gfp  (Addgene inc)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

doi: 10.1007/s00018-024-05323-y

Figure Lengend Snippet: INF2 R218Q induces the formation of multipolar spindles in MDCK cells. A Percentage of Cherry and INF2 R218Q cells in mitosis after 48 h of expression. More than 1000 cells were analyzed for each experimental condition in five independent experiments. B Distribution of Cherry and INF2 R218Q cells across different phases of the cell cycle. More than 200 cells were analyzed for each experimental condition, four independent experiments. C Percentage of mitotic Cherry and INF2 R218Q cells displaying multipolar spindles. 68 Cherry cells and 152 INF2 R218Q cells were examined; four independent experiments. D Images of INF2 R218Q mitotic cells stained for centrin, α-tubulin and γ-tubulin. Nuclei were visualized using DAPI. E Classification of INF2 R218Q mitotic cells based on the number of MTOCs expressed as percentage of total cells. MTOCs were identified by γ-tubulin and α-tubulin staining. Over 200 cells were examined; four independent experiments. F Top: Schematic of centriole arrangements. Bottom: centriole arrangement, as visualized with centrin, in INF2 R218Q mitotic cells categorized as clustered in two centrosomes (2 + 2), disengaged in one (2 + 1 + 1) or both (1 + 1 + 1 + 1) centrosomes, or with an abnormal number of centrioles, presented as a percentage of total cells. More than 170 cells were examined; three independent experiments. G Top panel: schematic of ER invasion of the spindle space in cells expressing pathogenic INF2. Bottom panels: INF2 L76P cells stably expressing GFP-sec61 and stained with SiR-DNA analyzed by videomicroscopy during mitosis. The arrowheads indicate regions of the mitotic spindle invaded by ER membranes. H The graph shows the percentage of wt INF2 and INF2 R218Q cells with the spindle space invaded by ER membranes. More than 80 cells were analyzed; three independent experiments. I Videomicroscopic analysis of INF2 R218Q cells stably expressing GFP-sec61 and stained with SiR-DNA during formation of multiple micronuclei. Scale bars, 5 μm. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: The DNA constructs expressing GFP fused to centrin-1 (# 72641), centrin-2 (# 41147), and centrin-3 (# 69746), CaM (# 47602), H2B (# 11680), sec61β (# 62008), HEC1 (# 114049) and α-tubulin (# 58197) were obtained from Addgene.

Techniques: Expressing, Staining, Stable Transfection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

doi: 10.1007/s00018-024-05323-y

Figure Lengend Snippet: INF2 R218Q induces nuclear accumulation of p53 and activation of caspase-3 in MDCK cells. A The graph depicts the time from prophase initiation to the end of cell division, as determined by time-lapse analysis, in control cells (n = 63) and in cells expressing pathogenic INF2 ending with mild (n = 48 cells) and severe (n = 36 cells) nuclear defects; three independent experiments were performed. MDCK cells stably expressing H2B-GFP were used in this experiment. B Immunoblot analysis of p53 in wt INF2 and INF2 R218Q cells. GAPDH was used as loading control. C Quantification of p53 levels in wt INF2 and R218Q cells. Five independent experiments were performed. D Image of p53 staining in an equatorial plane of wt INF2 and INF2 R218 cells. Cells were stained with anti-Cherry antibodies to help visualization of the exogenous proteins. E Percentage of cells with nuclear p53, considering staining in the main nucleus or micronuclei as positive. Over 350 cells were examined; three independent experiments. F Images of cleaved caspase-3 staining in apoptotic INF2 R218Q cells (top) and normal cells (bottom). Nuclei were visualized with DAPI. G Percentage of Cherry, wt INF2 and INF2 R218Q cells positive for cleaved caspase-3 after 48 h of expression. More than 700 cells were examined per experimental condition; three independent experiments. H The percentage of cells with normal, mild or severe nuclear morphology or in mitosis that died between 55 and 72 h of INF2 R218Q expression was determined by time-lapse microscopy in cells labeled with SiR-DNA. 130 cells were analyzed; three independent experiments. I Videomicroscopic analysis of a mitotic (left panels) and an INF2 R218Q cell with a severe phenotype (right panels) undergoing death and detachment from the substrate. Arrowheads indicate the dying cell. Nuclei were stained with SiR-DNA. DIC, differential interference contrast microscopy. Scale bars, 10 μm ( D , F ), 250 μm ( I ). n.s., not significant; **, p < 0.01; ***, p < 0.001

Article Snippet: The DNA constructs expressing GFP fused to centrin-1 (# 72641), centrin-2 (# 41147), and centrin-3 (# 69746), CaM (# 47602), H2B (# 11680), sec61β (# 62008), HEC1 (# 114049) and α-tubulin (# 58197) were obtained from Addgene.

Techniques: Activation Assay, Control, Expressing, Stable Transfection, Western Blot, Staining, Time-lapse Microscopy, Labeling, Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

doi: 10.1007/s00018-024-05323-y

Figure Lengend Snippet: INF2 R218Q requires integrity of its actin polymerization activity to produce nuclear abnormalities. A Dynamics of wt INF2 during mitosis in MDCK cells expressing GFP-tubulin. Cells were stained with SiR-DNA. B Images of INF2 KO MDCK cells expressing INF2 R218Q and R218Q IA. ( C ) Box plots showing the intensity of the perinuclear F-actin and Cherry corresponding to INF2-1 R218Q and INF2-1 R218Q IA MDCK KO cells. More than 150 cells were examined; three independent experiments. D Percentage of MDCK KO cells expressing INF2-1 R218Q IA and INF2-2 R218Q IA exhibiting mild or severe nuclear phenotypes. More than 350 cells were examined; three independent experiments. E Images of an equatorial plane of INF2 KO MDCK cells expressing INF2 A149D and INF2 3LA. F Percentage of cells expressing INF2 A149D and INF2 3LA displaying mild or severe nuclear phenotypes. More than 300 cells were examined across three independent experiments. The contrast in the Cherry channel was increased as indicated to identify the cells expressing mutant INF2. Nuclei were visualized with DAPI. Scale bars, 5 μm ( A ) and 10 μm ( B , E ). **, p < 0.01; ***, p < 0.001

Article Snippet: The DNA constructs expressing GFP fused to centrin-1 (# 72641), centrin-2 (# 41147), and centrin-3 (# 69746), CaM (# 47602), H2B (# 11680), sec61β (# 62008), HEC1 (# 114049) and α-tubulin (# 58197) were obtained from Addgene.

Techniques: Activity Assay, Expressing, Staining, Mutagenesis