Journal: PLOS Biology

Article Title: Polyphosphate modulates the stress-responsive formation of functional RNA-protein condensates in bacteria and mammalian cells

doi: 10.1371/journal.pbio.3003775

Figure Lengend Snippet: (A) Brightfield images of Hfq condensates reconstituted with the indicated Hfq concentrations in the presence or absence of 1× polyP-300 (in Pi units). Samples were prepared in 50 mM NaCl and 20 mM HEPES pH 7.0. (B) Phase diagram of Hfq and polyP-300 in the same buffer as (A). Formation of condensates was computationally determined by quantitative image analysis as detailed in the Methods section. Lines corresponding with specific polyP:Hfq ratios are highlighted. (C) Fluorescence and brightfield images of condensates reconstituted with 50 µM Hfq (supplemented with 4% Cy3-Hfq S65C) in the presence or absence of 1 µM FAM-labeled rA 30 or 100 µM AF647-labeled polyP-300. Samples were prepared in the same buffer as (A). (D) Electrophoretic mobility shift assays (EMSA). Lanes 1–6: 25 µM Hfq with 0, 0.5, 1, 2, 3, 4 µM FAM-rA 30 . Lanes 7–10 and lanes 11–14: 25 µM Hfq with 1 µM or 4 µM FAM-rA 30 , respectively, +25, 62.5, 125, 250 µM AF647-polyP300. Lane 15: 25 µM Hfq + 125 µM AF647-polyP300. A representative gel ( n = 2) is shown. Hfq was stained with Coomassie blue. Hfq-polyP-RNA complexes indicated by vertical arrowheads. (E) Left panel: Native western blot of bacterial lysates from MG1655 hfq::hfq-mCherry WT and Δ ppk at N+ and N-24 using antibodies against mCherry. Right panel: UV-bleached DAPI stain of native gel shown in left panel. PolyP is shown as dark areas. (F) Left panel: Native western blot of cell lysates from N-24 MG1655 hfq::hfq-mCherry WT (MG1655) using antibodies against mCherry. Lysates were treated with the indicated enzymes for 2 h prior to electrophoresis. Right panel: UV-bleached DAPI stain of native gel as shown in left panel. (G) Native western blot of N+ and N-24 lysates from hfq::hfq-mCherry rne::rne-mTQ2 WT (MG1655) relative to Δ ppk using antibodies against mCherry (Hfq) and GFP (RNase E). N-24 samples were left untreated (−) or digested with Ppx for 2 h (+) prior to electrophoresis. (H) Subcellular localization of Hfq-mCherry and RNase E-mTQ2 in N24 WT (MG1655) or Δ ppk . Scale bars: 5 µm. (I) Distribution of log2 ratios of RNase E intensities in Hfq foci (see Methods for details) vs. cellular background. Each data point shows a separate imaging field taken across two separate biological replicates. Scale bars: 5 µm. All underlying data can be found in .

Article Snippet: HfqS65C was labeled with Cy3 Maleimide Mono-Reactive Dye (Amersham) following the manufacturer’s protocol.

Techniques: Fluorescence, Labeling, Electrophoretic Mobility Shift Assay, Staining, Western Blot, Electrophoresis, Imaging

Journal: Journal of Cell Science

Article Title: Suppressing microtubule detyrosination augments adeno-associated virus 2 endosomal escape and gene delivery

doi: 10.1242/jcs.264190

Figure Lengend Snippet: Suppression of detyrosinated microtubules improves AAV2 motility and perinuclear accumulation. (A,B) Live-cell imaging and track displacement measurement of Cy3-tagged AAV2 in Huh7 cells in untreated control conditions, in cells overexpressing (OE) VASH2–GFP or TTL–GFP or in cells pretreated with parthenolide. Color-coded trajectories indicate blue for the start and red for the end of the tracks acquired for 60 s duration. Dashed lines highlight cell edge; solid lines highlight location of nucleus (A). Quantification of AAV2 track displacement obtained from movies in A (B). Results show mean±s.d. for ∼500 trajectories from n =4 cells analyzed for each condition from three replicates. (C–E) Time-dependent spatial distribution analysis of AAV2 in Huh7cells. TIRF imaging of AAV2-Cy3 (white) in cells fixed at 1 h, 2 h and 4 h after AAV2 endocytosis (C). Schematic representation of the division of the cytoplasmic region into three equal zones: outer (near plasma membrane), inner (perinuclear) and middle (between zone 1 and 2), to analyze the spatial distribution of AAV2 within cells (D). Percentage of AAV2 distribution across the zones over time in Huh7 cells (E). Bars represent the mean±s.d. for n =10 cells analyzed in each condition from three replicates. *** P <0.001 (unpaired two-tailed t -test). Scale bars: 10 µm (A); 20 µm (C).

Article Snippet: Briefly AAV2 (3×10 12 vgs; 20 μg) was incubated with Cy3 dye (Cytiva PA23001; 50 μM) in a conjugation buffer (0.1 M sodium bicarbonate, pH 9.3; Sigma-Aldrich) for 8 h at 4°C.

Techniques: Live Cell Imaging, Control, Imaging, Clinical Proteomics, Membrane, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Suppressing microtubule detyrosination augments adeno-associated virus 2 endosomal escape and gene delivery

doi: 10.1242/jcs.264190

Figure Lengend Snippet: Tyrosinated microtubules promote AAV2 endosomal escape. (A,B) Time-dependent colocalization analysis of AAV2 with late endosomes in Huh7 cells. Confocal imaging of Rab7–GFP (green) along with Cy3-tagged AAV2 (magenta) at different time points in Huh7 cells in untread conditions and pre-treated with parthenolide (A). Percentage of AAV2 colocalized with Rab7 decorated late endosomes over time analyzed from ten images ( n =10) of three replicates (B). (C,D) Confocal imaging (C) and quantification (D) of AAV2 (magenta) colocalization with lysosomes decorated by LAMP1 (green) (C). Whereas untreated control cells show significant accumulation of AAV2 in lysosomes over time, parthenolide-treated cells show no significant change in lysosomal colocalization with time analyzed from ten images ( n =10) of three replicates (D). In A and C, dashed lines highlight cell edge; solid lines highlight location of nucleus. (E–G) AAV2 endosomal escape measurement using Calcien fluorescence assay in Huh7 cells. Schematic representation of Calcein fluorescence assay. Endosomal Calcein fluorescence appears as a punctate signal whereas, AAV2 mediated endosomal rupture leads to diffused cytoplasmic Calcein fluorescence (E). Created in BioRender by Jayandharan, G. R., 2025. https://BioRender.com/te8cvym . This figure was sublicensed under CC-BY 4.0 terms. Live-cell imaging of Calcein (white) in AAV2 infected cells at 1, 2, 4 and 8 h post AAV2 endocytosis in untreated control, VASH2–GFP overexpression (OE), TTL–GFP overexpression, or parthenolide pretreated conditions (F). Quantification of cytoplasmic Calcein fluorescence over time ( n =22 from three replicates) (G). Bars represent the mean±s.d. a.u., arbitrary units. *** P <0.001; ns, not significant (unpaired two-tailed t -test). Scale bars: 10 µm (A,C); 20 µm (F).

Article Snippet: Briefly AAV2 (3×10 12 vgs; 20 μg) was incubated with Cy3 dye (Cytiva PA23001; 50 μM) in a conjugation buffer (0.1 M sodium bicarbonate, pH 9.3; Sigma-Aldrich) for 8 h at 4°C.

Techniques: Imaging, Control, Fluorescence, Live Cell Imaging, Infection, Over Expression, Two Tailed Test

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2 reduces the number of FGFR1 and FGF2 vesicles and their colocalization in U251 and SF126 cells. ( A ) U251 control cells with low endogenous SPRY2 level and U251 cells with SPRY2-OE were transfected with FGFR1 fused to enhanced green fluorescent protein (FGFR1-EGFP) and treated with cyanine 3-labeled FGF2 (FGF2-Cy3) for 30 min. Whole-cell analysis of confocal images revealed a reduction in FGFR1 (green) and FGF2 (red) vesicles per cell and their reduced colocalization (yellow) in response to SPRY2-OE. N = 18 experiments, mean ± SEM. *** p < 0.001, **** p < 0.0001. ( B ) SF126 control cells with high endogenous SPRY2 content and SF126 cells with shSPRY2 were transfected with FGFR1-EGFP and treated with FGF2-Cy3 for 30 min. Whole-cell analysis of confocal images revealed an increase in FGFR1 (green) and FGF2 (red) vesicles per cell as well as their enhanced colocalization (yellow) in response to shSPRY2. N = 15 experiments, mean ± SEM. *** p < 0.001, **** p < 0.0001. White, bold arrowheads indicate cell surface localization of FGFR1 and FGF2. Yellow arrowheads mark internalized FGFR1 vesicles colocalizing with FGF2. Scale bar = 4 µm.

Article Snippet: Subsequently, recombinant FGF2 was fluorescently labeled with cyanine 3 (Cy3) maleimide mono-reactive dye (Amersham, Darmstadt, Germany) according to the manufacturer’s protocol.

Techniques: Control, Transfection, Labeling, Cell Analysis

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2 inhibits colocalization of clathrin with FGFR1 and FGF2 in U251 and SF126 cells. ( A ) U251 control cells with low endogenous SPRY2 level and U251 cells with SPRY2-OE were transfected with FGFR1-EGFP, treated with FGF2-Cy3 for 30 min, and immunostained against clathrin. Whole-cell analysis of confocal images revealed no change in the number of clathrin vesicles per cell (blue) after SPRY2-OE. The colocalization of clathrin (blue) with FGFR1 (green; colocalization with clathrin = turquoise) and FGF2 (red; colocalization with clathrin = magenta) was reduced in response to SPRY2-OE. N = 6 experiments, mean ± SEM. * p < 0.05. ( B ) SF126 control cells with high endogenous SPRY2 content and SF126 cells with shSPRY2 were transfected with FGFR1-EGFP, treated with FGF2-Cy3 for 30 min, and immunostained against clathrin. The number of clathrin vesicles per cell (blue) was not altered with shSPRY2. The colocalization of clathrin (blue) with FGFR1 (green; colocalization with clathrin = turquoise) and FGF2 (red; colocalization with clathrin = magenta) was enhanced with shSPRY2. N = 5 experiments, mean ± SEM. ** p < 0.01. White, bold arrowheads indicate cell surface localization of FGFR1 and FGF2. Yellow arrowheads mark internalized FGFR1 and FGF2 vesicles colocalizing with clathrin. Scale bar = 4 µm.

Article Snippet: Subsequently, recombinant FGF2 was fluorescently labeled with cyanine 3 (Cy3) maleimide mono-reactive dye (Amersham, Darmstadt, Germany) according to the manufacturer’s protocol.

Techniques: Control, Transfection, Cell Analysis

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2 inhibits colocalization of caveolin-1 with FGFR1 and FGF2 in U251 and SF126 cells and reduces caveolin-1 vesicles. ( A ) U251 control cells with low endogenous SPRY2 level and U251 cells with SPRY2-OE were transfected with FGFR1-EGFP, treated with FGF2-Cy3 for 30 min, and immunostained against caveolin-1. Whole-cell analysis of confocal images revealed a reduction in the number of caveolin-1 vesicles per cell (blue) after SPRY2-OE. The colocalization of caveolin-1 (blue) with FGFR1 (green; colocalization with caveolin-1 = turquoise) and FGF2 (red; colocalization with caveolin-1 = magenta) was reduced in response to SPRY2-OE. N = 6 experiments, mean ± SEM. ** p < 0.01, *** p < 0.001. ( B ) SF126 control cells with high endogenous SPRY2 content and SF126 cells with shSPRY2 were transfected with FGFR1-EGFP, treated with FGF2-Cy3 for 30 min, and immunostained against caveolin-1. The number of caveolin-1 vesicles per cell (blue) was slightly but not significantly enhanced with shSPRY2. The colocalization of caveolin-1 (blue) with FGFR1 (green; colocalization with caveolin-1 = turquoise) and FGF2 (red; colocalization with caveolin-1 = magenta) was enhanced with shSPRY2. N = 5 experiments, mean ± SEM. * p < 0.05. White, bold arrowheads indicate cell surface localization of FGFR1 and FGF2. Yellow arrowheads mark internalized FGFR1 and FGF2 vesicles colocalizing with caveolin-1. Scale bar = 4 µm.

Article Snippet: Subsequently, recombinant FGF2 was fluorescently labeled with cyanine 3 (Cy3) maleimide mono-reactive dye (Amersham, Darmstadt, Germany) according to the manufacturer’s protocol.

Techniques: Control, Transfection, Cell Analysis

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2 decreases transferrin-647 uptake and colocalization of transferrin with FGFR1 and FGF2 in U251 and SF126 cells. ( A ) U251 control cells with low endogenous SPRY2 level and U251 cells with SPRY2-OE were transfected with FGFR1-EGFP and treated with FGF2-Cy3 for 30 min and with transferrin-647 for 15 min. Whole-cell analysis of confocal images revealed a reduction in the uptake of transferrin vesicles per cell (blue) after SPRY2-OE. The colocalization of transferrin (blue) with FGFR1 (green; colocalization with transferrin = turquoise) and FGF2 (red; colocalization with transferrin = magenta) was also reduced in response to SPRY2-OE. N = 6 experiments, mean ± SEM. ** p < 0.01, *** p < 0.001. ( B ) SF126 control cells with high endogenous SPRY2 content and SF126 cells with shSPRY2 were transfected with FGFR1-EGFP and treated with FGF2-Cy3 for 30 min and with transferrin-647 for 15 min. The uptake of transferrin vesicles per cell (blue) was increased with shSPRY2, and the colocalization of transferrin (blue) with FGFR1 (green; colocalization with transferrin = turquoise) and FGF2 (red; colocalization with transferrin = magenta) was enhanced with shSPRY2. N = 5 experiments, mean ± SEM. * p < 0.05, ** p < 0.01. White, bold arrowheads indicate cell surface localization of FGFR1 and FGF2. Yellow arrowheads mark FGFR1 and FGF2 vesicles colocalizing with transferrin. Scale bar = 4 µm.

Article Snippet: Subsequently, recombinant FGF2 was fluorescently labeled with cyanine 3 (Cy3) maleimide mono-reactive dye (Amersham, Darmstadt, Germany) according to the manufacturer’s protocol.

Techniques: Control, Transfection, Cell Analysis