Journal:

Article Title: Dynein-mediated Vesicle Transport Controls Intracellular Salmonella Replication

doi: 10.1091/mbc.E03-08-0614

Figure Lengend Snippet: SCV recruits functional Rab7. (A) Confocal fluorescence analysis of HeLa cells transiently transfected with GFP-Rab7 and infected with S. typhimurium SL1344. The SCV recruited GFP-Rab7 after 30 min of infection. Representative confocal images at 3 h and after o/n culture are shown. The insets in the top panels show a coat of GFP-Rab7 on the SCV that is representative for the accumulation of Rab7 observed at all time points. The majority of the SCV contained elongated bacteria after o/n culture, still colocalizing with GFP-Rab7 (bottom). Left, GFP-Rab7; middle, anti-Salmonella LPS; and right, merge. Bar, 10 μm. (B) Quantification of three experiments for colocalization of the SCV with GFP-Rab7. Values are given as mean percentage of colocalization ± SE. t = 0.5 h is the time point immediately after the 30 min infection (for each time point 100 cells were counted). (C) To determine whether the Rab7 recruited to the SCV is functional, the GTPase cycle of Rab7 was studied using FRAP. Representative examples of GFP-Rab7 cells are shown in a glow-over/under representation. Images are shown before bleach (prebleach), immediately after bleach and at three time points after bleach. The bleached area and the nucleus (N) are indicated. Top, SCV indicated with a rectangle and control late endocytic structures in the circle were bleached. Bottom, noninfected control cell was bleached in the circle. Bar, 10 μm. (D) Representative curves of fluorescent recovery in the bleached spot/area. The fluorescence (percentage) was related to the initial fluorescence set at 100%. t = 0 s is the first image after the bleach. (E) Quantification of the recovery time (t1/2) deduced from the curves including the SE. There is no difference between the t1/2 of SCV and late endocytic structures (control) even within the same cell. (F) Sucrose gradient fractionation profile of vesicles from cells infected with GFP-S. typhimurium SL1344. Lysosomes were detected by β-hexosaminidase activity and mature cathepsin D staining. The density (molar concentration sucrose) of the corresponding fractions measured with a refractomer is indicated (top). The localization of the SCV was determined with anti-GFP antibodies. LAMP-1, Rab7, and RILP localization were analyzed with specific antibodies. Bottom, corresponding Western blots for LAMP-1, RILP, Rab7, and GFP. The localization of the soluble fraction, lysosomes, and the SCV are indicated.

Article Snippet: Wild-type and mutant Rab7 cDNA, a kind gift from P. Chavier ( Meresse et al ., 1995 ; Meresse et al ., 1999a ), were subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) with an N-terminal myc-tag for immunodetection.

Techniques: Functional Assay, Fluorescence, Transfection, Infection, Fractionation, Activity Assay, Staining, Concentration Assay, Western Blot

Journal:

Article Title: Dynein-mediated Vesicle Transport Controls Intracellular Salmonella Replication

doi: 10.1091/mbc.E03-08-0614

Figure Lengend Snippet: Expression of RILP results in a replication block of Salmonella. (A) Confocal analysis of infected cells expressing GFP-Rab7 microinjected with RILP and H2B-GFP. From left to right, GFP-Rab7 and H2B-GFP, Salmonella SL1344 labeled with anti-LPS-Texas Red, RILP, and the merge. Bar, 10 μm. (B) Confocal analysis of cells infected with either SL1344 (top) or the 12023 Salmonella strain (bottom) and microinjected with RILP. Cells microinjected with RILP cDNA (and H2B-GFP cDNA as injection marker) show clustering of late endosomes and lysosomes. In these cells, the elongated bacteria are not formed after o/n incubation (top). Note that elongated bacteria occurred in control cells. Microinjected cells are indicated with an asterisk. Left, H2B-GFP and Salmonella (stained with anti-LPS) are shown. Middle, CD63 staining. Right, merge. Bar, 10 μm. Bottom, cells infected with the Salmonella 12023 strain. Microinjected cells are indicated with an asterisk. Left, Salmonella 12023 strain. Middle, RILP. Right, merge. Bar, 10 μm. (C) Quantification of five individual experiments (n = 30–40 microinjected cells and n = 100 control cells). Left, mean percentage of SCV-containing elongated bacteria or 10–0 single bacteria are indicated ± SE for both RILP and control cells infected with the Salmonella SL1344 strain. Right, SCV containing >30 bacteria/cell or 10–0 single bacteria/cell are indicated ± SE for both RILP and control cells infected with the Salmonella 12023 strain.

Article Snippet: Wild-type and mutant Rab7 cDNA, a kind gift from P. Chavier ( Meresse et al ., 1995 ; Meresse et al ., 1999a ), were subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) with an N-terminal myc-tag for immunodetection.

Techniques: Expressing, Blocking Assay, Infection, Labeling, Injection, Marker, Incubation, Staining

Journal: bioRxiv

Article Title: Direct observation of aggregate-triggered selective autophagy

doi: 10.1101/2021.04.21.440799

Figure Lengend Snippet: a) Coding strand of knock-in target site before exon 1 (black box) of the RAB7A locus. The sgRNA target site (orange) and PAM sequence (cyan) are indicated. Insert is EGFP followed by a linker sequence (grey box). DNA sequencing of the homozygous knock-in clone is aligned below. b) 1% agarose gel including PCR-amplification product of RAB7A locus of WT and EGFP-Rab7 KI lines.

Article Snippet: The HeLa GFP-RAB7 line was generated by transfecting HeLa cells (ATCC) with the donor plasmid pDONOR-GFP-RAB7 and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, #62988) bearing the appropriate targeting sequence (5’-TAGTTTGAAGGATGACCTCT-3’; pX459v2-RAB7a sg3).

Techniques: Knock-In, Sequencing, DNA Sequencing, Agarose Gel Electrophoresis, Amplification

Journal: bioRxiv

Article Title: Direct observation of aggregate-triggered selective autophagy

doi: 10.1101/2021.04.21.440799

Figure Lengend Snippet: a) Endogenous GFP-RAB7 KI HeLa cell line transiently showing mKeima-PIM aggregates. b) Endogenous GFP-RAB7 signal showing larger RAB7 positive vesicles at the perinuclear area and smaller vesicles in the periphery. c) Zoom of the cell in (a) showing aggregates at neutral pH (green) and low pH (red) and the correlation with RAB7. d) Selected frames from a time-lapse movie showing the gradual recruitment of RAB7 and concomitant decrease of mKeima ratio. e) Analysis of RAB7 intensity (cyan) and mKeima ratio (black) of a 10 typical acidification events, including the one shown in (d). Data shown are mean (solid line) ± s.d. (shaded area) of 3-10 events. f-g) Time-lapse imaging of GFP-RAB7 KI HeLa cells showing putative kiss-and-run events with RAB7 positive vesicles (g) and full fusion event of RAB7 positive vesicle with the autolysosome (f). Scale bars: 10 µm (a) and 2µm (b,d-g)

Article Snippet: The HeLa GFP-RAB7 line was generated by transfecting HeLa cells (ATCC) with the donor plasmid pDONOR-GFP-RAB7 and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, #62988) bearing the appropriate targeting sequence (5’-TAGTTTGAAGGATGACCTCT-3’; pX459v2-RAB7a sg3).

Techniques: Imaging

Journal: bioRxiv

Article Title: Direct observation of aggregate-triggered selective autophagy

doi: 10.1101/2021.04.21.440799

Figure Lengend Snippet: a) Monitoring a forming autolysosome in the perinuclear region (see schematic in b). Frames from a time-lapse movie depicting multiple rounds of transient associations with RAB7 positive vesicles (1.), subsequent fusion (2.) and full acidification (3.). Arrows and arrowheads indicate interactions and absence of interactions, respectively. b) Analysis of RAB7 intensity and mKeima ratio of (a). Scale bar: 2 µm

Article Snippet: The HeLa GFP-RAB7 line was generated by transfecting HeLa cells (ATCC) with the donor plasmid pDONOR-GFP-RAB7 and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, #62988) bearing the appropriate targeting sequence (5’-TAGTTTGAAGGATGACCTCT-3’; pX459v2-RAB7a sg3).

Techniques:

Journal: bioRxiv

Article Title: Direct observation of aggregate-triggered selective autophagy

doi: 10.1101/2021.04.21.440799

Figure Lengend Snippet: First, DFCP1 is recruited to aggregates (omegasome formation). DFCP1 puncta are present only for a short time but the timing of DFCP1 puncta presence relative to acidification is variable (patterned area). Just before acidification of the cargo, STX17 is recruited for ∼7 min and STX17 leaves the autophagosome after start of cargo acidification. RAB7 is gradually recruited to the autophagolysosome while cargo acidification is also taking place.

Article Snippet: The HeLa GFP-RAB7 line was generated by transfecting HeLa cells (ATCC) with the donor plasmid pDONOR-GFP-RAB7 and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, #62988) bearing the appropriate targeting sequence (5’-TAGTTTGAAGGATGACCTCT-3’; pX459v2-RAB7a sg3).

Techniques:

Journal: Aging (Albany NY)

Article Title: LncRNA PVT1 promotes exosome secretion through YKT6, RAB7, and VAMP3 in pancreatic cancer

doi: 10.18632/aging.103268

Figure Lengend Snippet: PVT1 and exosome secretion-associated factors are increased in PC cell lines. ( A ) The expression of PVT1 in PC cell lines. ( B ) The mRNA expression of YKT6 in PC cell lines. ( C ) The expression of RAB7 in PC cell lines. ( D ) The expression of VAMP3 in PC cell lines. * P < 0.05, data are expressed as the mean ± SD.

Article Snippet: The primary antibodies used were as following: ras-related protein Rab-7 (RAB7) (1:100), CD63 (1:200), YKT6 v-SNARE homolog (YKT6) (1:100), and vesicle-associated membrane protein 3 (VAMP3) (1:100) (Applied Biological Materials Inc., Richmond, BC, Canada).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: LncRNA PVT1 promotes exosome secretion through YKT6, RAB7, and VAMP3 in pancreatic cancer

doi: 10.18632/aging.103268

Figure Lengend Snippet: PVT1 affects the expression and localization of RAB7 in HS766T cells. ( A ) The mRNA expression of Rab GTPases genes in PVT1-overexpressing HS766T cells. ( B ) The protein expression of RAB7 in PVT1-overexpressing HS766T cells. ( C ) Analysis of RAB7 (green) and CD63 (red) in PVT1-overexpressing HS766T cells, as determined by confocal microscope. ( D ) The interaction between PVT1 and RAB7, as determined by RIP assay and qRT-PCR. ( E ) The correlation between PVT1 and RAB7, as determined by pull-down assay. ( F ) The knockdown efficiency of si-RAB7 in HS766T cells. ( G ) The concentration of exosome derived from HS766T cells with overexpression of PVT1 and knockdown of RAB7. * P < 0.05, data are expressed as the mean ± SD.

Article Snippet: The primary antibodies used were as following: ras-related protein Rab-7 (RAB7) (1:100), CD63 (1:200), YKT6 v-SNARE homolog (YKT6) (1:100), and vesicle-associated membrane protein 3 (VAMP3) (1:100) (Applied Biological Materials Inc., Richmond, BC, Canada).

Techniques: Expressing, Microscopy, Quantitative RT-PCR, Pull Down Assay, Concentration Assay, Derivative Assay, Over Expression

Journal: Aging (Albany NY)

Article Title: LncRNA PVT1 promotes exosome secretion through YKT6, RAB7, and VAMP3 in pancreatic cancer

doi: 10.18632/aging.103268

Figure Lengend Snippet: Primer information.

Article Snippet: The primary antibodies used were as following: ras-related protein Rab-7 (RAB7) (1:100), CD63 (1:200), YKT6 v-SNARE homolog (YKT6) (1:100), and vesicle-associated membrane protein 3 (VAMP3) (1:100) (Applied Biological Materials Inc., Richmond, BC, Canada).

Techniques: Sequencing