-2 or DMSO (CTR) for 45 min. After treatments, cells were fixed and stained with anti-insulin antibody. Scale bar: 5 μm. D Quantitative analysis of insulin granules in the TIRF zone in control and MLi-2 treated βtc3 cells. Data are normalized for the cell area and are reported as mean ± SD. Each dot represents the average granule density in one cell ( n = 22 cells). Two-way ANOVA: ** p < 0.01, **** p < 0.001. E Glucose-stimulated (20 mM) insulin secretion in βtc3 cells expressing LRRK2 WT or G2019S constructs. Insulin secretion was evaluated in the presence or absence of the LRRK2 kinase inhibitor GSK (200 nM) in G2019S-transfected cells ( n = 3 independent experiments). Data are expressed as percentage of insulin content and are reported as mean ± SD. Two-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.005

Article Snippet: Samples were imaged by a TIRF microscope (Axio Observer Z1, Zeiss), equipped with a 100 × 1.45 NA, oil immersion, objective and an Argon laser.

Techniques: Activity Assay, Incubation, Isolation, Staining, Control, Expressing, Construct, Transfection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LRRK2 kinase modulates glucose-stimulated insulin secretion via RAB8 phosphorylation and ciliogenesis

doi: 10.1007/s00018-025-05810-w

Figure Lengend Snippet: RAB8 is phosphorylated by LRRK2, and its phosphorylation promotes insulin release. A Full-length RFP-LRRK2 was expressed in βtc3 cells, and the recombinant protein was isolated on the RFP-selector resin (RFP-resin). A control-selector resin (Ctr-Resin) was used to detect unspecific binding. Interacting proteins were resolved by western blotting analysis with the anti-RAB8 antibody. B Western blot analysis of RAB8 phosphorylation on the threonine 72 residue at different time points after glucose stimulation (20 mM), in the presence or absence of LRRK2 kinase inhibitor MLi-2 (10 nM, 45 min pre-treatment) and C relative quantification ( n = at least 3 independent experiments). Actin was used as loading control. Data are expressed as Phospho-RAB8 over RAB8 and are reported as mean ± SD. Two-way ANOVA: ** p < 0.01; **** p < 0.001. D Glucose-stimulated (20 mM) insulin secretion in βtc3 cells transfected with WT RAB8 and T72A mutant. Data are expressed as percentage of insulin content and values are reported as mean ± SD ( n = 6 experiments). Two-way ANOVA: * p < 0.05; ***p < 0.005; **** p < 0.001. E Quantitative analysis of insulin granule trafficking in the TIRF zone in βtc3 cells cotransfected with GFP-insulin and WT RAB8 and T72A mutant at different time points after glucose (20 mM) and KCl (40 mM) stimulations. Data are normalized on cell area and are reported as mean ± SD. Two-way ANOVA. ** p < 0.01; **** p < 0.001 vs WT RAB8, same time point; °° p < 0.01, vs 0’ WT RAB8 ( n = at least 3 independent experiments). F Representative immunofluorescence images of βtc3 cells triple stained with DAPI (blue), anti-RAB8 (green) and anti-insulin (red) antibodies under basal (1 mM glucose) and stimulated (20 mM glucose) conditions. Scale bar: 5 μm

Article Snippet: Samples were imaged by a TIRF microscope (Axio Observer Z1, Zeiss), equipped with a 100 × 1.45 NA, oil immersion, objective and an Argon laser.

Techniques: Phospho-proteomics, Recombinant, Isolation, Control, Binding Assay, Western Blot, Residue, Quantitative Proteomics, Transfection, Mutagenesis, Immunofluorescence, Staining