Journal: Nucleic Acids Research

Article Title: Ablation of Mto1 in zebrafish exhibited hypertrophic cardiomyopathy manifested by mitochondrion RNA maturation deficiency

doi: 10.1093/nar/gkab228

Figure Lengend Snippet: Western blotting analysis of mitochondrial proteins. ( A , C ) Twenty micrograms of total proteins from mutant and WT zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies for 5 subunits of OXPHOS (mitochondrion-encoding Nd1, Co2 and Atp8 and nucleus-encoding Sdha and Atp5a), Tufm, Tfam and Tom20 as a loading control. Quantification of levels of OXPHOS subunits ( B ) and other mitochondrial proteins ( D ). Average contents of Nd1, Co2, Atp8, Atp5a, Sdha, Tufm and Tfam were normalized to the average content of Tom20 in mutant and WT zebrafish. The values for the mutant zebrafish are expressed as percentages of the average values for the WT zebrafish. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure .

Article Snippet: The antibodies used for this investigation were from Sigma [Mto1 (Sigma, HPA030232) and Gapdh (SAB2701826)], Abcam [Nd1 (ab74257), Sdha (ab151684), Atp5a (ab188107), Mtpap (ab154555) and Uqcrc2 (ab203832)] and Proteintech [Co2 (55070-1-AP), Atp8 (26723-1-AP), Ndufs1 (12444-1-AP), Cox5a (11448–1-AP), Atp5c (60284-1-Ig), Tfam (19998-1-AP), and Tufm (26730-1-AP) and Tom20 (1802-1-AP)].

Techniques: Western Blot, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Ablation of Mto1 in zebrafish exhibited hypertrophic cardiomyopathy manifested by mitochondrion RNA maturation deficiency

doi: 10.1093/nar/gkab228

Figure Lengend Snippet: The synergic effects between Mto1 and Mtpap on the polyadenylation of mRNAs. ( A, B ) Immunoprecipitation analysis of MTO1 with MTPAP. HEK 293T cells transiently expressing with or without MTO1-FLAG were solubilized with a lysis buffer and lysate proteins were immuno-precipitated with immunocapture buffer (left) (input) and FLAG-antibody (right) (IP), respectively. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting using anti-FLAG, anti-MTPAP and TOM20 antibodies, respectively. ( C ) Western blot analysis. Twenty micrograms of total proteins from mto1 −/− and WT zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies for Mtpap and Gapdh as a loading control. ( D ) Polyadenylation profiles of mitochondrial mRNAs upon mto1 −/− and WT zebrafish hearts. The 3′ termini of nd1, cytb , cox1 and cox3 mRNAs were assessed by RT-PCR amplifications from polyadenylated and oligoadenylated RNAs of mto1 −/− and WT zebrafish hearts and 3% agarose gel electrophoresis. Arrows indicated the positions of PCR products from the polyadenylated and oligoadenylated RNAs, respectively. ( E ) Quantification of poly(A) proportions of nd1, cytb , cox1 and cox3 transcripts in the WT and mto1 −/− zebrafish. ( F ) Poly(A) tail lengths from individually sequenced clones after 3’ end RACE analysis of nd1 transcripts in the mto1 −/− and WT zebrafish hearts. Graph details and symbols are explained in the legend to Figure .

Article Snippet: The antibodies used for this investigation were from Sigma [Mto1 (Sigma, HPA030232) and Gapdh (SAB2701826)], Abcam [Nd1 (ab74257), Sdha (ab151684), Atp5a (ab188107), Mtpap (ab154555) and Uqcrc2 (ab203832)] and Proteintech [Co2 (55070-1-AP), Atp8 (26723-1-AP), Ndufs1 (12444-1-AP), Cox5a (11448–1-AP), Atp5c (60284-1-Ig), Tfam (19998-1-AP), and Tufm (26730-1-AP) and Tom20 (1802-1-AP)].

Techniques: Immunoprecipitation, Expressing, Lysis, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Clone Assay

Journal: Biology Open

Article Title: Actin filaments accumulated in the nucleus remain in the vicinity of condensing chromosomes in the zebrafish early embryo

doi: 10.1242/bio.059783

Figure Lengend Snippet: Nuclear F-actin detected by phalloidin in fixed zebrafish early embryos. Zebrafish embryos were fixed at the 32-, 128-, 1000-cell, and sphere stage, and stained with Acti-stain™ 555 phalloidin and Hoechst. Low (A) and high (B) power views of single confocal sections are shown. (C) Relative intensity profile plots of lines indicated in B. The intensity is normalized using the average cytoplasmic intensity. Phalloidin signals were clearly observed in nuclei of the 32-, 128-, and 1000-cell stages. Scale bars: 50 μm (A), 10 μm (B).

Article Snippet: After fixation, embryos were washed three times with PBS and stained with 2 μg/ml Hoechst 33342 (Nacalai Tesque, 04929-82) and 20 nM Acti-stain™ 555 phalloidin (Cytoskeleton, PHDH1-A) in PBS containing 0.05% Triton X-100 at 4°C overnight in the dark.

Techniques: Staining

Journal: Biology Open

Article Title: Actin filaments accumulated in the nucleus remain in the vicinity of condensing chromosomes in the zebrafish early embryo

doi: 10.1242/bio.059783

Figure Lengend Snippet: Transcription inhibition delayed the nuclear actin decrease after the 1000-cell stage. (A) Zebrafish embryos were injected with α-amanitin to inhibit RNA-polymerase II-mediated transcription, or the vehicle (PBS), with Cy3-labeled morpholino antisense oligonucleotide for miR-430 transcripts as a transcription marker, and JF646-LANA as a chromatin marker. Every 90 s, 25 z-stack images with 4 μm intervals were acquired using a confocal microscope. Seven slices at each time point from the 128-cell to dome stage were depicted and the max intensity projection images are shown. (B) Embryos were grown to the 1000-cell stage and oblong stage before fixation and staining with Hoechst and Acti-stain™ 555 phalloidin. Single confocal sections are shown with N/C ratios of phalloidin intensity ( N =10 cells from three embryos). In the box plots, center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th to 75th percentiles;×indicates the means; and data points are plotted as closed circles (PBS, blue; α-amanitin, orange). P -values obtained with a Student's t -test (unpaired, two-tailed) are also shown (* P <0.05). At the 1000-cell stage, clear phalloidin signals are observed in the nuclei of both embryos treated without or with α-amanitin. At the oblong stage, phalloidin signals are not concentrated in the nucleus of embryos without α-amanitin but are still observed in the nucleus of α-amanitin-treated embryos. (C) Zebrafish embryos were injected with α-amanitin, or PBS, and then with UtrCH-sfGFP and H3K9ac Fab-Cy5. Every 90 s, 25 z-stack fluorescence images with 4 μm intervals were acquired. The mean values of N/C ratios and the number of nuclei (control: blue, +α-amanitin: orange) are plotted with the s.d. from the 128-cell to dome stage for two independent experiments/embryos. α-amanitin-injected embryos showed the higher N/C ratio of UtrCH-sfGFP value in later stages (sphere and dome). Single confocal sections at the sphere stage are shown on the right. UtrCH-sfGFP remained accumulated in some nuclei during the sphere stage (orange arrowheads). Scale bars: 10 μm (A) and 20 μm (B and C).

Article Snippet: After fixation, embryos were washed three times with PBS and stained with 2 μg/ml Hoechst 33342 (Nacalai Tesque, 04929-82) and 20 nM Acti-stain™ 555 phalloidin (Cytoskeleton, PHDH1-A) in PBS containing 0.05% Triton X-100 at 4°C overnight in the dark.

Techniques: Inhibition, Injection, Labeling, Marker, Microscopy, Staining, Two Tailed Test, Fluorescence

Journal: bioRxiv

Article Title: Lipid Dysregulation Unveil the Intricate Interplay of Lysosomal and Mitochondrial Changes in Frontotemporal Dementia with GRN Haploinsufficiency

doi: 10.1101/2024.01.22.576606

Figure Lengend Snippet: A. Protein Levels of LC3B-II and P62 Assessed by Western Blotting in Primary Human Fibroblasts. Primary human fibroblasts were cultured with or without a 6-hour STV pre-assay period and treated with CLQ at 30 µM for 5 hours. Western blotting was performed to assess the protein levels of LC3B-II and P62, and quantification are shown as bar plot graphs. B. Representative Images of LC3B Puncta (green) and DAPI (blue) Staining in Primary Human Fibroblasts. Primary human fibroblasts were treated with or without a 5-hour pre-assay period with CLQ at 30 µM. LC3B puncta (green) staining and DAPI (blue) staining were used to visualize autophagosomes and nuclei, respectively. Scale bar = 100 µm. C. Representative TEM images of primary human fibroblasts, showing autophagosomes (purple). Insets: Autophagosome. Scale bar is indicated in the figure. D. Protein Levels of LC3B-II and PINK1 Analyzed by Western Blotting in Cytoplasmic and Mitochondrial Fractions of Primary Human Fibroblasts. Primary human fibroblasts were treated with or without a 5-hour pre-assay period with CLQ at 30 µM. Western blotting was performed to analyze the protein levels of LC3B-II and PINK1 in both cytoplasmic and mitochondrial fractions. E. Representative Images of LC3B (green) and TOM20 (red) Staining in Primary Human Fibroblasts. Bar plot shows the quantification of the relative area of LC3B colocalized with TOM20. Scale bar = 10 µm. Healthy control fibroblasts(CTL), FTD-GRN patient fibroblast (GRN). Starving (STV), Chloroquine (CLQ), non treated (NT). Data are presented as means ± SEMs (n = 3). Statistical significance represented by *p < 0.05 and **p < 0.01, determined using Student’s t test.

Article Snippet: Primary Antibodies: PGRN (1μg/mL, AF2420, R&D systems), LAMP-1(1:10 000, ab25630, Abcam), LAMP-2 (1:1 000, #49067, Cell Signaling), LC3Bxp (1:1 000, #3868, Cell Signaling), SQSTM1/P62 (1:1 000, #5114, Cell Signaling), β-Tubulin (1:5 000, MA5-16308, Thermo Fisher) PINK1 (1:1 000, BC100-494, Novus Bio) TOM20 (1:1 000, 11802-1-AP, Proteintech), GAPDH (1:50 000, 60004-1-Ig, Proteintech), Lamin A+C (1:10 000, ab108595, Abcam), TDP-43 (1:1 000, ab104223, Abcam).

Techniques: Western Blot, Cell Culture, Staining