paraformaldehyde fixed hela cultures  (Thermo Fisher)

Journal: mBio

Article Title: Evidence for a post-invasion role of the Chlamydia trachomatis type III secreted effector TmeA in redirection of host plasma membrane-derived material

doi: 10.1128/mbio.01993-25

Figure Lengend Snippet: Evidence for TmeA function during late-cycle development. HeLa cells were infected at a multiplicity of infection (MOI) of 1 with WT serovar L2, and whole-culture RNA or protein was harvested at 12, 16, or 24 h post-infection. ( A ) TmeA-specific mRNA was detected via qRT-PCR and normalized to rpoD levels in triplicate replicates. MOMP-specific mRNA was similarly quantitated as a quality control. ND = none detected. ( B ) TmeA was visualized by immunoblot, where MOMP-specific signals were examined as positive controls. ( C ) HeLa cells were equally infected at an MOI of 0.5 with WT, Δ tmeA , cis- tmeA , or Δ tarp Chlamydia and cultured for 24 h. Cultures were methanol fixed and stained using Hsp60-specific and Alexa-conjugated primary and secondary antibodies, respectively. Inclusion images were acquired, and inclusion areas (µm 2 ) were quantitated using automated CX5 software. Violin plots depict data plots ( n = 1,000) from representative replicates. Mean values (red line) and SD (dotted lines) are shown, and one-way ANOVA with multiple comparisons was used to assess statistical significance (ns = not significant; ****, P < 0.0001). ( D ) Progeny EBs from triplicate HeLa cultures equally infected with WT, Δ tmeA , cis- tmeA , or Δ tarp Chlamydia were enumerated after 24 h cultivation of passaged material. Progeny count data are represented as means of primary culture replicates (closed circles) with corresponding SDs. Statistical significance was determined by one-way ANOVA with multiple comparisons (ns = not significant; ***, P < 0.001).

Article Snippet: Detection via fluorescence microscopy was accomplished using direct fluorescence of paraformaldehyde-fixed HeLa cultures probed with Alexa488-Transferrin (Invitrogen) or 1,1′-dioctadecyl-3,3,3',3′-tetramethylindocarbocyanine perchlorate (DiI; Invitrogen) or for cells expressing GFP-TmeA, NBD-deficient GFP-TmeA, or mCherry-TmeA ( ).

Techniques: Infection, Quantitative RT-PCR, Control, Western Blot, Cell Culture, Staining, Software

Journal: mBio

Article Title: Evidence for a post-invasion role of the Chlamydia trachomatis type III secreted effector TmeA in redirection of host plasma membrane-derived material

doi: 10.1128/mbio.01993-25

Figure Lengend Snippet: Secretion of TmeA by mature inclusions. HeLa cells were infected with Δ tmeA expressing pBOMB-TmeA-FT. Media were supplemented with inducer (+aTc) or mock treated (−aTc) at 12 h post-infection and processed at 24 h post-infection. ( A ) Cultures were fixed with paraformaldehyde followed by ice-cold methanol. TmeA and MOMP were detected using Flag (red) or MOMP (grn)-specific antibodies, respectively. Arrows indicate the area of inset and bar = 10 µm. ( B ) Immunoblot of whole-culture material probed with TmeA-specific antibodies or Hsp60 antibodies as a loading control. ( C ) Immunoblot analysis of endogenous TmeA was performed on Triton X114 aqueous (Aq) or detergent (Det) fractions of pure WT EBs or infected HeLa cultures harvested at 24 h post-infection. Antigen-specific antibodies were used to detect TmeA and Hsp60, and MOMP was visualized as a control for soluble and membrane-associated proteins, respectively. Fractionation of Δ tmeA -infected cells was performed as an antibody specificity control.

Article Snippet: Detection via fluorescence microscopy was accomplished using direct fluorescence of paraformaldehyde-fixed HeLa cultures probed with Alexa488-Transferrin (Invitrogen) or 1,1′-dioctadecyl-3,3,3',3′-tetramethylindocarbocyanine perchlorate (DiI; Invitrogen) or for cells expressing GFP-TmeA, NBD-deficient GFP-TmeA, or mCherry-TmeA ( ).

Techniques: Infection, Expressing, Western Blot, Control, Membrane, Fractionation

Journal: mBio

Article Title: Evidence for a post-invasion role of the Chlamydia trachomatis type III secreted effector TmeA in redirection of host plasma membrane-derived material

doi: 10.1128/mbio.01993-25

Figure Lengend Snippet: Surface DiI is trafficked to WT inclusions. HeLa cells were infected with C. trachomatis L2 and pulsed with DiI for 5 min at 24 h post-infection. ( A ) Epi-fluorescence images of DiI-pulsed cultures at the time of dye removal ( T = 0) or after incubation for 120 min. DiI is visualized as gray/red, while inclusions (I) and nuclei are shown in blue in merged images. Bar = 10 µm. ( B ) Confocal images of infected HeLa cells paraformaldehyde-fixed 120 min after DiI pulse. Saponin-permeabilized cells were counterstained with IncG-specific antibodies. White arrows indicate the inset region, and the yellow arrow indicates the plane for quantification of intensity profiles plotted as arbitrary units (au). The position of the IM is indicated on intensity profiles. Bar = 10 µm. ( C ) Inclusions ( n = 100) were scored for co-localization with DiI over times ranging from 0 to 240 min post DiI pulse. Data are presented as means of triplicate replicates (closed circles) with SD, and one-way ANOVA with multiple comparisons was used to assess significance (**, P < 0.001; ***, P < 0.0001). ( D ) DiI localization 60 min after DiI pulse of 24 h WT-infected cultures that were untreated (mock) or treated with 3 µg/mL Brefeldin A (BfA) added beginning at 4 h post-infection. DiI (red) and DAPI (blue) images are shown, and inclusions (I) are indicated in the merged images. Bar = 10 µm. ( E ) DiI co-localization with inclusions ( n = 100) was quantitated, and average values for triplicate samples (closed circles) are shown with error bars corresponding to SD. Statistical significance was tested using Student’s T test (ns = not significant).

Article Snippet: Detection via fluorescence microscopy was accomplished using direct fluorescence of paraformaldehyde-fixed HeLa cultures probed with Alexa488-Transferrin (Invitrogen) or 1,1′-dioctadecyl-3,3,3',3′-tetramethylindocarbocyanine perchlorate (DiI; Invitrogen) or for cells expressing GFP-TmeA, NBD-deficient GFP-TmeA, or mCherry-TmeA ( ).

Techniques: Infection, Fluorescence, Incubation

Journal: mBio

Article Title: Evidence for a post-invasion role of the Chlamydia trachomatis type III secreted effector TmeA in redirection of host plasma membrane-derived material

doi: 10.1128/mbio.01993-25

Figure Lengend Snippet: TmeA is required for delivery of surface-localized DiI to the chlamydial inclusion. ( A ) Representative fluorescence images of HeLa cells infected for 24 h with WT, Δ tmeA , or cis- tmeA and paraformaldehyde fixed 120 min after DiI pulse. Samples were saponin permeabilized and stained with IncG-specific antibodies and with DAPI to visualize nuclei. DiI (red) or IncG (green) signals are shown, and DAPI (blue) is shown in merged images. Insets from merged channels are included with gray-scale images. Bars = 10 µm. ( B ) Inclusions ( n = 100) were scored for the presence or absence of IM-localized DiI. Data are presented as means of triplicate samples (closed circles) with SD. One-way ANOVA with multiple comparisons was used to assess significance (***, P < 0.001; ****, P < 0.0001). ( C ) Quantitation of NBD-ceramide co-localization with inclusions ( n = 100) after 120 min in HeLa triplicate cultures (closed circles) infected for 24 h with C. trachomatis WT or Δ tmeA . Labeling of WT infections in the presence of 3 µg/mL BfA served as a positive control. Data are presented as means of triplicate samples (closed circles) with SD. One-way ANOVA with multiple comparisons was used to assess significance (ns = not significant; ***, P < 0.0001).

Article Snippet: Detection via fluorescence microscopy was accomplished using direct fluorescence of paraformaldehyde-fixed HeLa cultures probed with Alexa488-Transferrin (Invitrogen) or 1,1′-dioctadecyl-3,3,3',3′-tetramethylindocarbocyanine perchlorate (DiI; Invitrogen) or for cells expressing GFP-TmeA, NBD-deficient GFP-TmeA, or mCherry-TmeA ( ).

Techniques: Fluorescence, Infection, Staining, Quantitation Assay, Labeling, Positive Control

Journal: mBio

Article Title: Evidence for a post-invasion role of the Chlamydia trachomatis type III secreted effector TmeA in redirection of host plasma membrane-derived material

doi: 10.1128/mbio.01993-25

Figure Lengend Snippet: Clathrin is required for DiI trafficking to the chlamydial inclusion. HeLa cells were infected with WT L2 and cultured for 24 h. ( A ) Clathrin-dependent endocytosis was disrupted by treatment with 40 µM Pitstop 2 1 h prior and during DiI pulse. Samples were paraformaldehyde fixed after 120 min. For siRNA treatments ( B ), HeLa cultures were mock treated or transfected with clathrin-specific siRNA or scramble (Scr) control 24 h prior to infection with C. trachomatis L2. DiI labeling was carried out 24 h post-infection. ( C ) Dynamin or clathrin-dependent endocytosis was disrupted by treatment with 30 µM Dynasore or 40 µM Pitstop 2, respectively. Representative images showing DiI (red) or DAPI (blue) signal are shown from each experimental treatment, and inclusion (I) position is indicated. The presence or absence of DiI inclusion staining in 100 inclusions was assessed for quantitation comparing mock and inhibitor-treated cultures. Quantitative values are represented as overall (bars) and individual (closed circles) averages from triplicate cultures with SD. Statistical significance was assessed by Student’s T test when comparing two treatments or one-way ANOVA with multiple comparisons for >2 conditions (ns = not significant; *, P < 0.01; ***, P < 0.001; ****, P < 0.0001). ( D and E ) HeLa cultures were mock treated or transfected with clathrin-specific siRNA or scramble (Scr) control 24 h prior to infection with WT ( D ) or Δ tmeA ( E ) C. trachomatis . IFU-normalized material was passaged onto fresh HeLa for progeny EB enumeration at 24 h post-infection. Data are represented as means of triplicate samples (closed circles) with SDs. Statistical significance was determined by one-way ANOVA with multiple comparisons (ns = not significant, **, P < 0.01). ( F ) HeLa cells were infected with WT or Δ tarp for 24 h. Quantitation of DiI co-localization with inclusions ( n = 100) was determined 120 min after DiI pulse in 24 h infected cultures. Data are presented as means of triplicate samples (closed circles) with SD, and a Student’s t -test was used for significance (ns = not significant).

Article Snippet: Detection via fluorescence microscopy was accomplished using direct fluorescence of paraformaldehyde-fixed HeLa cultures probed with Alexa488-Transferrin (Invitrogen) or 1,1′-dioctadecyl-3,3,3',3′-tetramethylindocarbocyanine perchlorate (DiI; Invitrogen) or for cells expressing GFP-TmeA, NBD-deficient GFP-TmeA, or mCherry-TmeA ( ).

Techniques: Infection, Cell Culture, Transfection, Control, Labeling, Staining, Quantitation Assay

Journal: mBio

Article Title: Evidence for a post-invasion role of the Chlamydia trachomatis type III secreted effector TmeA in redirection of host plasma membrane-derived material

doi: 10.1128/mbio.01993-25

Figure Lengend Snippet: TmeA N-WASP-activating activity is required for delivery of surface-localized DiI to the chlamydial inclusion. ( A ) Quantitation of DiI co-localization with inclusions ( n = 100) after 120 min in HeLa cultures infected with WT for 24 h. Cultures were untreated (mock) or treated with 7.5 µM Wiskostatin (wisko) or 200 µM CK666 30 min prior to and during DiI pulse. ( B ) Representative images of Wiskostatin and CK666-treated cultures. Cultures were stained 120 min after DiI pulse and DiI (red) or DAPI (blue) signals are shown. Inclusion (I) position is indicated in the merged images. Bar = 10 µm. ( C ) Quantitation of DiI co-localization with Δ tmeA 24 h inclusions ( n = 100) where cultures were untreated or treated with 7.5 µM Wiskostatin (wisko) or 200 µM CK666 30 min prior to DiI pulse. ( D ) Inclusions ( n = 100) were scored for localization of DiI with WT, Δ tmeA , or tmeA -ΔNBD inclusions at 24 h post infection. ( E ) Representative fluorescence images of HeLa cells infected for 24 h tmeA ΔNBD and fixed 120 min after DiI pulse. DiI (red) or DAPI (blue) signals are shown with inclusion (I) position indicated in the merged images. All bar = 10 µm. All quantitative data are represented as means of triplicate samples (closed circles) with corresponding SDs. Statistical significance was determined by one-way ANOVA with multiple comparisons (ns = not significant, *, P < 0.01; **, P < 0.001; ***, P < 0.0001).

Article Snippet: Detection via fluorescence microscopy was accomplished using direct fluorescence of paraformaldehyde-fixed HeLa cultures probed with Alexa488-Transferrin (Invitrogen) or 1,1′-dioctadecyl-3,3,3',3′-tetramethylindocarbocyanine perchlorate (DiI; Invitrogen) or for cells expressing GFP-TmeA, NBD-deficient GFP-TmeA, or mCherry-TmeA ( ).

Techniques: Activity Assay, Quantitation Assay, Infection, Staining, Fluorescence

Journal: mBio

Article Title: Evidence for a post-invasion role of the Chlamydia trachomatis type III secreted effector TmeA in redirection of host plasma membrane-derived material

doi: 10.1128/mbio.01993-25

Figure Lengend Snippet: Ectopically expressed TmeA co-localizes with internalized DiI. ( A ) Epifluorescence images of uninfected HeLa cells ectopically expressing GFP, GFP-TmeA, or GFP-SINC for 24 h. Nuclei were detected after fixation with DAPI (blue). Arrows indicate area of inset and bar = 10 µm. ( B ) HeLa cells expressing GFP-TmeA were pulsed with DiI and incubated for 90 min followed by paraformaldehyde fixation. Localization was visualized by direct fluorescence, and representative images are shown. ( C ) GFP-TmeAΔNBD or GFP-SINC expressing HeLa cells were pulsed with DiI (red) and fixed for visualization at 90 min. Arrows indicate area of inset in merged images. Bars = 10 µm. ( D ) Corresponding Pearson co-localization coefficients of respective signals were calculated from 10 inclusions. The dashed line indicates a 0.5 coefficient cutoff.

Article Snippet: Detection via fluorescence microscopy was accomplished using direct fluorescence of paraformaldehyde-fixed HeLa cultures probed with Alexa488-Transferrin (Invitrogen) or 1,1′-dioctadecyl-3,3,3',3′-tetramethylindocarbocyanine perchlorate (DiI; Invitrogen) or for cells expressing GFP-TmeA, NBD-deficient GFP-TmeA, or mCherry-TmeA ( ).

Techniques: Expressing, Incubation, Fluorescence