Journal: Cells

Article Title: Globoside Is an Essential Intracellular Factor Required for Parvovirus B19 Endosomal Escape

doi: 10.3390/cells13151254

Figure Lengend Snippet: Endosome-to-Golgi retrograde transport of B19V and MVM. ( A ) UT7/Epo cells were treated with increasing concentrations of Retro-2 at 37 °C for 15 min before infection with B19V (10 5 geq/cell). After 30 min, cells were washed to remove unbound virus and 24 hpi NS1 mRNA was quantified by RT-qPCR. Knockdown of Rab9 in UT7/Epo cells by RNAi. Cells were transfected with either Rab9 siRNA, a scrambled siRNA, mock-transfected (Lipofectamine), or left untreated. Two days after transfection, knockdown efficiency was assayed by ( B ) Western blot or ( C ) RT-qPCR. ( D ) B19V infection in Rab9 knockdown UT7/Epo cells. Rab9 knockdown cells were infected with B19V (10 5 geq/cell), and NS1 mRNA was quantified 24 hpi by RT-qPCR. ( E ) Endosomal integrity in MVM-infected A9 cells. Cells were infected with MVM (2 × 10 3 geq/cell) in the presence of absence of tetramethylrhodamine-labeled dextran (DXT, 3 kDa, 0.1 mg/mL) at 37 °C for 30 min. Cells were then either fixed and stained with mAb B7 against MVM capsids (upper panel) or incubated at 37 °C for up to 5 h and analyzed directly using fluorescence microscopy (lower panels). Effect of BFA ( F ), GCA ( G ) and Retro-2 ( H ) on MVM infection in A9 cells. Drugs were added 15 min prior to MVM infection or added 5 hpi. Unbound virus was removed after 30 min and NS1 mRNA was quantified 24 hpi by RT-qPCR. ( I ) Rab9 knockdown efficiency in A9 cells was quantified by Western blot as described in ( B ). ( J ) MVM infection in Rab9 knockdown A9 cells. Rab9 knockdown cells were infected with MVM (2 × 10 3 geq/cell), and NS1 mRNA was quantified 24 hpi by RT-qPCR. Results are presented as the mean of two ( A , J ) or three ( C , D , F – H ) independent experiments ± standard deviation (SD). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The mouse mAb against Rab9 (MA3-067) was obtained from Thermo Fisher Scientific.

Techniques: Infection, Virus, Quantitative RT-PCR, Knockdown, Transfection, Western Blot, Labeling, Staining, Incubation, Fluorescence, Microscopy, Standard Deviation

Journal: Nature cell biology

Article Title: Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells

doi: 10.1038/ncb3372

Figure Lengend Snippet: USP19 recruits misfolded proteins to the ER and ER-associated late endosomes. (a) USP19 preferentially recruits GFP1–10 to membranes. The cytosol and membrane fractions from HEK293T cells co-transfected with the indicated constructs were analysed by immunoblotting. (b) Knockout of USP19 reduces membrane-associated GFP1–10. Control and USP19 CRISPR (KO) cells were transfected with GFP1–10. After fractionation, GFP1–10 in the membrane and cytosol fractions was analysed by immunoblotting. The graph shows the level of membrane-associated GFP1–10 relative to cytosolic GFP1–10 (mean ± s.e.m., n = 3 independent experiments, ∗∗P < 0.01). (c) USP19 recruits GFP1–10 to the ER membrane. COS7 cells co-transfected with GFP1–10 and FLAG-USP19 were permeabilized, stained with GFP and FLAG antibodies and analysed by confocal microscopy. Bottom panels show the outline of GFP1–10 and USP19 signal in the outlined area. Scale bar, 5 μm. (d,e) A photobleaching-based assay reveals vesicles containing MAPS cargos. (d) A schematic illustration of the photobleaching experiment shown in e. The marked area in d and e was photobleached with a 568nm laser to remove cytosolic and ER-associated mCh–GFP1–10 background. Arrows indicate a few examples of vesicles revealed after photobleaching. Scale bar, 5 μm. (f) MAPS vesicles were labelled with Rab9. Shown are two frames from a live-cell imaging experiment using cells transfected with mCe–USP19, mCi–Rab9, and mCh–GFP1–10 after photobleaching. Panels 4–6 are enlarged views of the indicated area in panel 1 after 20s. (g) Structure-illuminated microscopy analysis of MAPS vesicles. Cells transfected with mCe–USP19, Ubl4A–Venus and mCh–Rab9 were permeabilized, fixed and imaged. Note that Ubl4A–Venus is detected on the membranes of intraluminal vesicles, but not in the lumen of these vesicles. (h) Transmission electron microscopy analyses of MAPS vesicles. COS7 cells expressing FLAG-tagged GFP1–10 were permeabilized, fixed, and stained with anti-FLAG antibodies and immunogold-labelled secondary antibodies. Blue arrows show examples of luminal GFP1–10 signals. Arrowheads show PM-associated GFP1–10. The inset shows an example of GFP1–10 association with the limiting membrane on the luminal side (yellow arrows). Statistics source data for b can be found in Supplementary Table 2. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Article Snippet: For SIM microscopy, COS7 cells were permeabilized with digitonin and fixed with 2% paraformaldehyde, and stained with anti-Rab9 antibodies (Cell Signaling).

Techniques: Membrane, Transfection, Construct, Western Blot, Knock-Out, Control, CRISPR, Fractionation, Staining, Confocal Microscopy, Photobleaching Assay, Live Cell Imaging, Microscopy, Transmission Assay, Electron Microscopy, Expressing

Journal: Nature cell biology

Article Title: Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells

doi: 10.1038/ncb3372

Figure Lengend Snippet: Secretion of misfolded proteins through late endosomes. (a–c) The number of MAPS vesicles in cells correlates with the secretion efficiency. (a) Representative cells transfected with mCherry or mCh–GFP1–10 before and after photobleaching. (b) Representative cells transfected with mCh–GFP1–10 together with either mCi–USP19 WT or the mCi–USP19C506S (CS) mutant. (c) Quantification of MAPS vesicles in photobleached COS7 cells that were transfected as indicated. (d) Confocal analysis of v-SNAREs in live cells transfected with mCh–Rab9 and the indicated EGFP-tagged v-SNAREs. (e) Expression of late endosome-localized v-SNAREs promotes GFP1–10 secretion. Secretion of GFP1–10 was analysed in cells transfected with GFP1–10 and the indicated EGFP–v-SNAREs. (f) The graph shows the quantification result from experiments represented in e (mean ± s.e.m., n=3 independent experiments, ∗P <0.05). (g) A schematic illustration of the MAPS pathway. Statistics source data for f can be found in Supplementary Table 2. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Article Snippet: For SIM microscopy, COS7 cells were permeabilized with digitonin and fixed with 2% paraformaldehyde, and stained with anti-Rab9 antibodies (Cell Signaling).

Techniques: Transfection, Mutagenesis, Expressing

Journal: Arthritis Research & Therapy

Article Title: Inhibition of macropinocytosis blocks antigen presentation of type II collagen in vitro and in vivo in HLA-DR1 transgenic mice

doi: 10.1186/ar1964

Figure Lengend Snippet: Subcellular distribution of type II collagen (CII) in macrophages. (a) Macrophages were subjected to subcellular fractionation and Percoll fractions were analyzed for the expression of the plasma membrane-associated enzyme alkaline phosphodiesterase I (open diamonds), the lysosomal enzyme β-hexosaminidase (closed diamonds) and markers of late endosomes Rab7 (closed circles) and Rab9 (open circles); 27% Percoll alone is shown as fraction 0. Enzyme activity was measured as absorbance at 405 nm. Goat serum was used as a negative control (squares). (b) Macrophages were incubated in the absence (open circles) or presence of 200 μg/ml CII for 30 minutes and chased for 1 (open diamonds), 3 (closed diamonds), 5 (squares) and 24 h (closed circles) followed by subcellular fractionation and CII-specific ELISA. (c-d) Macrophages were pulse-chased with CII as above: (c) in the absence (closed squares) or presence of cytochalasin D (closed circles), amiloride (open squares) and 5-(N,N-dimethyl)amiloride (DMA; open circles); (d) in the presence of monodansylcadaverine (MDC; open diamonds) and filipin (closed diamonds) in the doses shown in the legend to Figure 1 or in the absence of CII and inhibitors (triangles). Cells were subjected to subcellular fractionation followed by CII-specific ELISA. Absorbance was measured at 405 nm. One of two experiments showing essentially the same results is shown. Error bars denote standard deviation.

Article Snippet: Plates were washed and incubated for 1 hour with goat anti-CII polyclonal antibody, goat anti-Rab7 and Rab9 polyclonal antibody (1: 200; Santa Cruz Biotechnology, Inc., Heidelberg, Germany).

Techniques: Fractionation, Expressing, Activity Assay, Negative Control, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: PLoS ONE

Article Title: Tumor Imaging and Targeting Potential of an Hsp70-Derived 14-Mer Peptide

doi: 10.1371/journal.pone.0105344

Figure Lengend Snippet: MCF7 tumor cells grown in glass-bottomed MatTek dishes for 48 h were fixed in paraformaldehyde, permeabilized and stained with primary antibodies specific for early (Rab4, Rab5), late (Rab7, Rab9) and recycling (Rab11) endosomes or lysosomes (LAMP1), followed by an appropriate Cy3-labeled secondary antibody (red). The nucleus was counter-stained with DAPI (blue). Representative three-frame composites from across the z-stack are shown. All of the vesicular markers assessed were found to be homogenously distributed throughout the cells. Objective 63×; scale bar 10 µm.

Article Snippet: Monoclonal antibodies recognizing Rab4, Rab5, Rab7, Rab9 and Rab11 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.) and antibodies recognizing LAMP1 and LAMP2 were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.).

Techniques: Staining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: The Amyotrophic Lateral Sclerosis M114T PFN1 Mutation Deregulates Alternative Autophagy Pathways and Mitochondrial Homeostasis

doi: 10.3390/ijms23105694

Figure Lengend Snippet: Deregulation of macroautophagy in M114T lymphoblasts. ( A – F ) Immunoblot analysis of protein extracts from lymphoblasts of healthy controls (white) and ALS patients carrying the M114T (black) and E117G (grey) PFN1 mutations. ( A ) Densitometry analyses show the amount of LC3II to total LC3 or (B) RAB9 levels standardized to GAPDH expression. Protein levels are represented as a percentage of the healthy controls. ( C , D ) Patient lymphoblasts were treated (+) or not (−) with etoposide autophagy inducer or ( E , F ) NH 4 Cl lysosomal inhibitor. (C, E, lower panel) Accumulation of LC3II and (D, F, lower panels) RAB9 after these treatments were evaluated by densitometry and presented as fold of induction. Results are means ± SEM for 3 to 8 independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001.

Article Snippet: Primary antibodies against GAPDH (D16H11, Cell Signaling Technology, Leiden, The Netherlands), HA (ab9110, Abcam, Amsterdam, The Netherlands), LC3B (NB100-2220, Novus Biologicals, Noyal Chatillon sur Seiche, France), MFN2 (M6319, Sigma-Aldrich, Saint Quentin Fallavier, France), PFN1 (ab124904, Abcam), RAB9 (11420-1-AP, ProteinTech, Manchester, UK) were made in rabbit.

Techniques: Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Amyotrophic Lateral Sclerosis M114T PFN1 Mutation Deregulates Alternative Autophagy Pathways and Mitochondrial Homeostasis

doi: 10.3390/ijms23105694

Figure Lengend Snippet: Deregulation of MFN2 mitochondrial marker in M114T patient lymphoblasts. ( A ) Anti-MFN2 was used to quantify mitochondrial levels in lymphoblast protein extracts. ( B ) Densitometry analyses of MFN2 protein levels for healthy controls (white), M114T patient (black) and E117G patient (grey), normalized to GAPDH levels and presented as a percentage of healthy controls. ( C , D ) NH 4 Cl or ( E , F ) Etoposide treatments were used to modulate the RAB9-mediated alternative mitophagy. ( D ) Accumulation/loss of MFN2 after NH 4 Cl treatment and ( F ) after etoposide treatment were represented for healthy controls (white), M114T patient (black) and E117G patient (grey). Results are means ± SEM of 4 to 8 independent experiments. * p < 0.05; ** p < 0.01.

Article Snippet: Primary antibodies against GAPDH (D16H11, Cell Signaling Technology, Leiden, The Netherlands), HA (ab9110, Abcam, Amsterdam, The Netherlands), LC3B (NB100-2220, Novus Biologicals, Noyal Chatillon sur Seiche, France), MFN2 (M6319, Sigma-Aldrich, Saint Quentin Fallavier, France), PFN1 (ab124904, Abcam), RAB9 (11420-1-AP, ProteinTech, Manchester, UK) were made in rabbit.

Techniques: Marker

Journal: International Journal of Molecular Sciences

Article Title: The Amyotrophic Lateral Sclerosis M114T PFN1 Mutation Deregulates Alternative Autophagy Pathways and Mitochondrial Homeostasis

doi: 10.3390/ijms23105694

Figure Lengend Snippet: Deregulation of autophagic and mitochondrial markers in M114T transfected cells. ( A ) Schematic representation of plasmid constructs to overexpress PFN1 fused to eGFP gene. ( B ) Mitochondrial respiration was measured in HEK293T expressing ePFN1 WT (white), ePFN1 C71G (dark grey), ePFN1 M114T (black), ePFN1 E117G (light grey) and ePFN1 G118V (grey) plasmid constructs using the MTT assay. To facilitate data interpretation, the mitochondrial respiration of PFN1 WT was adjusted to 100%. ( C ) Immunoblots were performed using anti-GFP, RAB9, MFN2 and GAPDH antibodies on HEK293T protein extracts. ( D ) Densitometry analyses of eGFP, ( E ) RAB9 and ( F ) MFN2 protein levels in HEK293T cells transfected with the 5 plasmid constructs were normalized to those of GAPDH and are presented relative to those of ePFN1 WT . Results are means ± SEM for at least 9 independent experiments. ( G ) NSC-34 were transfected with eGFP-PFN1 constructs expressing WT or various mutant forms of PFN1 (C71G, M114T, E117G or G118V) and triple immunofluorescence was performed to detect mitochondria (using CYCS in red) and RAB9A (in far-red) in cells with and without expression of eGFP tagged PFN1 constructs (in green). Nuclei are stained with DAPI (blue). Bar is 10 µm. ( H ) Fluorescent signal intensities of RAB9A and ( I ) CYCS were measured in cells expressing the eGFP-PFN1 constructs and are presented relative to those recorded in untransfected cells. Results are means ± SEM for 3 independent experiments with 20 to 70 transfected cells recorded for each plasmid condition. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001.

Article Snippet: Primary antibodies against GAPDH (D16H11, Cell Signaling Technology, Leiden, The Netherlands), HA (ab9110, Abcam, Amsterdam, The Netherlands), LC3B (NB100-2220, Novus Biologicals, Noyal Chatillon sur Seiche, France), MFN2 (M6319, Sigma-Aldrich, Saint Quentin Fallavier, France), PFN1 (ab124904, Abcam), RAB9 (11420-1-AP, ProteinTech, Manchester, UK) were made in rabbit.

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, MTT Assay, Western Blot, Mutagenesis, Immunofluorescence, Staining