Journal: Cancer Communications

Article Title: NHE7 upregulation potentiates the uptake of small extracellular vesicles by enhancing maturation of macropinosomes in hepatocellular carcinoma

doi: 10.1002/cac2.12515

Figure Lengend Snippet: NHE7 co‐localizes with Rab21 and promotes the maturation of macropinosomes. (A) Immunofluorescence staining of NHE7 (green), endosomal markers (red), EEA1, Rab7, Rab11a and Rab21, and the trans‐Golgi network marker Golgin‐97 in MHCC97L ( left ) and MHCCLM3 ( right ) cells. Cells were also stained with DAPI (blue). Coimmunofluorescence staining (B) and quantification (C) of NHE7 (green) and Rab21 (red) expression in MHCC97L nontargeting control (CTL‐KD) and NHE7 knockdown (NHE7‐KD) cells. HLE vector control (CTL‐SAM) and NHE7‐overexpressing (NHE7‐SAM) cells were subjected to coimmunofluorescence staining (D) and the signal was analyzed (E) The percentage of NHE7 co‐localized with Rab21 was quantified, and the mean AF568 fluorescence intensity was determined. Immunoblotting was performed to evaluate Rab21 expression in MHCC97L and HLE cells. Data are presented as the means ± SEMs. ** P < 0.01; *** P < 0.001. Abbreviations: AF, Alexa Fluor; CTL, ctrl; DAPI, 4′,6‐diamidino‐2‐phenylindole; EEA1, early endosome antigen 1; KD, knockdown; NHE7, Na(+)/H(+) exchanger 7; Rab21, Ras‐associated binding protein 21; SAM, synergistic activation mediator; SEMs, standard error of the means.

Article Snippet: Primary antibodies against NHE7 (1:100; #PA5‐61843, Invitrogen), early endosome antigen 1 (1:100; #ab70521, EEA1, Abcam), Rab7 (1:100; #95746, Cell Signaling Technology, Danvers, MA, USA), Rab11a (1:100; #STJ140068, St John's Laboratory, London, UK), Rab21 (1:100; #sc‐81917, Santa Cruz Biotechnology), or Golgin‐97 (1:100; #A21270, Invitrogen) was used to treat the cells.

Techniques: Immunofluorescence, Staining, Marker, Expressing, Control, Knockdown, Plasmid Preparation, Fluorescence, Western Blot, Binding Assay, Activation Assay

Journal: Advanced Science

Article Title: Delivering Antisense Oligonucleotides across the Blood‐Brain Barrier by Tumor Cell‐Derived Small Apoptotic Bodies

doi: 10.1002/advs.202004929

Figure Lengend Snippet: sCABs are transcytosed by BMECs to cross the BBB. a) TEM images showing a phagosome containing a sCAB in the BMEC of a mouse 10 min after an i.v. sCABs injection. The right panel shows a magnified view of the phagophore selected by the red square and arrow. Scale bar: 500 nm. b) TEM images showing the structure of TJs after sCABs passed through microvessels 15 min after an i.v. sCABs injection. The TJs in the red squares are shown at high magnifications on the right. The red arrow depicts sCABs that passed through BMECs. Scale bar: 500 nm. c) Representative fluorescence images showing the intact structure of TJs after sCABs treatment containing Cy5‐ASO. The right panels show magnified views of the area selected by the white square. Scale bar: 25 µm. d) Representative fluorescence microscopy images showing sCABs containing Cy5‐ASO phagocytized by b. End3 cells in the BBB model. Scale bar: 25 µm. e) Representative fluorescence images showing Cy5‐ASO delivered by sCABs but not the naked one was phagocytized by microglial cells in the BBB model 24 h after the incubation. The morphology of microglial cells as imaged with differentia linterference contrast (DIC). Scale bar: 25 µm. f) Fluorescence images showing GFP‐labeled sCABs containing Cy5‐ASO phagocytized by microglia 24 h after the incubation. The right panels show magnifications of the area selected by the white square. Scale bar: 10 µm. g–k) Representative fluorescence images showing the colocalization of sCABs containing Cy5‐ASO with special protein markers ((g) labeled with clathrin, (h) with caveolin‐1, (i) with EEA‐1, (h) with Rab11 involved in endocytosis, and (k) with Snap23). The colocalization of clathrin and caveolin‐1 was examined at 5 min after sCABs incubation, EEA‐1 and Rab11 at 10 min, and Snap23 at 20 min. The right panels show magnifications of the areas selected by the white square. Scale bar: 25 µm. Images are representative of three independent experiments.

Article Snippet: EEA‐1 (anti‐EEA‐1 antibody, BM4402, Boster, China), an intracellular marker of early endosomes, was tested at 10 min, as was Rab11 (anti‐Rab11B antibody, A04526‐1, Boster, China), a marker of recycling endosomes.

Techniques: Injection, Fluorescence, Microscopy, Incubation, Labeling

Journal: The Journal of Cell Biology

Article Title: Clarin-1 acts as a modulator of mechanotransduction activity and presynaptic ribbon assembly

doi: 10.1083/jcb.201404016

Figure Lengend Snippet: Clarin-1 is involved in vesicle recycling and endocytosis. (A) Bar graph of fast dye uptake recovery after tip link disruption by BAPTA (means ± SEM; number of independent experiments, n = 3). (B) Semiquantitative analysis of the total fluorescence per neuromast. Means ± SEM. ***, P < 0.001 versus control; +++, P < 0.001 versus clarin-1 (Clrn1) MO + cRNA; ˄˄˄, P < 0.001 versus clarin-1 MO + cRNAΔCT. Number of independent experiments, n > 5. (C–F) Representative images from the semiquantitative analysis in B. Long-term FM1-43 incorporation of 3 dpf larvae preincubated with BAPTA. (G–J) Representative images of at least five independent experiments. Expression of the early endosomal marker rab11a/b. Arrowheads denote apical accumulation of rab11a/b. Bar, 5 µm. a.u., arbitrary unit.

Article Snippet: Other antibodies used in this study were affinity-purified mouse anti-myosin7A ( ; ), rabbit anti–clarin-1 DrClrn1 (provided by J. Phillips, University of Oregon, Eugene, OR; ), mouse anti-cc2d2a (provided by R. Bachmann-Gagescu, University of Washington, Seattle, WA; ), mouse anti–ribeye b , rabbit anti-rab11a/b (GeneTex, Inc.), mouse anti-His tag (Thermo Fisher Scientific), mouse anti–acetylated tubulin (T6793; Sigma-Aldrich; RRID: AB_477585), mouse anti–HCS-1 and mouse anti-ZN12 (Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-MAGUK (75–030; Neuromab; RRID: AB_2091920), goat anti–-γ-tubulin (sc-12881; Santa Cruz Biotechnology, Inc.; RRID: AB_641317), and mouse anti-panactin (MAB1501; EMD Millipore; RRID: AB_2223041).

Techniques: Disruption, Fluorescence, Control, Expressing, Marker