single colonies  (Danaher Inc)

Journal: mBio

Article Title: Role of listeriolysin O and phospholipases C in L. monocytogenes intercellular protrusion dynamics, resolution, and autophagy avoidance

doi: 10.1128/mbio.01183-25

Figure Lengend Snippet: LLO and PLCs control formation of L. monocytogenes infectious foci. Plasma membrane-labeled HeLa cells (Lck-mTurquoise2, blue) were infected with WT, ∆ actA , ∆ hly , or ∆ plcAB L. monocytogenes that express RFP upon access to the cytosol (red). ( A ) Micrographs extracted from representative movies show the progression of infectious foci at four different time points (overlay). Corresponding binary images of fluorescent intracellular L. monocytogenes (Bi Lm , white) and binary images that encompass the focus regions (Bi Lm 2, white) are presented below the micrographs. Scale bars are 100 µm. Arrows point out areas of compacted bacterial growth. ( B i) Average L. monocytogenes focus size over time. (Bii) Average focus circularity (perfect circle = 1) over time. (Biii) Average L. monocytogenes growth over time. Error bars indicate the standard error of the mean. N = 3 independent experiments with 7–10 foci analyzed per strain and independent experiment. Data compared WT to mutant strains by a linear mixed-effects model (* P < 0.05). ns, not significant. (Bi) ∆ actA P = 0.0001, ∆ hly P ≤ 0.0001, ∆ plcAB P = 0.0062; (Bii) ∆ actA P ≤ 0.0001, ∆ hly P = 0.5872, ∆ plcAB P ≤ 0.0001; (Biii) ∆ actA P ≤ 0.0001, ∆ hly P = 0.0002, ∆ plcAB P = 0.0022. Note that the ∆ actA strain did not form infectious foci and circularity measurement reflected the shape of infected cells, which at the last time points, rupture and release bacteria into the cell culture medium.

Article Snippet: To generate single-colony stable HeLa cell (American Type Culture Collection #CCL-2) lines expressing monomeric fluorescent proteins (mTurquoise2, mVenus, or mScarlet-I) anchored to the inner leaflet of the plasma membrane through myristoylation and palmitoylation lipid modification sequence (Lck), the Lck-fluorescent protein (FP) coding sequences were amplified from their respective vectors and cloned into BamHI/EcoRI sites of the pLVX-M-puro vector using the primers BamHI-Lck.Fwd and Lck.Rev described in .

Techniques: Control, Clinical Proteomics, Membrane, Labeling, Infection, Mutagenesis, Bacteria, Cell Culture

Journal: mBio

Article Title: Role of listeriolysin O and phospholipases C in L. monocytogenes intercellular protrusion dynamics, resolution, and autophagy avoidance

doi: 10.1128/mbio.01183-25

Figure Lengend Snippet: LC3 is associated with cell-to-cell spread membrane protrusion remnants of WT L. monocytogenes . HeLa cells expressing plasma membrane marker were infected with WT RFP- L. monocytogenes . Cells were fixed, permeabilized, and labeled for LC3. ( A ) Micrographs of L. monocytogenes (green) and LC3 (red) at one z -plane. (Ai and ii) z -stack images were deconvolved using the Richardson-Lucy method. Magnified images show LC3 (red), L. monocytogenes (green), and plasma membrane (green) as single fluorochromes or overlays. Solid arrows indicate areas of co-localization of plasma membrane and LC3. Dashed arrows indicate areas of co-localization of L. monocytogenes and LC3.

Article Snippet: To generate single-colony stable HeLa cell (American Type Culture Collection #CCL-2) lines expressing monomeric fluorescent proteins (mTurquoise2, mVenus, or mScarlet-I) anchored to the inner leaflet of the plasma membrane through myristoylation and palmitoylation lipid modification sequence (Lck), the Lck-fluorescent protein (FP) coding sequences were amplified from their respective vectors and cloned into BamHI/EcoRI sites of the pLVX-M-puro vector using the primers BamHI-Lck.Fwd and Lck.Rev described in .

Techniques: Membrane, Expressing, Clinical Proteomics, Marker, Infection, Labeling

Journal: mBio

Article Title: Role of listeriolysin O and phospholipases C in L. monocytogenes intercellular protrusion dynamics, resolution, and autophagy avoidance

doi: 10.1128/mbio.01183-25

Figure Lengend Snippet: Entrapment of ∆ plcAB L. monocytogenes in LAMP1-positive autophagosome during cell-to-cell spread. HeLa cells were infected with RFP-WT, -∆ hly , or -∆ plcAB L. monocytogenes for 7–10 h. Cells were fixed, permeabilized, and labeled using primary antibodies against LC3 and LAMP1 and secondary fluorescent antibodies, and with 4′,6-diamidino-2-phenylindole (DAPI). ( A ) Micrographs of L. monocytogenes (green), LC3 (red), LAMP1 (white), and nuclei (DAPI, dark blue) as single fluorochromes or overlays. Scale bars are 25 µm. (Ai and ii) z -stack images were deconvolved using the Richardson-Lucy method. Magnified images correspond to boxed regions from panel A . Scale bars in magnified images are 5 µm. ( B ) Micrographs of L. monocytogenes (green), LC3 (red), LAMP1 (white), HeLa cell plasma membrane (light blue), and nuclei (dark blue) as single fluorochromes or overlays. Arrows indicate LC3 and LAMP-1 associated vacuoles containing multiple Δ plcAB L. monocytogenes . Scale bars are 5 µm. ( C ) Micrographs of L. monocytogenes (green), actin (red), LAMP1 (white), and nuclei (dark blue) as single fluorochromes or overlays. (Ci–iii) Boxed areas magnified. Arrows indicate LC3-associated vacuoles containing multiple Δ plcAB L. monocytogenes . Scale bars are 5 µm. Data are representative of N = 2 independent experiments with two to three internal replicates per experimental condition.

Article Snippet: To generate single-colony stable HeLa cell (American Type Culture Collection #CCL-2) lines expressing monomeric fluorescent proteins (mTurquoise2, mVenus, or mScarlet-I) anchored to the inner leaflet of the plasma membrane through myristoylation and palmitoylation lipid modification sequence (Lck), the Lck-fluorescent protein (FP) coding sequences were amplified from their respective vectors and cloned into BamHI/EcoRI sites of the pLVX-M-puro vector using the primers BamHI-Lck.Fwd and Lck.Rev described in .

Techniques: Infection, Labeling, Clinical Proteomics, Membrane