pacgfp c1 sec61β  (Addgene inc)

Journal: bioRxiv

Article Title: Live-cell imaging reveals nutrient-dependent dynamics of ER-mitochondria contact formation via PDZD8

doi: 10.1101/2025.04.17.649323

Figure Lengend Snippet: (A) Schematic representation of the MERCdRED system. (B) Diagram of the MERCdRED-expressing vector. Two components of the MERCdRED system, Tomm20-GB and RA-Sec61β, were co-expressed from a single vector using a P2A sequence, which enables post-translational self-cleavage. (C) Diagram illustrating the establishment of the MERCdRED cell line. (D) Comparison of MERCdRED signals between live cells and cells fixed with 2% paraformaldehyde (PFA) and 0.5% glutaraldehyde (GA). (E) Representative live images of the MERCdRED cells (MERCdRED shown in magenta) transfected with plasmids encoding an ER marker GFP-Sec61β (yellow) or a mitochondrial marker Tomm20-iRFP (cyan). The boxed regions of the top panels are shown at higher magnification in the corresponding lower panels. (F) Quantification of the MERCdRED intensity on mitochondria in images obtained as described (E). The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values. n = 29, 18 cells for the MERCdRED cells and the control cells (negative control) from five independent experiments. Statistical analysis was performed using two-tailed Student’s t-test. ****P < 0.0001

Article Snippet: The RA-Sec61β sequence was amplified by PCR from RA-NES (Addgene, catalog no. #61019) and pAc-GFP-Sec61β (Addgene, catalog no. #62008) with the pair of primers 5’-cga gct caa gct tcg aat tca tgg tga gca aga gcg agg a -3’ and 5’-tct gag gct agc ctt gta cag ctc gtc cat gc -3’, 5’-caa ggc tag cct cag atc tat gcc tgg tcc -3’ and 5’-gga gtg aat tgc ggc cgc cta cga acg agt gta ctt gcc c -3’, respectively, and subcloned into the EcoRI and NotI sites of pCIG vector with In-Fusion HD cloning kit, resulting in pCAG-RA-Sec61β.

Techniques: Expressing, Plasmid Preparation, Sequencing, Comparison, Transfection, Marker, Control, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Live-cell imaging reveals nutrient-dependent dynamics of ER-mitochondria contact formation via PDZD8

doi: 10.1101/2025.04.17.649323

Figure Lengend Snippet: (A) Schematic representation depicting the depletion of the MERCS-tethering protein complex PDZD8 and FKBP8 in MERCdRED cells. Fkbp8 gene was knocked down using lentivirus-mediated shRNA infection, and Pdzd8 gene was knocked out by treating cells with 1 μM 4-hydroxy (4-OH) tamoxifen, which induces Cre/loxP-dependent conditional knockout. (B) Immunoblot analysis of Pdzd8 f/f ::Cre ERT2 MEFs infected with lentivirus carrying control shRNA or Fkbp8 shRNA, treated with or without 0.5 μM 4-OHT. Cell lysates were subjected to immunoblotting with antibodies to PDZD8, FKBP8, and α-tubulin. (C) Representative live images of MERCdRED cells with or without depletion of Pdzd8 and/or Fkbp8 . GFP-Sec61β (yellow) or Tomm20-iRFP (cyan) was used as an ER marker or a mitochondrial marker, respectively. Pdzd8 conditional knockout was indicated as Pdzd8 cKO, and Fkbp8 knockdown was shown as Fkbp8 KD. The boxed regions of the top panels are shown at higher magnification in the corresponding lower panels. (D) Quantification of the MERCdRED intensity on mitochondria in images obtained as described (C). The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values. n = 17, 14, 15, 15 cells for control + control, Pdzd8 cKO + control, control + Fkbp8 KD, and Pdzd8 cKO + Fkbp8 KD, respectively, from two independent experiments. Statistical analysis was performed using Tukey’s multiple comparisons test. ****P < 0.0001, **P < 0.01, *P < 0.05.

Article Snippet: The RA-Sec61β sequence was amplified by PCR from RA-NES (Addgene, catalog no. #61019) and pAc-GFP-Sec61β (Addgene, catalog no. #62008) with the pair of primers 5’-cga gct caa gct tcg aat tca tgg tga gca aga gcg agg a -3’ and 5’-tct gag gct agc ctt gta cag ctc gtc cat gc -3’, 5’-caa ggc tag cct cag atc tat gcc tgg tcc -3’ and 5’-gga gtg aat tgc ggc cgc cta cga acg agt gta ctt gcc c -3’, respectively, and subcloned into the EcoRI and NotI sites of pCIG vector with In-Fusion HD cloning kit, resulting in pCAG-RA-Sec61β.

Techniques: shRNA, Infection, Knock-Out, Western Blot, Control, Marker, Knockdown

Journal: bioRxiv

Article Title: Live-cell imaging reveals nutrient-dependent dynamics of ER-mitochondria contact formation via PDZD8

doi: 10.1101/2025.04.17.649323

Figure Lengend Snippet: (A) Representative images from time-lapse imaging shown as Supplementary movie 1. Images were obtained at 0.5 Hz for 32 seconds (17 frames). MERCdRED-positive areas on mitochondria were shown in magenta, and Tomm20-iRFP (cyan) was used as a mitochondrial marker. Yellow arrowheads indicate newly emerging MERCdRED signals that appeared in the subsequent time frame relative to the previous representative frame. Data are representative of 20 cells from three independent experiments. (B) Representative tracking images of MERCdRED-positive puncta shown in Supplementary movie 1. Tracking images were generated using TrackMate (ImageJ plugin) across the images obtained in (A). The colors of trajectories indicate the speeds of MERCdRED-positive puncta (shown in white). (C) Relationship between the area and the speed of each MERCdRED punctum. The average speed and area of each punctum over the entire observation period were calculated from tracking images obtained in (B). (D) The average speed of small puncta (area < 0.05 μm 2 ; left side of the dotted line in (C)) and large puncta (area > 0.05 μm 2 ; right side of the dotted line in (C)) was calculated. Data are means ± s.e.m. of 762 small puncta and 300 large puncta in 20 cells from three independent experiments. Statistical analysis was performed using the two-tailed Mann-Whitney test. (E) Schematic representation depicting the nutritional starvation in the MERCdRED cells. The cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in fed condition, whereas they were incubated in EBSS for 5 hours in starved condition. (F) Representative images from live-cell imaging of MERCdRED cells in the fed or starved condition. GFP-Sec61β (yellow) or Tomm20-iRFP (cyan) was used as an ER marker or a mitochondrial marker, respectively. The boxed regions of the top panels are shown at higher magnification in the corresponding lower panels. (G) Quantification of the mitochondrial area in images obtained as described (F). The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values. n = 14, 13, 12, 12 cells for fed + control, starved + control, fed + Pdzd8 cKO, and starved + Pdzd8 cKO, respectively, from two independent experiments. Statistical analysis was performed using Tukey’s multiple comparisons test. n.s., not significant. (H) Quantification of the MERCdRED intensity on mitochondria in images obtained as described (F). The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values. n = 14, 13, 12, 12 cells for fed + control, starved + control, fed + Pdzd8 cKO, and starved + Pdzd8 cKO, respectively, from two independent experiments. Statistical analysis was performed using Tukey’s multiple comparisons test. ****P < 0.0001, ***P < 0.001, **P < 0.01.

Article Snippet: The RA-Sec61β sequence was amplified by PCR from RA-NES (Addgene, catalog no. #61019) and pAc-GFP-Sec61β (Addgene, catalog no. #62008) with the pair of primers 5’-cga gct caa gct tcg aat tca tgg tga gca aga gcg agg a -3’ and 5’-tct gag gct agc ctt gta cag ctc gtc cat gc -3’, 5’-caa ggc tag cct cag atc tat gcc tgg tcc -3’ and 5’-gga gtg aat tgc ggc cgc cta cga acg agt gta ctt gcc c -3’, respectively, and subcloned into the EcoRI and NotI sites of pCIG vector with In-Fusion HD cloning kit, resulting in pCAG-RA-Sec61β.

Techniques: Imaging, Marker, Generated, Two Tailed Test, MANN-WHITNEY, Incubation, Live Cell Imaging, Control

Journal: Virology

Article Title: Dengue virus induces mitochondrial elongation through impairment of Drp1-triggered mitochondrial fission

doi: 10.1016/j.virol.2016.10.022

Figure Lengend Snippet: A. and B. Western blot analysis of NS4b and NS3 protein levels was performed in subcellular fractions from uninfected and DENV-infected Huh7 cells (A.) and DCs (B.). After 48h of infection, cells were fractionated to separate cytosolic and mitochondrial fractions, and compared to total cell lysates. TOMM20 was used as a loading and purity control for mitochondrial fraction. FACL4 antibody served as a marker for MAMs. β-actin antibody was used as a loading control. Western blots are representative of three independent experiments. C. Immunofluorescence analysis of NS4b protein localization in uninfected and DENV-infected Huh7 cells after 48h (MOI of 1). Cells were transfected with pTagRFP-mito and pAcGFP-Sec61β. Images were taken with confocal LSM800 at ×63×1.5 magnification.

Article Snippet: The following plasmids were used in our study: pNS4b5-eGFP [ 48 ], pTagRFP-mito (Evrogen), pAcGFP-Sec61β (gift from Tom Rapoport, Addgene plasmid #15108), pEYFP-Drp1, pEYFP-Drp1 S616D, pEYFP-Drp1 S616A, pEYFP-Drp1 S637D, pEYFP-Drp1 S637A (Drp1 phosphomimetic and phosphodefective mutant plasmids were a gift from Dr. Luca Scorrano, University of Padua), pDrp1 K38A-CFP (gift from Dr. Gyorgy Hajnoczky, Thomas Jefferson University)).

Techniques: Western Blot, Infection, Control, Marker, Immunofluorescence, Transfection

Journal: Virology

Article Title: Dengue virus induces mitochondrial elongation through impairment of Drp1-triggered mitochondrial fission

doi: 10.1016/j.virol.2016.10.022

Figure Lengend Snippet: A. Supernatants from DENV infected Vero cells were collected after 24h of infection. Cells were treated with medium containing DMSO or CCCP 10 μM and supernatants were again collected after 6h post treatment (30h pi). To reverse CCCP treatment, DMSO and CCCP-treated cells were washed once with PBS and supernatant was collected (48h pi). DENV titers from collected supernatants were analyzed using an ELISpot assay. Data are representative of one out of two independent experiments, analyzed with unpaired Student-t test. Error bars represent SD of three biological replicates per condition. B. Immunofluorescence analysis of mitochondrial morphology of uninfected and DENV-infected Vero cells after indicated treatments. Cells were transfected with pTagRFP-mito, challenged with DENV (MOI of 1) and treated as indicated before being fixed and stained with antibody against DENV antigen. Images were taken with confocal LSM800 at ×63×1.5 magnification. Magnified images of boxed area show mitochondria stained with pTagRFP-mito for each condition. Images were converted to black and white using ImageJ. C. Huh7.5 cells were transfected with two independent sets of dsiRNAs against Mfn1 or Mfn2 (set (A), left and set (B), right) and infected with DENV (MOI of 0.1) for 24h. Western blots were probed with NS3 and NS5 antibodies and equal protein loading was assessed using an antibody against β-actin. Blot is representative of at least four independent experiments. D. For quantification of NS3 and NS5 protein levels, blots of four independent experiments were evaluated after normalization to β-actin and expressed as mean +/− SEM. Statistical analysis was performed using unpaired Student-t test. E. DENV titers from collected supernatants of Huh7.5 cells treated with dsiRNAs against Mfn1 or Mfn2 were analyzed using an ELISpot assay. DENV titers were transformed to log (FFU/ml) and displayed as mean +/− SEM. Statistical analysis was performed using paired t-test. Titers are representative of independent experiments (n=3).

Article Snippet: The following plasmids were used in our study: pNS4b5-eGFP [ 48 ], pTagRFP-mito (Evrogen), pAcGFP-Sec61β (gift from Tom Rapoport, Addgene plasmid #15108), pEYFP-Drp1, pEYFP-Drp1 S616D, pEYFP-Drp1 S616A, pEYFP-Drp1 S637D, pEYFP-Drp1 S637A (Drp1 phosphomimetic and phosphodefective mutant plasmids were a gift from Dr. Luca Scorrano, University of Padua), pDrp1 K38A-CFP (gift from Dr. Gyorgy Hajnoczky, Thomas Jefferson University)).

Techniques: Infection, Enzyme-linked Immunospot, Immunofluorescence, Transfection, Staining, Western Blot, Transformation Assay