Journal: bioRxiv

Article Title: Intercellular Communication via Mitotic Nanotubes is Influenced by Connexin-43 Trafficking and Actin Remodeling

doi: 10.64898/2026.02.08.704470

Figure Lengend Snippet: (A) Confocal immunocytochemical image of fixed cells stained to visualize endogenous Cx43 (green) and nuclei (blue). (B) Confocal live-cell image of cells transfected to express mEmerald-Cx43 (green) and the plasma membrane marker mCherry-CAAX (magenta). (C and C’) Confocal live image of cells transfected to express Halo-Cx43 (green), shown with (C) or without (C’) DIC overlay. (D and E) Transmission electron micrographs of a gap junction plaque (D) and an annular gap junction (E) in which the typical pentalaminar membrane morphology can be distinguished. In A-E, solid arrows represent gap junction plaques; dashed arrows represent the invaginated region of the gap junction plaque; arrowheads represent annular gap junctions. (F) Cells in various cell cycle stages were fixed and stained to visualize actin (magenta), Cx43 (green), and nuclei (blue). (F’) Enlargements of mitotic nanotubes in outlined areas in F to show association with Cx43-positive structures (arrows). (G) Cellular distribution of Cx43-positive structures across cell cycle stages. The bar graph represents the mean total area of Cx43 structures per compartment per cell ± SEM. Across 3 experiments, 79 cells were analyzed, with n ≥ 8 per cell cycle stage. (J) Micrograph of a membrane extension that forms a bridge between two cells. The extension is closed at the distal end (J’) where it makes contact with another cell. Contact sites shown in A (boxes J’ and J’’) were magnified to show the typical gap junction plaque membrane. (J’) Contact site between the closed end of the membrane extension and the adjacent cell. (J’’) Contact site between an area along the length of the membrane extension and the tip of another membrane extension. Scale bars = 5µm (A, F, F’), 10µm (B, C, C’), 100nm (D, E), 2µm (J), 100nm (J’, J’’).

Article Snippet: Plasmids encoding the gap junction markers mEmerald-Cx43 (Addgene, Watertown, MA, plasmid #54055) and Halo-Cx43 were gifts from Michael Davidson and John O’Brien, respectively.

Techniques: Staining, Transfection, Clinical Proteomics, Membrane, Marker, Transmission Assay

Journal: bioRxiv

Article Title: Intercellular Communication via Mitotic Nanotubes is Influenced by Connexin-43 Trafficking and Actin Remodeling

doi: 10.64898/2026.02.08.704470

Figure Lengend Snippet: (A) Time-lapse of gap junction plaque turnover in dividing and non-dividing cells. Gap junction plaques were monitored between pairs of interphase cells that later underwent mitosis (Dividing Cells) and pairs of interphase cells that did not divide over ≥ 16 hours of imaging (Nondividing Cells). Large plaques were present at cell-cell contacts at t=0 in both cell pair types (arrows). However, plaque internalization occurred more frequently, resulting in more annular gap junctions in the cytoplasm (arrowheads) of dividing cells than in non-dividing cells. In the montage shown, new annular gap junctions were similarly distributed between the cytoplasm of both cells (Cells 1 and 2). However, among the dividing cells, annular gap junctions were predominantly released into the cell that initially underwent mitosis, compared with the cell that divided later (Cell 4). After 80 minutes, little to no gap junction plaques remained at the surface of the dividing cells, whereas plaques between non-dividing cells showed no apparent change in size. (B) Time-lapse of gap junction dynamics as cells enter, undergo, and exit mitosis. Cells initially in interphase (t = 0:00) were observed to change shape, from flat to round (t = 9:20) and then divide to form daughter cells (t = 10:55) that flattened as they entered interphase (t = 14:40). New gap junction plaques were observed between daughter cells (t=14:40; also enlarged and shown with green channel only). (C) Gap junction plaque internalization into interphase cells in B. The gap junction plaque area has been enlarged, and the mEmerald channel (Cx43) is shown only. The mEmerald channel (Cx43) is shown only at 5-minute intervals for the first 1:20. Solid arrows follow the release of one annular gap junction over time, while dashed arrows follow the release and fission of a second annular gap junction. (D) Mitotic cell nanotubes from an area in B (t = 9:10) that has been enlarged to show Cx43-positive structures (arrows) visible along the mitotic nanotube as well as at both ends of the nanotube. (E) Mitotic nanotube changes shown with three-dimensional rotation of the images in A (t = 8:45 - 9:10). Note the change in Cx43-positive structure distribution within nanotubes over time and that the mitotic nanotubes remained above the substrate. Cells shown were synchronized, transfected to express mEmerald-Cx43 (green) and mCherry-CAAX (magenta), and imaged at 5-minute intervals. Scale bars = 5µm (A, C, D); 10µm (B, E).

Article Snippet: Plasmids encoding the gap junction markers mEmerald-Cx43 (Addgene, Watertown, MA, plasmid #54055) and Halo-Cx43 were gifts from Michael Davidson and John O’Brien, respectively.

Techniques: Imaging, Transfection

Journal: bioRxiv

Article Title: Intercellular Communication via Mitotic Nanotubes is Influenced by Connexin-43 Trafficking and Actin Remodeling

doi: 10.64898/2026.02.08.704470

Figure Lengend Snippet: (A) Mitotic nanotubes in the area between a rounding mitotic cell and a neighboring cell. The solid arrow (0-34 min) represents a linear Cx43-positive structure within the mitotic nanotube. With time, the Cx43-positive structure elongates (0-26 min), fragments (30 min), and moves toward the dividing cell (34 min) before being lost from view (38 min). (B) Transfer of a Cx43-positive structure via mitotic nanotubes. A Cx43-positive structure (arrowhead), which resembles an annular gap junction, moved between two cells and changed shape within the mitotic nanotube before entering the cytoplasm. Some Cx43-positive material remained at one end of the mitotic nanotube (arrows). (C) Movement of a Cx43-positive structure along a mitotic nanotube. A mitotic-nanotube-associated Cx43 structure (arrows), initially at one end of the mitotic nanotube (0 min), moves along a straight path toward the other cell (1.5-10.5 min) and eventually enters the cytoplasm of the other cell (13.5 min). In this figure, cells were transfected to express mEmerald-Cx43 (green) and mCherry-CAAX (magenta) and confocal Z-stacks (step size = 0.25µm) were captured at 4-minute (A) or 1.5-minute intervals (B, C). Scale bars = 5µm (A, C); 2.5µm (B).

Article Snippet: Plasmids encoding the gap junction markers mEmerald-Cx43 (Addgene, Watertown, MA, plasmid #54055) and Halo-Cx43 were gifts from Michael Davidson and John O’Brien, respectively.

Techniques: Transfection

Journal: Nature Communications

Article Title: Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex

doi: 10.1038/s41467-024-50411-z

Figure Lengend Snippet: Fluorescent scFv immuno-probes generated in this work

Article Snippet: After adherence, cells were transfected with mammalian expression plasmids encoding mEmerald-tagged GFAP (Addgene #54107 or Flag-tagged human calbindin (Origene # RC201358) using Lipofectamine 2000 (Thermo Fisher # 11668500) or Lipofectamine 3000 (Thermo Fisher # L3000001) transfection reagent following the manufacturer’s protocol.

Techniques: Generated, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex

doi: 10.1038/s41467-024-50411-z

Figure Lengend Snippet: a Representative confocal images ( n = 3 experiments in each category) of different sections from the cerebellum labeled with: a calbindin-specific scFv probe conjugated with Alexa Fluor 488, a VGluT1-specific scFv probe conjugated with Alexa Fluor 532, a GFAP-specific scFv probe conjugated with 5-TAMRA, a K v 1.2-specific scFv probe conjugated with Alexa Fluor 594, and a parvalbumin-specific scFv probe conjugated with Alexa Fluor 647. The double dotted lines delineate the Purkinje cell layer. (see Supplementary Figs. and for larger fields of view). CB calbindin, VGluT1 vesicular glutamate transporter 1, GFAP glial fibrillary acidic protein, K v 1.2 potassium voltage-gated channel subfamily A member 2, PV parvalbumin, TAM 5-TAMRA. b Workflow of multicolor imaging enabled by scFv probes and linear unmixing (see the text). c Representative maximum intensity projection of the multicolor fluorescence image stack acquired by linear unmixing of confocal images ( n = 3 experiments). The signal of each fluorescent dye was pseudo-colored for better visualization. d Enlarged boxed inset from ( c ). The arrow indicates a Bergmann fiber (GFAP-positive) adjacent to the main dendrite of a Purkinje cell. Arrowhead indicates sites where axons form a pinceau structure labeled by the K v 1.2-specific scFv probe.

Article Snippet: After adherence, cells were transfected with mammalian expression plasmids encoding mEmerald-tagged GFAP (Addgene #54107 or Flag-tagged human calbindin (Origene # RC201358) using Lipofectamine 2000 (Thermo Fisher # 11668500) or Lipofectamine 3000 (Thermo Fisher # L3000001) transfection reagent following the manufacturer’s protocol.

Techniques: Labeling, Imaging, Fluorescence

Journal: Nature Communications

Article Title: Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex

doi: 10.1038/s41467-024-50411-z

Figure Lengend Snippet: a The high-resolution EM volume acquired from the cerebellar lobule, Crus 1 with multicolor immunofluorescence from scFv probes separated by linear unmixing ( n = 1 experiment). The multicolor fluorescence data was co-registered with the high-resolution EM data. The Neuroglancer link to access the dataset is provided in the source data file. Numbers 1–4 indicate approximate regions where the ultrastructure was examined at high resolution ( n = 12 experiments). Owing to the absence of detergent in immunofluorescence labeling, fine ultrastructure was preserved throughout the EM volume, such as in the molecular layer (1), in the Purkinje cell layer (2), in the glomeruli in the granule cell layer (3), and in the granule cell bodies (4). b Demonstration of the overlay between fluorescence signals and EM ultrastructure. Left panel shows the multicolor six-channel fluorescent image of slice 250 ( n = 848 slices) of the spatially transformed fluorescence image volume. The middle panel shows three fluorescence channels corresponding to the labeling of CB, GFAP, and Hoechst overlaid onto the EM micrograph of slice 250. Right panel shows four fluorescent channels corresponding to the labeling of VGluT1, K v 1.2, PV, and Hoechst overlaid onto the EM micrograph of slice 250. Other examples of fluorescence overlay are shown in Supplementary Fig. .

Article Snippet: After adherence, cells were transfected with mammalian expression plasmids encoding mEmerald-tagged GFAP (Addgene #54107 or Flag-tagged human calbindin (Origene # RC201358) using Lipofectamine 2000 (Thermo Fisher # 11668500) or Lipofectamine 3000 (Thermo Fisher # L3000001) transfection reagent following the manufacturer’s protocol.

Techniques: Immunofluorescence, Fluorescence, Labeling, Transformation Assay

Journal: Nature Communications

Article Title: Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex

doi: 10.1038/s41467-024-50411-z

Figure Lengend Snippet: a 2D CLEM image showing the fluorescence signal (green) of the calbindin-specific scFv probe overlapping with the cell body of a Purkinje cell. b EM image showing 2D segmentation (green) of the calbindin-positive Purkinje cell ( n = 1). c 3D reconstruction of the Purkinje cell labeled in a ( n = 1), with the cell body in dark green and a dendritic branch in light green; three parallel fibers (red) make synapses on three spine heads of the dendritic branch (arrow indicates a parallel fiber (PF); arrowhead indicates a synapse). d EM image showing 2D segmentation of the synapse (arrowhead) between a parallel fiber (red) and a spine head of the dendritic branch (green) ( n = 1). e 2D CLEM image showing fluorescence signals (red) of the GFAP-specific scFv probe overlapping with the cell body of a velate astrocyte in the granule cell layer ( n = 1). f EM image showing 2D segmentation (red) of the velate astrocyte in ( e ) ( n = 1). g 3D reconstruction of the velate astrocyte (red) labeled in ( e ) and two nearby granule cells (GC1 and GC2, light and dark blue) ( n = 2); the astrocyte extends a veil-like glial process (arrowhead) between the two granule cells. h EM image showing 2D segmentation of the glial process (arrowhead) between GC1 and GC2 ( n = 1). i 2D CLEM image showing fluorescence signals (red) of the GFAP-specific scFv probe overlapping with a Bergmann fiber ( n = 1). j EM image showing 2D segmentation (red) of the Bergmann fiber in ( i ) ( n = 1). k 3D reconstruction of two Bergmann glial cells (BG1 and BG2) ( n = 2) traced from their Bergmann fibers labeled by the GFAP-specific scFv probe. l EM image showing 2D segmentation of the cell body of BG2 ( n = 1) and a nearby basket cell ( n = 1), noting the lack of infoldings in BG2’s nuclear membrane compared to the basket cell. n indicates an example.

Article Snippet: After adherence, cells were transfected with mammalian expression plasmids encoding mEmerald-tagged GFAP (Addgene #54107 or Flag-tagged human calbindin (Origene # RC201358) using Lipofectamine 2000 (Thermo Fisher # 11668500) or Lipofectamine 3000 (Thermo Fisher # L3000001) transfection reagent following the manufacturer’s protocol.

Techniques: Fluorescence, Labeling, Membrane

Journal: bioRxiv

Article Title: Muscle specific stress fibers give rise to sarcomeres and are mechanistically distinct from stress fibers in non-muscle cells

doi: 10.1101/235424

Figure Lengend Snippet: (A) Electron microscopy (EM) schematic of a cardiac sarcomere from adult mouse. Electron dense regions on border of sarcomere are Z-discs (Z), while the core of the sarcomere is composed of thin actin filaments and thick myosin II filaments (A). Multiple sarcomeres aligned adjacently form a myofibril (lower mag EM, right). (B) hiCM allowed to spread for 24 hours following plating. Notice the clear stress fiber and sarcomere-like actin organization at the front and rear of the cell in box 1 and 2, respectively. (C) Spread U2-OS cell (left) and HeLa cell (right) displaying prominent actin arc stress fibers behind leading edge of cell (yellow arrow). (D) Percentage of hiCMs, U2-OS, and HeLa cells with actin arc stress fibers. hiCMs; 1372 cells over 3 experiments. U2OS; 37 cells over 4 experiments. HeLa; 186 cells over 4 experiments. (E) Wide-field time lapse of hiCM transfected with Lifeact-mEmerald to visualize actin. MSF at front of hiCM undergoes retrograde flow and acquires sarcomeres (arrow). (F) Laser-scanning confocal microscopy of MSF to sarcomere transition. High magnification montage on right from blue box in low mag still image (left) (G) Still of U2-OS cell expressing Lifeact-mEmerald (left). Kymograph (right) taken from purple line of left image. Note robust movement of actin arc stress fibers (yellow arrow). (H) Still of hiCM expressing Lifeact-mEmerald (left). Kymograph (right) taken from purple line of left image. Note slower movement of MSF in hiCM compared to actin arcs in U2-OS cell , and stationary nature of sarcomeres. (I) Quantification of actin stress fiber translocations rates in U2-OS cells and hiCMs. U2OS; 5 cells over 4 experiments. hiCMs; 12 cells over 3 experiments. Scale Bars; (A) 500 nm high mag, 2 μm low mag (B), (C), (E) middle and right, (F), (G) 10 μm, (E) left 5 μm.

Article Snippet: Plasmid encoding NMIIB-(N-terminal)-mEmerald was purchased from Addgene (54192; Addgene, Cambridge, MA).

Techniques: Electron Microscopy, Transfection, Confocal Microscopy, Expressing

Journal: bioRxiv

Article Title: Muscle specific stress fibers give rise to sarcomeres and are mechanistically distinct from stress fibers in non-muscle cells

doi: 10.1101/235424

Figure Lengend Snippet: (A) SIM showing NMIIA (left) and NMIIB (right) localized to the edge of hiCMs with MSFs. (B) Line-scans starting from edge of hiCMs showing peak localizations of NMIIA (black) and NMIIB (red) NMIIA: 15 cells, 2 experiments; NMIIB: 32 cells, 4 experiments. (C) hiCM transfected with NMIIA-mEmerald (N-terminal motors) and stained for endogenous NMIIB (left). High-mag views of NMIIA-NMIIB co-filaments (right, top). Line scans across NMII co-filaments above, from N-terminal motors (purple) and C-terminal rod domains (green). (D) Quantification of NMII cofilament length. Histogram displays the distribution of NMII co-filament lengths (motor-domain to motor-domain) NUMBER OF FILAMENTS. (E) Actin of representative control, NMIIA KD (siRNA MYH9), and NMIIB KD (siRNA MYH10) hiCMs allowed to spread for 24hrs. NMIIA KD hiCMs display disorganized sarcomeres, while NMIIB KD hiCMs display no actin-based sarcomeres. (F) Representative western blots of 2 experiments showing knockdown of NMIIA (siRNA MYH9) and NMIIB (siRNA MYH10). (G) Percentage of control, NMIIA KD (siRNA MYH9), and NMIIB KD (siRNA MYH10) hiCMs with actin based sarcomeres at 24hrs spread. Control: 49 cells, 6 experiments; NMIIA KD: 34 cells, 3 experiments; NMIIB KD: 59 cells, 4 experiments. (H) Line-scans of NMIIA and NMIIB from NMIIA (MYH9 KD) and NMIIB (MYH10 KD) KD hiCMs respectively. NMIIA KD: 16 cells 2 experiments; NMIIB KD: 42 cells, 4 experiments. Note how both NMIIA and NMIIB KD cells lose peak localization at leading edge. Scale bars; (A) 5 μm, (C) left 10 μm, (C) right 200 nm, (E) 5 μm.

Article Snippet: Plasmid encoding NMIIB-(N-terminal)-mEmerald was purchased from Addgene (54192; Addgene, Cambridge, MA).

Techniques: Transfection, Staining, Western Blot

Journal: bioRxiv

Article Title: Muscle specific stress fibers give rise to sarcomeres and are mechanistically distinct from stress fibers in non-muscle cells

doi: 10.1101/235424

Figure Lengend Snippet: (A) hiCM from transfected with NMIIA-mEmerald (N-terminal motors) and stained for NMIIB rods. Schematic depicts visualization strategy. Low mag of hiCM left, and high mag examples bottom right. (B) hiCM transfected with PCMII-mEGFP (N-terminal motors) and stained for endogenous NMIIB rods. Schematic depicts visualization strategy. Low mag of hiCM left, and high mag examples bottom right. (C) Low mag view of P3 mouse heart tissue stained for βCMII (motors) and NMIIB (rods). As in hiCMs, NMIIB is restricted from sarcomere structures but is localized adjacently to sarcomeres. Arrow indicates area of possible co-filaments. High mag example shown at right is taken from P3 mouse tissue imaged on Zeiss 880 with AiryScan from similar area indicated by arrow on low mag image. Schematic indicates visualization strategy. (D) High mag examples of NMIIA (left) and NMIIB-βCMII co-filaments from human hypertrophic cardiomyopathy patients imaged on Zeiss 880 with AiryScan. Cartoon schematic indicates imaging strategy. Scale bars: (A), (B), 10 μm (left); (A), (B), (C), (D), 200 nm (right).

Article Snippet: Plasmid encoding NMIIB-(N-terminal)-mEmerald was purchased from Addgene (54192; Addgene, Cambridge, MA).

Techniques: Transfection, Staining, Imaging

Journal: ACS central science

Article Title: Virus-like Nonvirus Cationic Liposome for Efficient Gene Delivery via Endoplasmic Reticulum Pathway.

doi: 10.1021/acscentsci.9b01052

Figure Lengend Snippet: Figure 1. Synthesis of a novel cationic liposome and its gene transfection efficiency. (A) Three-dimensional schematic of the antimicrobial peptide pardaxin. The pardaxin sequence, NH2-G-F-F-A-L-I-P-K-I-I-S-S-P-L-F-K-T-L-L-S-A-V-G-S-A-L-S-S-G-G-Q-E, is composed of two α-helices with a proline residue acting as a hinge between the two helices. The structural image was acquired from the NCBI protein data bank (https://www.ncbi. nlm.nih.gov/protein/1502235A). (B,C) Schematic and characterization of the Lipo-Par and Lipo-Non cationic liposomes. The scale bar of (C) represents 500 μm. (D,E) pEGFP transfection efficiency of the different cationic liposomes in different cell lines is shown. The mass ratio of the Lipofectamine 2000 (Lipo-2000) to pEGFP was 1.5:1. The mass ratio of the Lipo-Par or Lipo-Non to pEGFP was 6:1. (E) Results from the quantitative analysis of (D) using ImageJ. The scale bar represents 500 μm. (F) Agarose gel electrophoresis results. The mass ratios of the liposomes to pEGFP, from left to right, are 0:1, 1:1, 2:1, 3:1, 4:1, 5:1, and 6:1. When the ratio was 6:1, the DNA was tightly bound to the two liposomes. (G) DiD-lipo and FITC-DNA complexes were synthesized. The sequence of nonbiological functional FITC-DNA is 5′-FITC- CAGACCGACTGGATCT-3′. The mass ratio of the liposomes to the DNA was 6:1. The liposome is green, DNA is red, and the overlapping parts are yellow. The scale bar represents 500 μm. (H,I) FITC-DNA delivery of different cationic liposomes after 24 h treatment is shown. Cells were cultured with liposomes/DNA complexes for 2 h and cultured with fresh media for an additional 22 h. The scale bar of (H) represents 200 μm. (I) Results from the quantitative analysis of (H).

Article Snippet: An EGFP plasmid (pEGFP) was obtained that encoded only the reporter gene EGFP. mCherry-ER was a gift from Michael Davidson (Addgene plasmid #55041). mEmerald-tubulin-6 was a gift from Michael Davidson (Addgene plasmid #54291).

Techniques: Transfection, Sequencing, Residue, Liposomes, Agarose Gel Electrophoresis, Synthesized, Functional Assay, Cell Culture

Journal: ACS central science

Article Title: Virus-like Nonvirus Cationic Liposome for Efficient Gene Delivery via Endoplasmic Reticulum Pathway.

doi: 10.1021/acscentsci.9b01052

Figure Lengend Snippet: Figure 2. Internalization mechanism of the Lipo-Pars. (A) Schematic of Lipo-Par and Lipo-Non internalization. Common internalization pathways include those that are clathrin mediated, caveolin mediated, and macropinocytosis. The novel cationic liposome Lipo-Par enters cells mainly through the caveolin-mediated pathway, which is known to follow a nonlysosomal route. (B) Results from the investigation into Lipo-Par and Lipo- Non internalization. Cells were pretreated for 30 min with different chemical inhibitors to block relevant internalization pathways (CPZ, 25 μM; filipin, 5 μg/mL; indomethacin, 50 μM; amiloride, 50 μM; colchicine, 25 μM; and M-β-CD, 10 μg/mL) and then incubated with DiD-labeled Lipo-Pars or Lipo-Nons for 2 h. Images of representative single cells are enlarged. The lipid raft, a key component of caveosomes and clathrin- coated vesicles, plays a key role in both Lipo-Par and Lipo-Non uptake. Each liposome can enter cells in different ways, but the internalization of the Lipo-Pars was mediated mainly by caveolin and that of the Lipo-Nons was mediated mainly by clathrin. The scale bar represents 100 μm. (C) Results from the quantitative analysis of the flow cytometry experiments (values are from the values of the non-Blank group minus the value of the Blank group (1.1%) in the Figure S2A). Cells were pretreated for 30 min with different chemical inhibitors and transfected with pEGFP for 2 h,

Article Snippet: An EGFP plasmid (pEGFP) was obtained that encoded only the reporter gene EGFP. mCherry-ER was a gift from Michael Davidson (Addgene plasmid #55041). mEmerald-tubulin-6 was a gift from Michael Davidson (Addgene plasmid #54291).

Techniques: Blocking Assay, Incubation, Labeling, Cytometry, Transfection

Journal: ACS central science

Article Title: Virus-like Nonvirus Cationic Liposome for Efficient Gene Delivery via Endoplasmic Reticulum Pathway.

doi: 10.1021/acscentsci.9b01052

Figure Lengend Snippet: Figure 4. Effect of ER stress on gene transfection. (A−C) Results from the test used to determine whether intracellular calcium level changes affected gene transfection. In (A), the cells were pretreated with 10 μM BAPTA-AM, a cell permeable calcium chelating agent, for 30 min, followed by incubation with liposome/DNA complexes for 2 h at 37 °C. In (B), the cells were pretreated with 5 μM or 10 μM BAPTA-AM for 30 min and then transfected with pEGFP. After transfection, the cells were incubated with 5 μM or 10 μM BAPTA-AM for an additional 22 h. In (C), the results from the quantitative analysis of (B) using ImageJ are shown. The scale bar of (A) represents 200 nm, and the scale bar of (B) represents 500 μm. The experiment was done three times. (D) Uptake of the liposomes when the intracellular calcium level was disrupted. Cells were pretreated with 10 μM BAPTA-AM for 30 min, followed by incubation with Lipo-Pars or Lipo-Nons (green) for 2 h at 37 °C. After liposome uptake, the cells were incubated with BAPTA-AM for an additional 1, 4, 10, and 22 h. The scale bar represents 100 μm. (E) Results from experiments used to determine whether ER stress affected gene transfection. Cells were pretreated with BFA (2 μg/mL) and transfected for 2 h. The scale bar represents 500 μm. The experiment was done three times. (F) Results from experiments used to determine how weakened ER stress

Article Snippet: An EGFP plasmid (pEGFP) was obtained that encoded only the reporter gene EGFP. mCherry-ER was a gift from Michael Davidson (Addgene plasmid #55041). mEmerald-tubulin-6 was a gift from Michael Davidson (Addgene plasmid #54291).

Techniques: Transfection, Incubation, Liposomes

Journal: ACS central science

Article Title: Virus-like Nonvirus Cationic Liposome for Efficient Gene Delivery via Endoplasmic Reticulum Pathway.

doi: 10.1021/acscentsci.9b01052

Figure Lengend Snippet: Figure 6. Microtubules play a critical role in the efficient internalization of the Lipo-Pars. (A) Distribution of the liposomes when microtubules were depolymerized by 25 μM colchicine. Liposomes (green) are distributed randomly throughout the cells. The scale bar is 20 μm. (B−D) Results from the gene transfection comparison when the microtubules were depolymerized. Cells were pretreated with 25 μM colchicine, followed by pEGFP transfection for 2 h at 37 °C. After transfection, the cells were cultured for an additional 22 h. Flow cytometry was used to analyze transfection efficiency quantitatively, and the results are shown in (C) and (D). The scale bar of (B) is 500 μm. The experiment was done once. (E,F) 3D high-resolution analysis of the colocalization of the Lipo-Pars with the microtubules (MT). Plasmid coding mEmerald-tubulin was transfected using Lipofectamine 2000 and the Lipo-Pars (10 μg/mL) were allowed to internalize for 1 h (E) or 2 h (F) at 37 °C. Cells were fixed with 4% paraformaldehyde and observed using high-resolution laser confocal microscopy (LSM 880 with Airyscan, Carl Zeiss Jena, Germany). Representative parts of the cells are enlarged. Multichannel photos were taken on the same focal plane. The scale bar is 50 μm. (G) High-resolution colocalization analysis of the Lipo-Pars and microfilaments. Polymeric F-actin (red) was dyed using phalloidin−tetramethylrhodamine B isothiocyanate (Sigma-Aldrich) following the protocol. Multichannel photos were taken on the same focal plane. The scale bar represents 50 μm.

Article Snippet: An EGFP plasmid (pEGFP) was obtained that encoded only the reporter gene EGFP. mCherry-ER was a gift from Michael Davidson (Addgene plasmid #55041). mEmerald-tubulin-6 was a gift from Michael Davidson (Addgene plasmid #54291).

Techniques: Liposomes, Transfection, Comparison, Cell Culture, Flow Cytometry, Plasmid Preparation, Confocal Microscopy

Journal: ACS central science

Article Title: Virus-like Nonvirus Cationic Liposome for Efficient Gene Delivery via Endoplasmic Reticulum Pathway.

doi: 10.1021/acscentsci.9b01052

Figure Lengend Snippet: Figure 7. High levels of Lipo-Par transfection are independent of mitosis. (A) Schematic of the distribution of the microtubules and the ER during mitosis showing that Lipo-Pars are moving along the microtubules quickly to deliver genes to the nucleus. (B,C) pEGFP transfection when mitosis was inhibited by PTX (1 μg/mL). The scale bar represents 500 μm. The experiment was done three times. (D) Colocalization of the Lipo-Pars and the ER when mitosis was inhibited by PTX (1 μg/mL). The scale bar represents 50 μm. (E,F) Results from the experiments conducted to determine whether the Lipo-Pars or Lipo-Nons disrupted the cell cycle. Double-stranded DNA was stained with propidium iodide (PI), and flow cytometry was performed according to the product specification (cell cycle and apoptosis analysis kit, Beyotime). The percentage of cells in G2 is shown in (F). The experiment was done once. (G) Colocalization of the Lipo-Pars to the ER when cells were in mitosis. The merged parts are yellow. The scale bar represents 50 μm. (H) When the cells are in mitosis, the DNA carried by the Lipo-Pars is more likely to enter the nucleus. In telophase, the blue stain of the nucleus appeared more intense, and more of the foreign FITC-DNA (red) had entered the nucleus due to the incompleteness of the nuclear membrane. The images were captured after the cells were treated with Lipo-Par/DNA complexes for 10 or 60 min. The scale bar represents 50 μm. (I) Results from the high-resolution analysis of the distribution of the FITC-DNA carried by the Lipo-Pars when cells were in telophase. The plasmid coding mCherry-(calreticulin and KDEL) was trans- fected using Lipofectamine 2000. DNA is green, and the ER is red. Representative parts of the cells are enlarged. The scale bar represents 20 μm.

Article Snippet: An EGFP plasmid (pEGFP) was obtained that encoded only the reporter gene EGFP. mCherry-ER was a gift from Michael Davidson (Addgene plasmid #55041). mEmerald-tubulin-6 was a gift from Michael Davidson (Addgene plasmid #54291).

Techniques: Transfection, Staining, Cytometry, Membrane, Plasmid Preparation