automated axio observer z1 microscope  (Carl Zeiss)

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