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Cortex Biochem Inc goat anti- c. trachomatis momp

(A) HeLa cells were infected with C. <t>trachomatis</t> L2 for 24 hrs and then fixed and stained with antibodies to GBF1 (red) and BIG1 (green). Bacteria and host DNA were detected using DAPI (blue). The cis and trans polarity of the Golgi was maintained in C. trachomatis L2-infected cells. N, host nucleus. *, inclusion. Scale bar = 5 µm. (B) HeLa cells were transfected with Arf1-GFP for 18 hrs, infected with C. trachomatis L2 for 24 hrs in the absence or presence of 10 µM BFA, and then fixed and stained with antibodies to GBF1 (red). Enlargements of boxed regions are shown to the right. Images represent a single z slice from confocal images. The exposure time for each filter set for all images was identical. Arf1-GFP localized to the region between two closely apposed inclusions (white arrow) and to a thin rim around the inclusion (red arrow) whereas GBF1 was excluded from these regions. *, inclusion. Scale bar = 5 µm. (C) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (D) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then labeled with BODIPY FL-Ceramide to visualize SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (E) HeLa cells were infected with C. trachomatis for 24 hrs, treated with 10 µM BFA or GCA during the last 3 hrs of infection, and then labeled with BODIPY FL-Ceramide to analyze SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (F) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then analyzed for progeny formation as described in . Values (mean ± standard error) are shown as percentage of control siRNA-treated samples. No significant decrease in progeny formation was observed. IFU, inclusion forming units.

Goat Anti C. Trachomatis Momp, supplied by Cortex Biochem Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development"

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002198

(A) HeLa cells were infected with C. trachomatis L2 for 24 hrs and then fixed and stained with antibodies to GBF1 (red) and BIG1 (green). Bacteria and host DNA were detected using DAPI (blue). The cis and trans polarity of the Golgi was maintained in C. trachomatis L2-infected cells. N, host nucleus. *, inclusion. Scale bar = 5 µm. (B) HeLa cells were transfected with Arf1-GFP for 18 hrs, infected with C. trachomatis L2 for 24 hrs in the absence or presence of 10 µM BFA, and then fixed and stained with antibodies to GBF1 (red). Enlargements of boxed regions are shown to the right. Images represent a single z slice from confocal images. The exposure time for each filter set for all images was identical. Arf1-GFP localized to the region between two closely apposed inclusions (white arrow) and to a thin rim around the inclusion (red arrow) whereas GBF1 was excluded from these regions. *, inclusion. Scale bar = 5 µm. (C) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (D) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then labeled with BODIPY FL-Ceramide to visualize SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (E) HeLa cells were infected with C. trachomatis for 24 hrs, treated with 10 µM BFA or GCA during the last 3 hrs of infection, and then labeled with BODIPY FL-Ceramide to analyze SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (F) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then analyzed for progeny formation as described in . Values (mean ± standard error) are shown as percentage of control siRNA-treated samples. No significant decrease in progeny formation was observed. IFU, inclusion forming units.

Figure Legend Snippet: (A) HeLa cells were infected with C. trachomatis L2 for 24 hrs and then fixed and stained with antibodies to GBF1 (red) and BIG1 (green). Bacteria and host DNA were detected using DAPI (blue). The cis and trans polarity of the Golgi was maintained in C. trachomatis L2-infected cells. N, host nucleus. *, inclusion. Scale bar = 5 µm. (B) HeLa cells were transfected with Arf1-GFP for 18 hrs, infected with C. trachomatis L2 for 24 hrs in the absence or presence of 10 µM BFA, and then fixed and stained with antibodies to GBF1 (red). Enlargements of boxed regions are shown to the right. Images represent a single z slice from confocal images. The exposure time for each filter set for all images was identical. Arf1-GFP localized to the region between two closely apposed inclusions (white arrow) and to a thin rim around the inclusion (red arrow) whereas GBF1 was excluded from these regions. *, inclusion. Scale bar = 5 µm. (C) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (D) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then labeled with BODIPY FL-Ceramide to visualize SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (E) HeLa cells were infected with C. trachomatis for 24 hrs, treated with 10 µM BFA or GCA during the last 3 hrs of infection, and then labeled with BODIPY FL-Ceramide to analyze SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (F) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then analyzed for progeny formation as described in . Values (mean ± standard error) are shown as percentage of control siRNA-treated samples. No significant decrease in progeny formation was observed. IFU, inclusion forming units.

Techniques Used: Infection, Staining, Transfection, Western Blot, Labeling

(A) HeLa cells were treated with the indicated siRNA for 3 days, infected with C. trachomatis L2 for 24 hrs, then fixed and stained with antibodies to 14-3-3β (green) to identify the inclusion membrane and GBF1 (red). Bacteria and host DNA were detected using DAPI (blue). The exposure time for each filter set for all images was identical. White arrows point to breaks in the inclusion membrane where the bacteria are released into cytoplasm in GBF1-depleted cells. Inclusions formed in BIG1 and/or BIG2 depleted cells remain intact. (B) HeLa cells were depleted of GBF1 for 3 days, infected with C. trachomatis L2 for 24 hrs, then fixed and stained with antibodies to MOMP (green) to identify bacteria and vimentin (red). Bacteria and host DNA were detected using DAPI (blue). The exposure time for each filter set for all images was identical. White arrows point to the region on the inclusion that is devoid of vimentin staining and where bacteria are released into the cytoplasm. N, host nucleus; *, inclusion. MOMP, C. trachomatis major outer membrane protein. Scale bar = 5 µm.

Figure Legend Snippet: (A) HeLa cells were treated with the indicated siRNA for 3 days, infected with C. trachomatis L2 for 24 hrs, then fixed and stained with antibodies to 14-3-3β (green) to identify the inclusion membrane and GBF1 (red). Bacteria and host DNA were detected using DAPI (blue). The exposure time for each filter set for all images was identical. White arrows point to breaks in the inclusion membrane where the bacteria are released into cytoplasm in GBF1-depleted cells. Inclusions formed in BIG1 and/or BIG2 depleted cells remain intact. (B) HeLa cells were depleted of GBF1 for 3 days, infected with C. trachomatis L2 for 24 hrs, then fixed and stained with antibodies to MOMP (green) to identify bacteria and vimentin (red). Bacteria and host DNA were detected using DAPI (blue). The exposure time for each filter set for all images was identical. White arrows point to the region on the inclusion that is devoid of vimentin staining and where bacteria are released into the cytoplasm. N, host nucleus; *, inclusion. MOMP, C. trachomatis major outer membrane protein. Scale bar = 5 µm.

Techniques Used: Infection, Staining

(A) HeLa cells transfected with CERT-GFP for 18 hrs were left uninfected or infected with C. trachomatis L2 for 24 hrs. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). N, host nucleus; *, inclusions. Scale bar = 5 µm. (B) HeLa cells transfected with CERT-GFP were infected with C. trachomatis serovar D for 24 hrs and then fixed and stained with antibodies to IncA (red) to identify the inclusion membrane. Bacteria and host DNA were detected using DAPI (blue). Enlargements (inset) of boxed regions are shown to the right. *, inclusions. Scale bar = 5 µm. (C–E) HeLa cells were transfected with CERT-GFP and HcRedVAP-A for 18 hrs and infected with C. trachomatis L2 for (C) 2, (D) 8, or (E) 24 hrs. (C) Cells were stained with DAPI to visualize the nascent inclusions (red arrows). (D and E) Enlargements (inset) of boxed regions are shown to the right. At 8 and 24 hpi, CERT-GFP and HcRedVAP-A colocalize on the inclusion membrane and exhibit a patchy distribution. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). N, host nucleus; *, inclusions. Scale bar = 5 µm, except with insets from panels B, C, and D where scale bar = 2.5 µm.

Figure Legend Snippet: (A) HeLa cells transfected with CERT-GFP for 18 hrs were left uninfected or infected with C. trachomatis L2 for 24 hrs. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). N, host nucleus; *, inclusions. Scale bar = 5 µm. (B) HeLa cells transfected with CERT-GFP were infected with C. trachomatis serovar D for 24 hrs and then fixed and stained with antibodies to IncA (red) to identify the inclusion membrane. Bacteria and host DNA were detected using DAPI (blue). Enlargements (inset) of boxed regions are shown to the right. *, inclusions. Scale bar = 5 µm. (C–E) HeLa cells were transfected with CERT-GFP and HcRedVAP-A for 18 hrs and infected with C. trachomatis L2 for (C) 2, (D) 8, or (E) 24 hrs. (C) Cells were stained with DAPI to visualize the nascent inclusions (red arrows). (D and E) Enlargements (inset) of boxed regions are shown to the right. At 8 and 24 hpi, CERT-GFP and HcRedVAP-A colocalize on the inclusion membrane and exhibit a patchy distribution. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). N, host nucleus; *, inclusions. Scale bar = 5 µm, except with insets from panels B, C, and D where scale bar = 2.5 µm.

Techniques Used: Transfection, Infection, Staining

HeLa cells were infected with C. trachomatis L2, treated with the indicated concentration of HPA-12 at 1–24 hpi, and then (A) fixed and stained with antibodies to MOMP (red) and with DAPI (blue) to visualize bacteria or (B) analyzed for progeny formation. Values (mean ± standard error) are shown as percentage of DMSO treated samples. Data are representative of 3 independent experiments. ***p<0.001 compared to DMSO treated cells (ANOVA). (C) HeLa cells were transfected with CERT-GFP and HA-CKIγ2 for 18 hrs, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to HA (red). The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Ectopic expression of HA-CKIγ2 decreased inclusion size but did not affect CERT-GFP recruitment to the inclusion membrane. Scale bar = 5 µm. (D) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (E) HeLa cells were depleted of CERT for 3 days and then labeled with BODIPY FL-Ceramide to analyze SM accumulation in the Golgi. The exposure time of all images was identical. CERT depletion reduced SM accumulation in the Golgi. Scale bar = 5 µm, (F) HeLa cells were depleted of CERT for 3 days, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to MOMP (green) to identify the inclusion. Bacteria and host DNA were detected using DAPI (blue). The exposure time for all images was identical. CERT depletion reduced inclusion size. Red arrows point to inclusions. Scale bar = 5 µm. (G) HeLa cells were depleted of CERT for 3 days, infected with C. trachomatis L2 and analyzed for inclusion size and progeny formation. Values (mean ± standard error) are shown as percentage of control siRNA samples. CERT depletion significantly reduced inclusion size and progeny formation. Data are representative of 2 independent experiments. ***p<0.001 for CERT siRNA-treated cells compared to control siRNA-treated cells (ANOVA). N, host nucleus. IFU, inclusion forming units. Scale bar = 5 µm.

Figure Legend Snippet: HeLa cells were infected with C. trachomatis L2, treated with the indicated concentration of HPA-12 at 1–24 hpi, and then (A) fixed and stained with antibodies to MOMP (red) and with DAPI (blue) to visualize bacteria or (B) analyzed for progeny formation. Values (mean ± standard error) are shown as percentage of DMSO treated samples. Data are representative of 3 independent experiments. ***p<0.001 compared to DMSO treated cells (ANOVA). (C) HeLa cells were transfected with CERT-GFP and HA-CKIγ2 for 18 hrs, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to HA (red). The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Ectopic expression of HA-CKIγ2 decreased inclusion size but did not affect CERT-GFP recruitment to the inclusion membrane. Scale bar = 5 µm. (D) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (E) HeLa cells were depleted of CERT for 3 days and then labeled with BODIPY FL-Ceramide to analyze SM accumulation in the Golgi. The exposure time of all images was identical. CERT depletion reduced SM accumulation in the Golgi. Scale bar = 5 µm, (F) HeLa cells were depleted of CERT for 3 days, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to MOMP (green) to identify the inclusion. Bacteria and host DNA were detected using DAPI (blue). The exposure time for all images was identical. CERT depletion reduced inclusion size. Red arrows point to inclusions. Scale bar = 5 µm. (G) HeLa cells were depleted of CERT for 3 days, infected with C. trachomatis L2 and analyzed for inclusion size and progeny formation. Values (mean ± standard error) are shown as percentage of control siRNA samples. CERT depletion significantly reduced inclusion size and progeny formation. Data are representative of 2 independent experiments. ***p<0.001 for CERT siRNA-treated cells compared to control siRNA-treated cells (ANOVA). N, host nucleus. IFU, inclusion forming units. Scale bar = 5 µm.

Techniques Used: Infection, Concentration Assay, Staining, Transfection, Expressing, Western Blot, Labeling

(A) HeLa cells were transfected with CERT-GFP, CERT (D324A)-GFP, or CERT (G67E)-GFP, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with an antibody to p230 (red) to identify the trans -Golgi. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Mutation of the PI4P binding (G67E) or VAP-A binding (D324A) domains did not affect CERT-GFP recruitment to the inclusion. Scale bar = 5 µm. (B) HeLa cells expressing CERT-GFP were infected with C. trachomatis L2, treated with 50 µM Exo1 (Arf1 inhibitor) for 1–24 hpi, and then fixed and stained with antibodies to MOMP (red) to identify bacteria. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). CERT-GFP localization to the inclusion was unaffected by Arf1 inhibition. Scale bar = 5 µm. (C) HeLa cells expressing CERT-GFP were infected with C. trachomatis L2, treated with 5 µM HPA-12 for 1–24 hpi, and then fixed and stained with antibodies to MOMP (red) to identify bacteria or to p230 (red) to identify the trans -Golgi. The exposure time for each filter set of all images was identical. Inhibition of CERT transfer and/or ceramide binding activity by HPA-12 treatment resulted in loss of CERT accumulation on the inclusion membrane. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Scale bar = 5 µm. N, host nucleus; *, inclusion.

Figure Legend Snippet: (A) HeLa cells were transfected with CERT-GFP, CERT (D324A)-GFP, or CERT (G67E)-GFP, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with an antibody to p230 (red) to identify the trans -Golgi. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Mutation of the PI4P binding (G67E) or VAP-A binding (D324A) domains did not affect CERT-GFP recruitment to the inclusion. Scale bar = 5 µm. (B) HeLa cells expressing CERT-GFP were infected with C. trachomatis L2, treated with 50 µM Exo1 (Arf1 inhibitor) for 1–24 hpi, and then fixed and stained with antibodies to MOMP (red) to identify bacteria. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). CERT-GFP localization to the inclusion was unaffected by Arf1 inhibition. Scale bar = 5 µm. (C) HeLa cells expressing CERT-GFP were infected with C. trachomatis L2, treated with 5 µM HPA-12 for 1–24 hpi, and then fixed and stained with antibodies to MOMP (red) to identify bacteria or to p230 (red) to identify the trans -Golgi. The exposure time for each filter set of all images was identical. Inhibition of CERT transfer and/or ceramide binding activity by HPA-12 treatment resulted in loss of CERT accumulation on the inclusion membrane. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Scale bar = 5 µm. N, host nucleus; *, inclusion.

Techniques Used: Transfection, Infection, Staining, Mutagenesis, Binding Assay, Expressing, Inhibition, Activity Assay

(A) HeLa cells were infected with C. trachomatis L2 for 24 hrs, treated with 5 µM HPA-12 for the last 3 hrs of infection, and then labeled with BODIPY FL-Ceramide to analyze SM accumulation by the inclusion. As a control for decreased SM acquisition by the inclusion, cells were also treated with 10 µM BFA or 25 µg/ml D609. The exposure time of all images was identical. Dashed red lines demarcate inclusions. The residual fluorescence likely represents Golgi staining. Scale bar = 5 µm. (B) Quantitation of SM acquisition following treatment with HPA-12, D609, or BFA. Values (mean ± standard error) are shown as percentage of mean fluorescence intensities relative to DMSO-treated samples. ***p<0.001 (ANOVA). HPA-12, D609, and BFA-treated cells displayed a significant decrease in fluorescence intensity of the inclusion and its contents compared to DMSO-treated samples.

Figure Legend Snippet: (A) HeLa cells were infected with C. trachomatis L2 for 24 hrs, treated with 5 µM HPA-12 for the last 3 hrs of infection, and then labeled with BODIPY FL-Ceramide to analyze SM accumulation by the inclusion. As a control for decreased SM acquisition by the inclusion, cells were also treated with 10 µM BFA or 25 µg/ml D609. The exposure time of all images was identical. Dashed red lines demarcate inclusions. The residual fluorescence likely represents Golgi staining. Scale bar = 5 µm. (B) Quantitation of SM acquisition following treatment with HPA-12, D609, or BFA. Values (mean ± standard error) are shown as percentage of mean fluorescence intensities relative to DMSO-treated samples. ***p<0.001 (ANOVA). HPA-12, D609, and BFA-treated cells displayed a significant decrease in fluorescence intensity of the inclusion and its contents compared to DMSO-treated samples.

Techniques Used: Infection, Labeling, Fluorescence, Staining, Quantitation Assay

(A) HeLa cells co-transfected for 18 hrs with CERT-GFP and C-terminally 3xFLAG-tagged SMS1/SMS2 (upper 2 rows) or SMS1-V5 and C-terminally 3xFLAG-tagged SMS2 (bottom row) were infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to FLAG (red) and/or to V5 (green). Single channel images of uninfected cells are shown to the right. Enlargements of the boxed regions (inset) in infected samples are shown to the right of infected set. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). CERT and SMS1 localization at the inclusion are distinct while CERT and SMS2 partially overlap. SMS2 localization partially overlaps with SMS1 around the inclusion, however SMS2 also localizes to the inclusion. At longer exposure times, SMS2 plasma membrane localization is evident. Scale bar = 5 µm, except the insets where scale bar = 2.5 µm. (B) HeLa cells transfected with SMS1-V5 (green) or SMS2-V5 (green) infected with C. trachomatis serovar D for 24 hrs and then fixed and stained with antibodies to IncA (red) to identify the inclusion membrane. Enlargements of the boxed regions (inset) are shown to the right. Images represent a single z slice from confocal images. The exposure time for each filter set for all images was identical. SMS2 but not SMS1 partially overlaps with IncA on the inclusion. Scale bar = 5 µm, except the insets where scale bar = 2.5 µm. (C) HeLa cells transfected for 18 hrs with CERT-GFP, SMS1-V5, or SMS2-V5 were infected with C. trachomatis L2, treated with 10 µM BFA or Nocodazole for 1–24 hpi, and then fixed and stained with antibodies to V5 (green) and to p230 (red) to identify the trans -Golgi. BFA and Nocodazole disrupted SMS1 localization around the inclusion but had no effect on SMS2 or CERT localization at the inclusion. Images represent a single z slice from confocal images. Scale bar = 5 µm. N, host nucleus; *, inclusion.

Figure Legend Snippet: (A) HeLa cells co-transfected for 18 hrs with CERT-GFP and C-terminally 3xFLAG-tagged SMS1/SMS2 (upper 2 rows) or SMS1-V5 and C-terminally 3xFLAG-tagged SMS2 (bottom row) were infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to FLAG (red) and/or to V5 (green). Single channel images of uninfected cells are shown to the right. Enlargements of the boxed regions (inset) in infected samples are shown to the right of infected set. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). CERT and SMS1 localization at the inclusion are distinct while CERT and SMS2 partially overlap. SMS2 localization partially overlaps with SMS1 around the inclusion, however SMS2 also localizes to the inclusion. At longer exposure times, SMS2 plasma membrane localization is evident. Scale bar = 5 µm, except the insets where scale bar = 2.5 µm. (B) HeLa cells transfected with SMS1-V5 (green) or SMS2-V5 (green) infected with C. trachomatis serovar D for 24 hrs and then fixed and stained with antibodies to IncA (red) to identify the inclusion membrane. Enlargements of the boxed regions (inset) are shown to the right. Images represent a single z slice from confocal images. The exposure time for each filter set for all images was identical. SMS2 but not SMS1 partially overlaps with IncA on the inclusion. Scale bar = 5 µm, except the insets where scale bar = 2.5 µm. (C) HeLa cells transfected for 18 hrs with CERT-GFP, SMS1-V5, or SMS2-V5 were infected with C. trachomatis L2, treated with 10 µM BFA or Nocodazole for 1–24 hpi, and then fixed and stained with antibodies to V5 (green) and to p230 (red) to identify the trans -Golgi. BFA and Nocodazole disrupted SMS1 localization around the inclusion but had no effect on SMS2 or CERT localization at the inclusion. Images represent a single z slice from confocal images. Scale bar = 5 µm. N, host nucleus; *, inclusion.

Techniques Used: Transfection, Infection, Staining

(A) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (B) HeLa cells were depleted of SMS1 or SMS2 for 3 days and then labeled with BODIPY FL-Ceramide to analyze SM accumulation in the Golgi. The exposure time of all images was identical. SMS1 but not SMS2 depletion reduced BODIPY FL lipid accumulation in the Golgi. Scale bar = 5 µm. (C and D) HeLa cells were depleted of SMS1 or SMS2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with an antibody to MOMP (red). Bacteria and host DNA were detected using DAPI (blue). (D) SMS1 and SMS2-depleted cells were analyzed for inclusion size and progeny formation. Values (mean ± standard error) are shown as percentage of control siRNA samples. SMS1 and SMS2 depletion reduced inclusion size and production of infectious progeny. Data are representative of 2 independent experiments. ***p<0.001, all samples compared to control siRNA treatment (ANOVA). N, host nucleus; red arrows point to inclusions. IFU, inclusion forming units. Scale bar = 5 µm.

Figure Legend Snippet: (A) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (B) HeLa cells were depleted of SMS1 or SMS2 for 3 days and then labeled with BODIPY FL-Ceramide to analyze SM accumulation in the Golgi. The exposure time of all images was identical. SMS1 but not SMS2 depletion reduced BODIPY FL lipid accumulation in the Golgi. Scale bar = 5 µm. (C and D) HeLa cells were depleted of SMS1 or SMS2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with an antibody to MOMP (red). Bacteria and host DNA were detected using DAPI (blue). (D) SMS1 and SMS2-depleted cells were analyzed for inclusion size and progeny formation. Values (mean ± standard error) are shown as percentage of control siRNA samples. SMS1 and SMS2 depletion reduced inclusion size and production of infectious progeny. Data are representative of 2 independent experiments. ***p<0.001, all samples compared to control siRNA treatment (ANOVA). N, host nucleus; red arrows point to inclusions. IFU, inclusion forming units. Scale bar = 5 µm.

Techniques Used: Western Blot, Labeling, Infection, Staining

Image Search Results

( A ) Immunofluorescence images of ectocervical (top) and endocervical (bottom) organoids, uninfected (left) or infected (right) for 48 hours with Chlamydia, stained for KRT5 (green), major outer membrane protein (MOMP) (red), KRT8 (gray), and DAPI (blue). ( B ) UMAP projection of single cells from ecto- and endocervical organoids, colored by infection status: uninfected (UI), infected (Inf), and bystander (Bstd). ( C and D ) UMAP showing reclustered ectocervical squamous epithelial population from (B), colored by infection status (C) and subtype identity (D). ( E ) Proportion of UI, Bstd, and Inf cells in each ectocervical squamous subtype. ( F to G ) UMAP showing reclustered endocervical columnar epithelia from (B), colored by infection status (F) and subtype (G). ( H ) Proportion of UI, Bstd, and Inf cells in each endocervical columnar subtype. ( I ) Heatmap of differentially regulated TFs between ecto- and endocervix across infection conditions; color bar depicts the TF activity scores from high (deep pink) to low (blue). ( J ) Violin plot of gene set enrichment scores for the GO term defense response to bacterium across epithelial compartments and infection states; statistical significance assessed by Wilcoxon rank-sum test with Holm-adjusted P values ( ****P ≤ 0.0001). ( K ) The relative expression of IFN-related genes across ecto- and endocervical subclusters; dot size represents the % of cells expressing a particular gene, and the color bar indicates the intensity of scaled mean expression levels ranging from high (red) to low (blue). ( L ) Gene-weighted density UMAP projections showing expression of STAT1 , STAT2 , and IRF9 across epithelial cells in (B). ( M ) Violin plot showing ISG15 expression across ecto- and endocervical organoids in uninfected, bystander, and infected states. ( N ) IHC images showing CDH1 (green), ISG15 (red), MOMP (gray), and DAPI (blue) in ecto- and endocervical organoids, uninfected (left) or infected (right). Yellow arrows mark infected cells; arrowheads indicate ISG15 + bystander cells.

Journal: Science Advances

Article Title: Single-cell atlas of cervical organoids uncovers epithelial immune heterogeneity and intercellular cross-talk during Chlamydia infection

doi: 10.1126/sciadv.ady1640

Figure Lengend Snippet: ( A ) Immunofluorescence images of ectocervical (top) and endocervical (bottom) organoids, uninfected (left) or infected (right) for 48 hours with Chlamydia, stained for KRT5 (green), major outer membrane protein (MOMP) (red), KRT8 (gray), and DAPI (blue). ( B ) UMAP projection of single cells from ecto- and endocervical organoids, colored by infection status: uninfected (UI), infected (Inf), and bystander (Bstd). ( C and D ) UMAP showing reclustered ectocervical squamous epithelial population from (B), colored by infection status (C) and subtype identity (D). ( E ) Proportion of UI, Bstd, and Inf cells in each ectocervical squamous subtype. ( F to G ) UMAP showing reclustered endocervical columnar epithelia from (B), colored by infection status (F) and subtype (G). ( H ) Proportion of UI, Bstd, and Inf cells in each endocervical columnar subtype. ( I ) Heatmap of differentially regulated TFs between ecto- and endocervix across infection conditions; color bar depicts the TF activity scores from high (deep pink) to low (blue). ( J ) Violin plot of gene set enrichment scores for the GO term defense response to bacterium across epithelial compartments and infection states; statistical significance assessed by Wilcoxon rank-sum test with Holm-adjusted P values ( ****P ≤ 0.0001). ( K ) The relative expression of IFN-related genes across ecto- and endocervical subclusters; dot size represents the % of cells expressing a particular gene, and the color bar indicates the intensity of scaled mean expression levels ranging from high (red) to low (blue). ( L ) Gene-weighted density UMAP projections showing expression of STAT1 , STAT2 , and IRF9 across epithelial cells in (B). ( M ) Violin plot showing ISG15 expression across ecto- and endocervical organoids in uninfected, bystander, and infected states. ( N ) IHC images showing CDH1 (green), ISG15 (red), MOMP (gray), and DAPI (blue) in ecto- and endocervical organoids, uninfected (left) or infected (right). Yellow arrows mark infected cells; arrowheads indicate ISG15 + bystander cells.

Article Snippet: The following primary antibodies were used for immunofluorescence: mouse anti–acetylated tubulin–Alexa Fluor 647 (1:300, Santa Cruz Biotechnology, sc-23950-AF647), mouse anti–E-cadherin–Alexa Fluor 488 (1:50, BD Biosciences, 560061), mouse anti–E-cadherin (1:50, BD Biosciences, 610181), rabbit anti–KRT5–Alexa Fluor 488 (1:300, Abcam, ab193894), mouse anti-MUC5B (1:200, Abcam, ab77995), rabbit anti-MUC21 (1:200, ProteinAtlas, HPA052028), rabbit anti-KRT8 (1:200, Abcam, ab59400), mouse-anti-KRT6 (1:50, Abcam, ab18586), recombinant rabbit anti-PAX8 (1:200, Abcam, ab239363), goat anti– C. trachomatis major outer membrane protein (1:500, Bio-Rad, 1990-0804), rabbit anti–HLA-DQA1 antibody (EPR7300) (1:200, Abcam, ab128959), rabbit anti-ISG15 polyclonal antibody (1:200, Proteintech,15981-1-AP), and for labeling the DNA, 4′,6-diamidino-2-phenylindole (DAPI, Roche, 10236276001) were used.

Techniques: Immunofluorescence, Infection, Staining, Membrane, Activity Assay, Expressing

Inhibition of Ct infections by spilanthol (SPL): ( a ) Confocal microscopy imaging of cells infected with Chlamydia trachomatis (Ct) serovar D and SPL vehicle control (DMSO) at 24 h (upper) or 48 h post infection (hpi) (lower) or with SPL followed immediately by infection with Ct serovar D, and similarly imaged at 24 or 48 hpi. Chlamydial inclusions were visualized with anti-chlamydial major outer membrane protein (MOMP) antibody (red) and the image was overlayed with phase contrast image of cells. ( b ) Similar to panel ( a ) with the difference that SPL (120 μM) was added at 24 hpi and then the cells were imaged after additional incubation for 24 h (48 hpi). ( c ) Quantification of the effects of SPL on Ct infection by Western blot analysis of chlamydial HSP60 protein (*** p < 0.001). Control represents cells treated with heat-inactivated Ct and 0.1% DMSO. ( d ) Transmission electron microscopy (TEM) imaging (9,300×) comparing Ct inclusions of SPL (120 μM) treated and vehicle (DMSO) treated cells at 24 hpi. The small darker features within the inclusion are the elementary bodies (EBs) while the larger lighter gray features are the reticulate bodies (RBs). ( e ) The effects of SPL (120 μM) on the proliferation of HeLa cells in the absence of Ct infection were quantified by analyzing the percent confluence over time using the IncuCyte ZOOM live-cell imager. The data at 24 and 48 h were extracted and plotted as bar graph. There was no statistically significant difference between control and SPL treatment conditions at either one of the timepoints. ( f ) As in panel ( a ), with the difference of infection being performed with Ct serovar A. All data representative of a minimum two biological replicates.

Journal: Antioxidants

Article Title: Discovery of Spilanthol Endoperoxide as a Redox Natural Compound Active against Mammalian Prx3 and Chlamydia trachomatis Infection

doi: 10.3390/antiox9121220

Figure Lengend Snippet: Inhibition of Ct infections by spilanthol (SPL): ( a ) Confocal microscopy imaging of cells infected with Chlamydia trachomatis (Ct) serovar D and SPL vehicle control (DMSO) at 24 h (upper) or 48 h post infection (hpi) (lower) or with SPL followed immediately by infection with Ct serovar D, and similarly imaged at 24 or 48 hpi. Chlamydial inclusions were visualized with anti-chlamydial major outer membrane protein (MOMP) antibody (red) and the image was overlayed with phase contrast image of cells. ( b ) Similar to panel ( a ) with the difference that SPL (120 μM) was added at 24 hpi and then the cells were imaged after additional incubation for 24 h (48 hpi). ( c ) Quantification of the effects of SPL on Ct infection by Western blot analysis of chlamydial HSP60 protein (*** p < 0.001). Control represents cells treated with heat-inactivated Ct and 0.1% DMSO. ( d ) Transmission electron microscopy (TEM) imaging (9,300×) comparing Ct inclusions of SPL (120 μM) treated and vehicle (DMSO) treated cells at 24 hpi. The small darker features within the inclusion are the elementary bodies (EBs) while the larger lighter gray features are the reticulate bodies (RBs). ( e ) The effects of SPL (120 μM) on the proliferation of HeLa cells in the absence of Ct infection were quantified by analyzing the percent confluence over time using the IncuCyte ZOOM live-cell imager. The data at 24 and 48 h were extracted and plotted as bar graph. There was no statistically significant difference between control and SPL treatment conditions at either one of the timepoints. ( f ) As in panel ( a ), with the difference of infection being performed with Ct serovar A. All data representative of a minimum two biological replicates.

Article Snippet: Antibodies were obtained from the following sources: goat anti- C. trachomatis MOMP (Meridian Life Sciences, Saco, ME, USA), rhodamine red X conjugated anti-goat secondary antibody (Jackson Laboratories, West Grove, PA, USA), HRP conjugated anti-rabbit secondary antibody (Cell Signaling Technologies, Danvers, MA, USA), rabbit anti-HSP60 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HRP conjugated anti-mouse secondary antibody (Cell Signaling Technologies, Danvers, MA, USA).

Techniques: Inhibition, Confocal Microscopy, Imaging, Infection, Control, Membrane, Incubation, Western Blot, Transmission Assay, Electron Microscopy

SPL disrupts the formation of F-actin ring and stability of Ct inclusions by increasing mitochondrial oxidative state. ( a ) Confocal imaging analysis of mitochondrial protein sulfenylation induced by Ct and SPL (120 μM) alone or when used in combination. Mitochondrial sulfenylated proteins were imaged with DCP-NEt 2 C; ( b ) Flow cytometry analysis of DCP-NEt 2 C labeled cells to further validate and quantify the effects of Ct and/or SPL treatments. Statistical significance is indicated by the effects size as medium (*, 0.4 < x < 0.7) or large (**, 0.7 < x < 1.2) ( n = 2); ( c ) Disruption of F-actin cytoskeletal organization and Ct inclusions by MitoPQ, a mitochondria-targeted ROS generator. F-actin was detected with Alexa Fluor 488-phalloidin (green) and chlamydial inclusions were detected using anti-MOMP antibody (red); ( d – f ) The same imaging analysis monitoring Ct inclusions and F-actin was performed to investigate the effect of SPL on Ct inclusions and F-actin network ( d ), the rescue of SPL detrimental effects by narciclasine ( e ), and to demonstrate that SPL disrupts the F-actin organization and the ring structure surrounding the Ct inclusion regardless of the time of addition to cells relative to Ct infection (control, Ct infection 24 hpi, 0.1% DMSO). All imaging data are representative of a minimum two biological replicates and were collected at 40× magnification. White arrows point to the F-actin ring.

Journal: Antioxidants

Article Title: Discovery of Spilanthol Endoperoxide as a Redox Natural Compound Active against Mammalian Prx3 and Chlamydia trachomatis Infection

doi: 10.3390/antiox9121220

Figure Lengend Snippet: SPL disrupts the formation of F-actin ring and stability of Ct inclusions by increasing mitochondrial oxidative state. ( a ) Confocal imaging analysis of mitochondrial protein sulfenylation induced by Ct and SPL (120 μM) alone or when used in combination. Mitochondrial sulfenylated proteins were imaged with DCP-NEt 2 C; ( b ) Flow cytometry analysis of DCP-NEt 2 C labeled cells to further validate and quantify the effects of Ct and/or SPL treatments. Statistical significance is indicated by the effects size as medium (*, 0.4 < x < 0.7) or large (**, 0.7 < x < 1.2) ( n = 2); ( c ) Disruption of F-actin cytoskeletal organization and Ct inclusions by MitoPQ, a mitochondria-targeted ROS generator. F-actin was detected with Alexa Fluor 488-phalloidin (green) and chlamydial inclusions were detected using anti-MOMP antibody (red); ( d – f ) The same imaging analysis monitoring Ct inclusions and F-actin was performed to investigate the effect of SPL on Ct inclusions and F-actin network ( d ), the rescue of SPL detrimental effects by narciclasine ( e ), and to demonstrate that SPL disrupts the F-actin organization and the ring structure surrounding the Ct inclusion regardless of the time of addition to cells relative to Ct infection (control, Ct infection 24 hpi, 0.1% DMSO). All imaging data are representative of a minimum two biological replicates and were collected at 40× magnification. White arrows point to the F-actin ring.

Techniques: Imaging, Flow Cytometry, Labeling, Disruption, Infection, Control

$SPL E shows more potent inhibition of Ct infection ( a ) Chemical structure and ESI-TOF MS data of SPL E synthesized as described in Materials and Methods. MS data also shows the presence of SPL E dimer formed under the ESI-TOF MS conditions; ( b ) Confocal microscopy imaging of cells infected with Ct and SPL E vehicle (0.1% DMSO) at 24 hpi (control) or with SPL E followed immediately by infection with Ct and similarly imaged at 24 hpi. Chlamydial inclusions were visualized with anti-chlamydial major outer membrane protein (MOMP) antibody (red) and the image was overlayed with phase contrast images of cells; ( c ) Inhibition of Ct infection by SPL E was quantified by calculating the % area stained with anti-MOMP and representing the data as a fraction of vehicle control (n = 3 biological replicates, 3–6 imaging areas per biological replicate); ( d ) The effects of SPL E (120 μM) on the proliferation of HeLa cells in the absence of Ct infection were quantified by analyzing the percent confluence over time using the IncuCyte ZOOM live-cell imager. The data at 24 and 48 h were extracted and plotted as bar graph. There was no statistically significant difference between control and SPL treatment conditions at either one of the time points (n = 3). ( e ) Disruption of F-actin cytoskeletal organization and Ct inclusions by SPL E (120 μM). F-actin was detected with Alexa Fluor 488-phalloidin (green) and chlamydial inclusions were detected using anti-MOMP antibody (red). All imaging data are representative of a minimum two biological replicates and were collected at 40x magnification. White arrows point to the F-actin ring.$

Journal: Antioxidants

Article Title: Discovery of Spilanthol Endoperoxide as a Redox Natural Compound Active against Mammalian Prx3 and Chlamydia trachomatis Infection

doi: 10.3390/antiox9121220

Figure Lengend Snippet: SPL E shows more potent inhibition of Ct infection ( a ) Chemical structure and ESI-TOF MS data of SPL E synthesized as described in Materials and Methods. MS data also shows the presence of SPL E dimer formed under the ESI-TOF MS conditions; ( b ) Confocal microscopy imaging of cells infected with Ct and SPL E vehicle (0.1% DMSO) at 24 hpi (control) or with SPL E followed immediately by infection with Ct and similarly imaged at 24 hpi. Chlamydial inclusions were visualized with anti-chlamydial major outer membrane protein (MOMP) antibody (red) and the image was overlayed with phase contrast images of cells; ( c ) Inhibition of Ct infection by SPL E was quantified by calculating the % area stained with anti-MOMP and representing the data as a fraction of vehicle control (n = 3 biological replicates, 3–6 imaging areas per biological replicate); ( d ) The effects of SPL E (120 μM) on the proliferation of HeLa cells in the absence of Ct infection were quantified by analyzing the percent confluence over time using the IncuCyte ZOOM live-cell imager. The data at 24 and 48 h were extracted and plotted as bar graph. There was no statistically significant difference between control and SPL treatment conditions at either one of the time points (n = 3). ( e ) Disruption of F-actin cytoskeletal organization and Ct inclusions by SPL E (120 μM). F-actin was detected with Alexa Fluor 488-phalloidin (green) and chlamydial inclusions were detected using anti-MOMP antibody (red). All imaging data are representative of a minimum two biological replicates and were collected at 40x magnification. White arrows point to the F-actin ring.

Techniques: Inhibition, Infection, Synthesized, Confocal Microscopy, Imaging, Control, Membrane, Staining, Disruption

Top row: representative field of extrusions following enrichment procedures, at 20× magnification. A Brightfield image showing 14 extrusions (white arrowheads). Inset: enlargement of the extrusion marked with white asterisk. B Micrograph of the same field of extrusions shown in A , labeled with DAPI (blue) to show the lack of nuclei in extrusions. A single host cell with nucleus is shown (yellow arrow). Middle row: representative micrographs of an isolated extrusion, visualized live with fluorescent probes for: C host plasma membrane (PM) (FM4-64, red), D GFP-expressing C. trachomatis (GFP, green), E nuclei (DAPI, blue), F merge of C - E. Bottom row: for comparison, micrographs of HeLa cells infected with GFP-expressing C. trachomatis L2 for 48 hpi, and visualized live with fluorescent probes for: G plasma membrane (FM4-64, red), H GFP-expressing C. trachomatis (green), I nuclei (DAPI, blue), J merge of G - I . Scale bar, 10 μm.

Journal: bioRxiv

Article Title: Extrusions promote engulfment and Chlamydia survival within macrophages

doi: 10.1101/041079

Figure Lengend Snippet: Top row: representative field of extrusions following enrichment procedures, at 20× magnification. A Brightfield image showing 14 extrusions (white arrowheads). Inset: enlargement of the extrusion marked with white asterisk. B Micrograph of the same field of extrusions shown in A , labeled with DAPI (blue) to show the lack of nuclei in extrusions. A single host cell with nucleus is shown (yellow arrow). Middle row: representative micrographs of an isolated extrusion, visualized live with fluorescent probes for: C host plasma membrane (PM) (FM4-64, red), D GFP-expressing C. trachomatis (GFP, green), E nuclei (DAPI, blue), F merge of C - E. Bottom row: for comparison, micrographs of HeLa cells infected with GFP-expressing C. trachomatis L2 for 48 hpi, and visualized live with fluorescent probes for: G plasma membrane (FM4-64, red), H GFP-expressing C. trachomatis (green), I nuclei (DAPI, blue), J merge of G - I . Scale bar, 10 μm.

Article Snippet: Antibodies/dyes were obtained from the following sources: Phalloidin 633, donkey anti-goat 488 from Invitrogen (Waltham, MA), DAPI, goat anti-mouse 488 from Thermo Fisher (Waltham, MA), anti-GFP 488, FM4–64 from Molecular Probes (Eugene, OR), MitoTracker Green, Annexin-V 568 from Life Technologies (Carlsbad, CA), mouse anti- Chlamydia FITC conjugate from Meridian Diagnostics (Cincinnati, OH), goat anti- C. trachomatis MOMP from Virostat (Portland, ME), mouse anti- C. trachomatis LPS ad mouse anti-CT223 donated by Bob Suchland (University of Washington, WA).

Techniques: Labeling, Isolation, Expressing, Infection

Top row: representative micrographs of an isolated extrusion, visualized live with fluorescent probes for: A C. trachomatis (GFP, green), B phosphatidylserine (PS; annexin V-568, red), C nuclei (DAPI, blue), D merge of A-C. Second row: representative live extrusions showing: E C. trachomatis (mKate2, red), F mitochondria (mitotracker-488, green), G nuclei (DAPI, blue), H merge of E-G. Third row: micrographs of uninfected HeLa cells visualized live with probes for: I plasma membrane (PM) (FM4-64, red), J mitochondria (mitotracker-488, green), K nuclei (DAPI, blue), L merge of I-K. Fourth row: representative micrographs of HeLa cells infected with mKate2-expressing C. trachomatis for 48 h, and visualized live for: M C. trachomatis (mKate2, red), N mitochondria (mitotracker-488, green), O nuclei (DAPI, blue), P merge of M-O. Q Positive association of mitochondria, PS, and nuclei, was quantitatively enumerated from over 75 images of extrusions. Data points show mean + SEM, n = 3. R Transmission electron micrograph of a representative, isolated C. trachomatis extrusion at 0 hpe (hours post extrusion). Scale bar, 10 μm for A-P ; 1 μm for R . Inset within R of enlarged region of extrusion showing presence of a double membrane on the periphery of the extrusion.

Journal: bioRxiv

Article Title: Extrusions promote engulfment and Chlamydia survival within macrophages

doi: 10.1101/041079

Figure Lengend Snippet: Top row: representative micrographs of an isolated extrusion, visualized live with fluorescent probes for: A C. trachomatis (GFP, green), B phosphatidylserine (PS; annexin V-568, red), C nuclei (DAPI, blue), D merge of A-C. Second row: representative live extrusions showing: E C. trachomatis (mKate2, red), F mitochondria (mitotracker-488, green), G nuclei (DAPI, blue), H merge of E-G. Third row: micrographs of uninfected HeLa cells visualized live with probes for: I plasma membrane (PM) (FM4-64, red), J mitochondria (mitotracker-488, green), K nuclei (DAPI, blue), L merge of I-K. Fourth row: representative micrographs of HeLa cells infected with mKate2-expressing C. trachomatis for 48 h, and visualized live for: M C. trachomatis (mKate2, red), N mitochondria (mitotracker-488, green), O nuclei (DAPI, blue), P merge of M-O. Q Positive association of mitochondria, PS, and nuclei, was quantitatively enumerated from over 75 images of extrusions. Data points show mean + SEM, n = 3. R Transmission electron micrograph of a representative, isolated C. trachomatis extrusion at 0 hpe (hours post extrusion). Scale bar, 10 μm for A-P ; 1 μm for R . Inset within R of enlarged region of extrusion showing presence of a double membrane on the periphery of the extrusion.

Techniques: Isolation, Infection, Expressing, Transmission Assay

HeLa cells were infected with mkate expressing C. trachomatis for 2 h, then rinsed and incubated at 37C for 18 hpi. A mKate2-expressing C. trachomatis (red). B Mitochondria stained with MitoTracker Green FM (green). C DAPI staining of host cell nuclei (blue). D Merge of panels A-C . Scale bar, 10 μm.

Journal: bioRxiv

Article Title: Extrusions promote engulfment and Chlamydia survival within macrophages

doi: 10.1101/041079

Figure Lengend Snippet: HeLa cells were infected with mkate expressing C. trachomatis for 2 h, then rinsed and incubated at 37C for 18 hpi. A mKate2-expressing C. trachomatis (red). B Mitochondria stained with MitoTracker Green FM (green). C DAPI staining of host cell nuclei (blue). D Merge of panels A-C . Scale bar, 10 μm.

Techniques: Infection, Expressing, Incubation, Staining

Top row: C. trachomatis extrusions were incubated onto HeLa cells for 2 h, then rinsed, and allowed to incubate at 37C for 18 hpi. Cells were fixed and stained for immunofluorescence analysis. A GFP-expressing Chlamydia (green). B Evans blue counterstain of HeLa cytoplasm (red). C DAPI staining of host cell nuclei (blue). D Merge of panels A-C. Bottom row: HeLa cells infected with GFP- Chlamydia 18 hpi. E GFP-expressing Chlamydia (green). F Evans blue counterstain of HeLa cytoplasm (red). G DAPI staining of host cell nuclei (blue). H Merge of panels E-G . Scale bar, 10 μm.

Journal: bioRxiv

Article Title: Extrusions promote engulfment and Chlamydia survival within macrophages

doi: 10.1101/041079

Figure Lengend Snippet: Top row: C. trachomatis extrusions were incubated onto HeLa cells for 2 h, then rinsed, and allowed to incubate at 37C for 18 hpi. Cells were fixed and stained for immunofluorescence analysis. A GFP-expressing Chlamydia (green). B Evans blue counterstain of HeLa cytoplasm (red). C DAPI staining of host cell nuclei (blue). D Merge of panels A-C. Bottom row: HeLa cells infected with GFP- Chlamydia 18 hpi. E GFP-expressing Chlamydia (green). F Evans blue counterstain of HeLa cytoplasm (red). G DAPI staining of host cell nuclei (blue). H Merge of panels E-G . Scale bar, 10 μm.

Techniques: Incubation, Staining, Immunofluorescence, Expressing, Infection

C. trachomatis L2 extrusions were isolated and enriched from infected HeLa cells at 48 hpi, and incubated in cell culture media for 0 to 8 h at 37°C. At times indicated, extrusions were broken by brief sonication, and bacteria were plated onto poly-D lysine coated coverglass for fixation and immunofluorescence staining. Chlamydia were stained with an anti-MOMP antibody (green) and visualized by fluorescence microscopy at 100× magnification for illumination and differentiation of C. trachomatis RB and EB developmental forms. A Representative image of ringlike RB (marked with white arrow) and smaller EB (marked with yellow arrow). Scale bar, 1 μm. B Quantitative analysis of the relative ratio of EB and RB from 24 h and 48 h infected cells (left two columns), and experimental extrusion samples, (right four columns). Ratios were calculated from number of total bacteria per field per time group. Labels inside bars represent percentages of RB. Data show mean percentages ± SEM, n = 3. **** denotes a p value < 0.0001.

Journal: bioRxiv

Article Title: Extrusions promote engulfment and Chlamydia survival within macrophages

doi: 10.1101/041079

Figure Lengend Snippet: C. trachomatis L2 extrusions were isolated and enriched from infected HeLa cells at 48 hpi, and incubated in cell culture media for 0 to 8 h at 37°C. At times indicated, extrusions were broken by brief sonication, and bacteria were plated onto poly-D lysine coated coverglass for fixation and immunofluorescence staining. Chlamydia were stained with an anti-MOMP antibody (green) and visualized by fluorescence microscopy at 100× magnification for illumination and differentiation of C. trachomatis RB and EB developmental forms. A Representative image of ringlike RB (marked with white arrow) and smaller EB (marked with yellow arrow). Scale bar, 1 μm. B Quantitative analysis of the relative ratio of EB and RB from 24 h and 48 h infected cells (left two columns), and experimental extrusion samples, (right four columns). Ratios were calculated from number of total bacteria per field per time group. Labels inside bars represent percentages of RB. Data show mean percentages ± SEM, n = 3. **** denotes a p value < 0.0001.

Techniques: Isolation, Infection, Incubation, Cell Culture, Sonication, Immunofluorescence, Staining, Fluorescence, Microscopy

Top row: Isolated Chlamydia extrusions were incubated with bone-marrow derived macrophages for 1 h, then rinsed, and fresh macrophage media was plated onto cells for duration of assay; 0–72 h at 37°C. At stages indicated, cells were fixed and stained to visualize: GFP-expressing C. trachomatis (green), macrophage nuclei (DAPI, blue), and actin (phalloidin-647 purple). Representative images of macrophages containing C. trachomatis extrusions show distinct stages of their interaction: A early, 0–4 h, presence of intact extrusions; B middle, 4–24 hpi, broken-down extrusions, with Chlamydia still visible within macrophage; C late, 48–72 hpi, no visibly intact extrusions, but Chlamydia still visible within macrophage. In other experiments, isolated extrusions were briefly sonicated to release Chlamydia , and bacteria were incubated with macrophages for 0–72 h at 37°C. Representative examples are shown at similar stages as for extrusions: D early, some bacteria present in macrophages; E middle, very few to no bacteria seen within macrophage; F late, no bacteria seen in macrophages. G Transmission electron micrograph of a representative macrophage containing an engulfed C. trachomatis extrusion following 1 h co-incubation (shown by yellow arrow). Scale bar, 1 μm. H Three dimensional view of a macrophage containing an engulfed extrusion. Host cell actin (purple), nuclei (blue), and GFP Chlamydia (GFP). Scale bar, 10 μm for all panels except G .

Journal: bioRxiv

Article Title: Extrusions promote engulfment and Chlamydia survival within macrophages

doi: 10.1101/041079

Figure Lengend Snippet: Top row: Isolated Chlamydia extrusions were incubated with bone-marrow derived macrophages for 1 h, then rinsed, and fresh macrophage media was plated onto cells for duration of assay; 0–72 h at 37°C. At stages indicated, cells were fixed and stained to visualize: GFP-expressing C. trachomatis (green), macrophage nuclei (DAPI, blue), and actin (phalloidin-647 purple). Representative images of macrophages containing C. trachomatis extrusions show distinct stages of their interaction: A early, 0–4 h, presence of intact extrusions; B middle, 4–24 hpi, broken-down extrusions, with Chlamydia still visible within macrophage; C late, 48–72 hpi, no visibly intact extrusions, but Chlamydia still visible within macrophage. In other experiments, isolated extrusions were briefly sonicated to release Chlamydia , and bacteria were incubated with macrophages for 0–72 h at 37°C. Representative examples are shown at similar stages as for extrusions: D early, some bacteria present in macrophages; E middle, very few to no bacteria seen within macrophage; F late, no bacteria seen in macrophages. G Transmission electron micrograph of a representative macrophage containing an engulfed C. trachomatis extrusion following 1 h co-incubation (shown by yellow arrow). Scale bar, 1 μm. H Three dimensional view of a macrophage containing an engulfed extrusion. Host cell actin (purple), nuclei (blue), and GFP Chlamydia (GFP). Scale bar, 10 μm for all panels except G .

Techniques: Isolation, Incubation, Derivative Assay, Staining, Expressing, Sonication, Transmission Assay

A Murine macrophage incubated with extrusion at 24 hpi, showing establishment of an inclusion. B Macrophage incubated with extrusions at 0 hpi, showing no CT223 staining around the periphery of the inclusion. C Extrusion engulfment 0 hpi showing what appears to be a small abberant body within the macrophage. D Free Chlamydia engulfment by macrophage showing survival of small population of Chlamydia 24 hpi. For all images in figure: From left: GFP-expressing C. trachomatis (Green), inclusion membrane staining with antibody to CT223 (red), nuclei stained with DAPI (blue), actin staining with phalloidin-633 (purple), and merge of 4 colors. Scale bar, 10 μm.

Journal: bioRxiv

Article Title: Extrusions promote engulfment and Chlamydia survival within macrophages

doi: 10.1101/041079

Figure Lengend Snippet: A Murine macrophage incubated with extrusion at 24 hpi, showing establishment of an inclusion. B Macrophage incubated with extrusions at 0 hpi, showing no CT223 staining around the periphery of the inclusion. C Extrusion engulfment 0 hpi showing what appears to be a small abberant body within the macrophage. D Free Chlamydia engulfment by macrophage showing survival of small population of Chlamydia 24 hpi. For all images in figure: From left: GFP-expressing C. trachomatis (Green), inclusion membrane staining with antibody to CT223 (red), nuclei stained with DAPI (blue), actin staining with phalloidin-633 (purple), and merge of 4 colors. Scale bar, 10 μm.

Techniques: Incubation, Staining, Expressing

Extrusions, or sonicated extrusions (free Chlamydia ), were incubated onto murine bone marrow derived macrophages and incubated at 37°C for up to 8 h. At times indicated, cells were sonicated to release bacteria, and C. trachomatis infectivity was quantitatively measured by performing IFU assays on McCoy cells. Data points show mean ± SEM, n = 3. **** denotes a p value < 0.0001, ** denotes a p value < 0.01.

Journal: bioRxiv

Article Title: Extrusions promote engulfment and Chlamydia survival within macrophages

doi: 10.1101/041079

Figure Lengend Snippet: Extrusions, or sonicated extrusions (free Chlamydia ), were incubated onto murine bone marrow derived macrophages and incubated at 37°C for up to 8 h. At times indicated, cells were sonicated to release bacteria, and C. trachomatis infectivity was quantitatively measured by performing IFU assays on McCoy cells. Data points show mean ± SEM, n = 3. **** denotes a p value < 0.0001, ** denotes a p value < 0.01.

Techniques: Sonication, Incubation, Derivative Assay, Infection

A Overview of the experimental strategy depicting extrusion isolation, infection times in macrophages, and collection of released C. trachomatis EB. B Extrusions, or sonicated extrusions, were used to infect macrophages, and cell supernatants were collected immediately after rinsing (t = 0) and at 48 h. The infectivity of Chlamydia in macrophage supernatants was determined by IFU assays on HeLa cells, at 24 h. Data points show mean ± SEM, n = 3. ** denotes a p value < 0.01.

Journal: bioRxiv

Article Title: Extrusions promote engulfment and Chlamydia survival within macrophages

doi: 10.1101/041079

Figure Lengend Snippet: A Overview of the experimental strategy depicting extrusion isolation, infection times in macrophages, and collection of released C. trachomatis EB. B Extrusions, or sonicated extrusions, were used to infect macrophages, and cell supernatants were collected immediately after rinsing (t = 0) and at 48 h. The infectivity of Chlamydia in macrophage supernatants was determined by IFU assays on HeLa cells, at 24 h. Data points show mean ± SEM, n = 3. ** denotes a p value < 0.01.

Techniques: Isolation, Infection, Sonication

Upon release from infected epithelial cells, Chlamydia -containing extrusions are engulfed by macrophages. Migration of these macrophages, followed by eventual escape of Chlamydia from them, can result in the dissemination of infectious C. trachomatis to more distant sites, e.g., away from inflammatory foci surrounding the primary site of infection, to draining lymph nodes, or to new hosts. Extrusions may alternatively mediate some of these outcomes without the need for hijacking macrophages.

Journal: bioRxiv

Article Title: Extrusions promote engulfment and Chlamydia survival within macrophages

doi: 10.1101/041079

Figure Lengend Snippet: Upon release from infected epithelial cells, Chlamydia -containing extrusions are engulfed by macrophages. Migration of these macrophages, followed by eventual escape of Chlamydia from them, can result in the dissemination of infectious C. trachomatis to more distant sites, e.g., away from inflammatory foci surrounding the primary site of infection, to draining lymph nodes, or to new hosts. Extrusions may alternatively mediate some of these outcomes without the need for hijacking macrophages.

Techniques: Infection, Migration

Fig. 1. Schematic view of the constructs used in this study: (a and b) denote full- length MOMP and chimeric MOMP, respectively, expressed in E. coli (pET101/D- TOPO vector), (c and d) denote full-length MOMP and chimeric MOMP, respectively, expressed in plants (pGreen0229 vector).

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 1. Schematic view of the constructs used in this study: (a and b) denote full- length MOMP and chimeric MOMP, respectively, expressed in E. coli (pET101/D- TOPO vector), (c and d) denote full-length MOMP and chimeric MOMP, respectively, expressed in plants (pGreen0229 vector).

Article Snippet: Three selected transgenic lines (numbers 9, 15, and 25) were used in further analysis and stable integration of the transgene in these lines was demonstrated for up to six generations using the polyclonal antibody against C. trachomatis MOMP (Acris Antibodies; Fig. 7a).

Techniques: Construct, Plasmid Preparation

Fig. 2. Northern blot analysis of plants transformed with the full-length MOMP construct. Plants 1 and 2 show the presence of MOMP mRNA transcripts. WT denotes untransformed wild type plant. All three tested transgenic plants were PCR positive.

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 2. Northern blot analysis of plants transformed with the full-length MOMP construct. Plants 1 and 2 show the presence of MOMP mRNA transcripts. WT denotes untransformed wild type plant. All three tested transgenic plants were PCR positive.

Techniques: Northern Blot, Transformation Assay, Construct, Transgenic Assay

Fig. 3. (a) Topology and primary structure of the Ct serovar E MOMP as adopted from Findlay et al. [26]: squares, amino acids residues found in membrane spanning helices; circles, amino acid residues found in extramembraneous parts of the protein. The domains selected for design of the chimeric MOMP are shown in red; (b) the putative ﬂexible conformation that can be obtained using the (Gly4Ser)2Gly4 linker (shown in black). The amino acid residues that differ between MOMP serovar E (shown) and serovar D in the VS2 and VS4 loops are given in blue; (c) the more rigid conformation that can be obtained using the (Gly4Ser)2Gly4 linker (shown in black). The amino acid residues that differ between MOMP serovar E (shown) and serovar D in the VS2 and VS4 loops are given in blue. The green C-terminal tag contain a V5 epitope and a His6 puriﬁcation tag, as expressed in Escherichia coli but not in plants (see Fig. 1). Fig. 4. (a) PCR analysis of the assembled MOMP chimeric construct. Ch denotes PCR product from a vector containing the assembled chimera, N denotes the PCR negative control, L denotes the DNA size marker. The ampliﬁed product has the expected size of 351 bp. (b) Western blot analysis of recombinant His-tagged chimeric MOMP protein expressed in Escherichia coli and puriﬁed using Ni–NTA chromatography. A band of the expected size (17 kDa) was detected using mouse monoclonal antibodies to Chlamydia trachomatis MOMP (Acris Antibodies). Ch denotes the chimeric MOMP protein, L denotes the protein size marker.

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 3. (a) Topology and primary structure of the Ct serovar E MOMP as adopted from Findlay et al. [26]: squares, amino acids residues found in membrane spanning helices; circles, amino acid residues found in extramembraneous parts of the protein. The domains selected for design of the chimeric MOMP are shown in red; (b) the putative ﬂexible conformation that can be obtained using the (Gly4Ser)2Gly4 linker (shown in black). The amino acid residues that differ between MOMP serovar E (shown) and serovar D in the VS2 and VS4 loops are given in blue; (c) the more rigid conformation that can be obtained using the (Gly4Ser)2Gly4 linker (shown in black). The amino acid residues that differ between MOMP serovar E (shown) and serovar D in the VS2 and VS4 loops are given in blue. The green C-terminal tag contain a V5 epitope and a His6 puriﬁcation tag, as expressed in Escherichia coli but not in plants (see Fig. 1). Fig. 4. (a) PCR analysis of the assembled MOMP chimeric construct. Ch denotes PCR product from a vector containing the assembled chimera, N denotes the PCR negative control, L denotes the DNA size marker. The ampliﬁed product has the expected size of 351 bp. (b) Western blot analysis of recombinant His-tagged chimeric MOMP protein expressed in Escherichia coli and puriﬁed using Ni–NTA chromatography. A band of the expected size (17 kDa) was detected using mouse monoclonal antibodies to Chlamydia trachomatis MOMP (Acris Antibodies). Ch denotes the chimeric MOMP protein, L denotes the protein size marker.

Techniques: Membrane, Construct, Plasmid Preparation, Negative Control, Marker, Western Blot, Recombinant, Chromatography, Bioprocessing

Fig. 5. Evaluation of the anti-chimeric MOMP antiserum produced in rabbits. The puriﬁed recombinant MOMP chimera was analyzed by immunoblotting using anti- chimeric MOMP serum (S), afﬁnity puriﬁed anti-chimeric MOMP antibodies (A) and pre-serum (P). L denotes the protein size marker.

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 5. Evaluation of the anti-chimeric MOMP antiserum produced in rabbits. The puriﬁed recombinant MOMP chimera was analyzed by immunoblotting using anti- chimeric MOMP serum (S), afﬁnity puriﬁed anti-chimeric MOMP antibodies (A) and pre-serum (P). L denotes the protein size marker.

Techniques: Produced, Recombinant, Western Blot, Marker

Fig. 6. Immunoﬂuorescence slides demonstrating antibody reactivity toward Chlamydia trachomatis elementary bodies and its full-length MOMP protein (bright ﬂuorescent dots). (a) Anti-MOMP chimera antibodies (post-serum), produced in rabbits injected with MOMP chimera, showing high speciﬁc reactivity against inactivated Ct elementary bodies. (b) Rabbit pre-serum lacking MOMP reactivity. (c) Minimal ﬂuorescence of the secondary anti-rabbit IgG antibody conjugate itself in the absence of rabbit serum. Magniﬁcation was 400 in (a) and 200 in (b and c), respectively.

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 6. Immunoﬂuorescence slides demonstrating antibody reactivity toward Chlamydia trachomatis elementary bodies and its full-length MOMP protein (bright ﬂuorescent dots). (a) Anti-MOMP chimera antibodies (post-serum), produced in rabbits injected with MOMP chimera, showing high speciﬁc reactivity against inactivated Ct elementary bodies. (b) Rabbit pre-serum lacking MOMP reactivity. (c) Minimal ﬂuorescence of the secondary anti-rabbit IgG antibody conjugate itself in the absence of rabbit serum. Magniﬁcation was 400 in (a) and 200 in (b and c), respectively.

Techniques: Produced, Injection

Fig. 8. (a) Semiquantitative analysis of the content of chimeric MOMP in transformed carrots. Kar and 313 denote two different transgenic lines in cultivars Karotan and Napoli, respectively. Comparison of the intensity of the stained bands in the transgenic plants and controls (puriﬁed and accurately quantiﬁed chimeric MOMP) allowed the estimation of the approximate MOMP chimera protein concentration in the carrots. (b) Immunoblot showing the speciﬁcity of the antiserum raised against E. coli-produced chimeric Ct MOMP protein when used for probing extracts from carrot lines 350 and 604 (in the Karotan background) expressing the same protein. L denotes the molecular weight standards, WT are extract from wild type Karotan carrots, and PC are E. coli-produced positive controls (2.5 and 7.5 lg protein, respectively). The asterisks indicate the MOMP chimera dimer.

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 8. (a) Semiquantitative analysis of the content of chimeric MOMP in transformed carrots. Kar and 313 denote two different transgenic lines in cultivars Karotan and Napoli, respectively. Comparison of the intensity of the stained bands in the transgenic plants and controls (puriﬁed and accurately quantiﬁed chimeric MOMP) allowed the estimation of the approximate MOMP chimera protein concentration in the carrots. (b) Immunoblot showing the speciﬁcity of the antiserum raised against E. coli-produced chimeric Ct MOMP protein when used for probing extracts from carrot lines 350 and 604 (in the Karotan background) expressing the same protein. L denotes the molecular weight standards, WT are extract from wild type Karotan carrots, and PC are E. coli-produced positive controls (2.5 and 7.5 lg protein, respectively). The asterisks indicate the MOMP chimera dimer.

Techniques: Transformation Assay, Transgenic Assay, Comparison, Staining, Protein Concentration, Western Blot, Produced, Expressing, Molecular Weight

$Fig. 7. (a) Western blot detection of constitutively expressed chimeric MOMP in Arabidopsis leaf extracts from T6 generation plants using polyclonal antibody against full-length C. trachomatis MOMP (Acris Antibodies). L denotes the protein size marker; 9, 15, and 25 denote three different transgenic lines of Arabidopsis; WT denotes non-transformed wild type Arabidopsis; A corresponds to 5 ll unfraction- ated plant extract and B corresponds to 15 ll unfractionated plant extract; (b) Southern blot analysis of four Arabidopsis lines transformed with the chimeric MOMP construct (lines 9, 12, 15, and 25). Two different DNA digests of each line were produced by using the Dra I and Nde I restriction enzymes and probed with random primer 32P-labeled chimera MOMP oligonucleotides. The restriction enzymes chosen did not digest the MOMP chimera transgene itself. The number of observed bands corresponds to the copy number of the transgene.$

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 7. (a) Western blot detection of constitutively expressed chimeric MOMP in Arabidopsis leaf extracts from T6 generation plants using polyclonal antibody against full-length C. trachomatis MOMP (Acris Antibodies). L denotes the protein size marker; 9, 15, and 25 denote three different transgenic lines of Arabidopsis; WT denotes non-transformed wild type Arabidopsis; A corresponds to 5 ll unfraction- ated plant extract and B corresponds to 15 ll unfractionated plant extract; (b) Southern blot analysis of four Arabidopsis lines transformed with the chimeric MOMP construct (lines 9, 12, 15, and 25). Two different DNA digests of each line were produced by using the Dra I and Nde I restriction enzymes and probed with random primer 32P-labeled chimera MOMP oligonucleotides. The restriction enzymes chosen did not digest the MOMP chimera transgene itself. The number of observed bands corresponds to the copy number of the transgene.

Techniques: Western Blot, Marker, Transgenic Assay, Transformation Assay, Plant Extract, Southern Blot, Construct, Produced, Labeling

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells were infected with C. trachomatis L2 for 24 hrs and then fixed and stained with antibodies to GBF1 (red) and BIG1 (green). Bacteria and host DNA were detected using DAPI (blue). The cis and trans polarity of the Golgi was maintained in C. trachomatis L2-infected cells. N, host nucleus. *, inclusion. Scale bar = 5 µm. (B) HeLa cells were transfected with Arf1-GFP for 18 hrs, infected with C. trachomatis L2 for 24 hrs in the absence or presence of 10 µM BFA, and then fixed and stained with antibodies to GBF1 (red). Enlargements of boxed regions are shown to the right. Images represent a single z slice from confocal images. The exposure time for each filter set for all images was identical. Arf1-GFP localized to the region between two closely apposed inclusions (white arrow) and to a thin rim around the inclusion (red arrow) whereas GBF1 was excluded from these regions. *, inclusion. Scale bar = 5 µm. (C) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (D) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then labeled with BODIPY FL-Ceramide to visualize SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (E) HeLa cells were infected with C. trachomatis for 24 hrs, treated with 10 µM BFA or GCA during the last 3 hrs of infection, and then labeled with BODIPY FL-Ceramide to analyze SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (F) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then analyzed for progeny formation as described in . Values (mean ± standard error) are shown as percentage of control siRNA-treated samples. No significant decrease in progeny formation was observed. IFU, inclusion forming units.

Article Snippet: Antibodies were obtained from the following sources: mouse anti- Chlamydia FITC conjugate (Meridian Diagnostics), goat anti- C. trachomatis MOMP (Cortex Biochem), mouse anti-GAPDH (Chemicon), mouse anti-GBF1 (BD Transduction Laboratories), rabbit anti-BIG1 (Santa Cruz), rabbit anti-BIG2 (Bethyl Laboratories, Inc.), chicken anti-CERT (Sigma), rabbit anti-14-3-3β (Santa Cruz), mouse anti-vimentin (Sigma), mouse anti-FLAG (Sigma), rabbit anti-V5 (Sigma), mouse anti-HA (Covance), rabbit anti-p58 (Sigma), mouse anti-ceramide IgM (Clone15B4), rabbit anti-calnexin (Cell Signaling), rabbit anti-goat IgG horseradish peroxidase (HRP) (Calbiochem), goat anti-rabbit IgG HRP (Amersham Biosciences), goat anti-mouse HRP (Amersham Biosciences), donkey anti-goat Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), chicken anti-mouse 594 (Molecular Probes), goat anti-mouse IgG1 Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), Texas red-conjugated donkey anti-chicken IgY (Jackson Laboratories), and donkey anti-rabbit Alexa 488 (Molecular Probes).

Techniques: Infection, Staining, Transfection, Western Blot, Labeling

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells were treated with the indicated siRNA for 3 days, infected with C. trachomatis L2 for 24 hrs, then fixed and stained with antibodies to 14-3-3β (green) to identify the inclusion membrane and GBF1 (red). Bacteria and host DNA were detected using DAPI (blue). The exposure time for each filter set for all images was identical. White arrows point to breaks in the inclusion membrane where the bacteria are released into cytoplasm in GBF1-depleted cells. Inclusions formed in BIG1 and/or BIG2 depleted cells remain intact. (B) HeLa cells were depleted of GBF1 for 3 days, infected with C. trachomatis L2 for 24 hrs, then fixed and stained with antibodies to MOMP (green) to identify bacteria and vimentin (red). Bacteria and host DNA were detected using DAPI (blue). The exposure time for each filter set for all images was identical. White arrows point to the region on the inclusion that is devoid of vimentin staining and where bacteria are released into the cytoplasm. N, host nucleus; *, inclusion. MOMP, C. trachomatis major outer membrane protein. Scale bar = 5 µm.

Techniques: Infection, Staining

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells transfected with CERT-GFP for 18 hrs were left uninfected or infected with C. trachomatis L2 for 24 hrs. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). N, host nucleus; *, inclusions. Scale bar = 5 µm. (B) HeLa cells transfected with CERT-GFP were infected with C. trachomatis serovar D for 24 hrs and then fixed and stained with antibodies to IncA (red) to identify the inclusion membrane. Bacteria and host DNA were detected using DAPI (blue). Enlargements (inset) of boxed regions are shown to the right. *, inclusions. Scale bar = 5 µm. (C–E) HeLa cells were transfected with CERT-GFP and HcRedVAP-A for 18 hrs and infected with C. trachomatis L2 for (C) 2, (D) 8, or (E) 24 hrs. (C) Cells were stained with DAPI to visualize the nascent inclusions (red arrows). (D and E) Enlargements (inset) of boxed regions are shown to the right. At 8 and 24 hpi, CERT-GFP and HcRedVAP-A colocalize on the inclusion membrane and exhibit a patchy distribution. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). N, host nucleus; *, inclusions. Scale bar = 5 µm, except with insets from panels B, C, and D where scale bar = 2.5 µm.

Techniques: Transfection, Infection, Staining

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: HeLa cells were infected with C. trachomatis L2, treated with the indicated concentration of HPA-12 at 1–24 hpi, and then (A) fixed and stained with antibodies to MOMP (red) and with DAPI (blue) to visualize bacteria or (B) analyzed for progeny formation. Values (mean ± standard error) are shown as percentage of DMSO treated samples. Data are representative of 3 independent experiments. ***p<0.001 compared to DMSO treated cells (ANOVA). (C) HeLa cells were transfected with CERT-GFP and HA-CKIγ2 for 18 hrs, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to HA (red). The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Ectopic expression of HA-CKIγ2 decreased inclusion size but did not affect CERT-GFP recruitment to the inclusion membrane. Scale bar = 5 µm. (D) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (E) HeLa cells were depleted of CERT for 3 days and then labeled with BODIPY FL-Ceramide to analyze SM accumulation in the Golgi. The exposure time of all images was identical. CERT depletion reduced SM accumulation in the Golgi. Scale bar = 5 µm, (F) HeLa cells were depleted of CERT for 3 days, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to MOMP (green) to identify the inclusion. Bacteria and host DNA were detected using DAPI (blue). The exposure time for all images was identical. CERT depletion reduced inclusion size. Red arrows point to inclusions. Scale bar = 5 µm. (G) HeLa cells were depleted of CERT for 3 days, infected with C. trachomatis L2 and analyzed for inclusion size and progeny formation. Values (mean ± standard error) are shown as percentage of control siRNA samples. CERT depletion significantly reduced inclusion size and progeny formation. Data are representative of 2 independent experiments. ***p<0.001 for CERT siRNA-treated cells compared to control siRNA-treated cells (ANOVA). N, host nucleus. IFU, inclusion forming units. Scale bar = 5 µm.

Techniques: Infection, Concentration Assay, Staining, Transfection, Expressing, Western Blot, Labeling

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells were transfected with CERT-GFP, CERT (D324A)-GFP, or CERT (G67E)-GFP, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with an antibody to p230 (red) to identify the trans -Golgi. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Mutation of the PI4P binding (G67E) or VAP-A binding (D324A) domains did not affect CERT-GFP recruitment to the inclusion. Scale bar = 5 µm. (B) HeLa cells expressing CERT-GFP were infected with C. trachomatis L2, treated with 50 µM Exo1 (Arf1 inhibitor) for 1–24 hpi, and then fixed and stained with antibodies to MOMP (red) to identify bacteria. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). CERT-GFP localization to the inclusion was unaffected by Arf1 inhibition. Scale bar = 5 µm. (C) HeLa cells expressing CERT-GFP were infected with C. trachomatis L2, treated with 5 µM HPA-12 for 1–24 hpi, and then fixed and stained with antibodies to MOMP (red) to identify bacteria or to p230 (red) to identify the trans -Golgi. The exposure time for each filter set of all images was identical. Inhibition of CERT transfer and/or ceramide binding activity by HPA-12 treatment resulted in loss of CERT accumulation on the inclusion membrane. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Scale bar = 5 µm. N, host nucleus; *, inclusion.

Techniques: Transfection, Infection, Staining, Mutagenesis, Binding Assay, Expressing, Inhibition, Activity Assay

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells were infected with C. trachomatis L2 for 24 hrs, treated with 5 µM HPA-12 for the last 3 hrs of infection, and then labeled with BODIPY FL-Ceramide to analyze SM accumulation by the inclusion. As a control for decreased SM acquisition by the inclusion, cells were also treated with 10 µM BFA or 25 µg/ml D609. The exposure time of all images was identical. Dashed red lines demarcate inclusions. The residual fluorescence likely represents Golgi staining. Scale bar = 5 µm. (B) Quantitation of SM acquisition following treatment with HPA-12, D609, or BFA. Values (mean ± standard error) are shown as percentage of mean fluorescence intensities relative to DMSO-treated samples. ***p<0.001 (ANOVA). HPA-12, D609, and BFA-treated cells displayed a significant decrease in fluorescence intensity of the inclusion and its contents compared to DMSO-treated samples.

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

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells co-transfected for 18 hrs with CERT-GFP and C-terminally 3xFLAG-tagged SMS1/SMS2 (upper 2 rows) or SMS1-V5 and C-terminally 3xFLAG-tagged SMS2 (bottom row) were infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to FLAG (red) and/or to V5 (green). Single channel images of uninfected cells are shown to the right. Enlargements of the boxed regions (inset) in infected samples are shown to the right of infected set. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). CERT and SMS1 localization at the inclusion are distinct while CERT and SMS2 partially overlap. SMS2 localization partially overlaps with SMS1 around the inclusion, however SMS2 also localizes to the inclusion. At longer exposure times, SMS2 plasma membrane localization is evident. Scale bar = 5 µm, except the insets where scale bar = 2.5 µm. (B) HeLa cells transfected with SMS1-V5 (green) or SMS2-V5 (green) infected with C. trachomatis serovar D for 24 hrs and then fixed and stained with antibodies to IncA (red) to identify the inclusion membrane. Enlargements of the boxed regions (inset) are shown to the right. Images represent a single z slice from confocal images. The exposure time for each filter set for all images was identical. SMS2 but not SMS1 partially overlaps with IncA on the inclusion. Scale bar = 5 µm, except the insets where scale bar = 2.5 µm. (C) HeLa cells transfected for 18 hrs with CERT-GFP, SMS1-V5, or SMS2-V5 were infected with C. trachomatis L2, treated with 10 µM BFA or Nocodazole for 1–24 hpi, and then fixed and stained with antibodies to V5 (green) and to p230 (red) to identify the trans -Golgi. BFA and Nocodazole disrupted SMS1 localization around the inclusion but had no effect on SMS2 or CERT localization at the inclusion. Images represent a single z slice from confocal images. Scale bar = 5 µm. N, host nucleus; *, inclusion.

Techniques: Transfection, Infection, Staining

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (B) HeLa cells were depleted of SMS1 or SMS2 for 3 days and then labeled with BODIPY FL-Ceramide to analyze SM accumulation in the Golgi. The exposure time of all images was identical. SMS1 but not SMS2 depletion reduced BODIPY FL lipid accumulation in the Golgi. Scale bar = 5 µm. (C and D) HeLa cells were depleted of SMS1 or SMS2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with an antibody to MOMP (red). Bacteria and host DNA were detected using DAPI (blue). (D) SMS1 and SMS2-depleted cells were analyzed for inclusion size and progeny formation. Values (mean ± standard error) are shown as percentage of control siRNA samples. SMS1 and SMS2 depletion reduced inclusion size and production of infectious progeny. Data are representative of 2 independent experiments. ***p<0.001, all samples compared to control siRNA treatment (ANOVA). N, host nucleus; red arrows point to inclusions. IFU, inclusion forming units. Scale bar = 5 µm.

Techniques: Western Blot, Labeling, Infection, Staining

Number of C. trachomatis S45 inclusions (mono- and mixed infections [ca-PEDV]) at 24 h pi (A) and at 48 h pi (B). Statistically significant differences between mono- and dual infections are indicated (*): (A) and (B) p = 0.001; chi-square test.

Journal: Veterinary Microbiology

Article Title: Mixed infections in vitro with different Chlamydiaceae strains and a cell culture adapted porcine epidemic diarrhea virus

doi: 10.1016/j.vetmic.2004.10.023

Figure Lengend Snippet: Number of C. trachomatis S45 inclusions (mono- and mixed infections [ca-PEDV]) at 24 h pi (A) and at 48 h pi (B). Statistically significant differences between mono- and dual infections are indicated (*): (A) and (B) p = 0.001; chi-square test.

Article Snippet: C. trachomatis S45 inclusions were labeled by a polyclonal goat anti- C. trachomatis -MOMP antibody (1:100, Chemicon International, Temecula, USA) and a Cy3-conjugated rabbit anti-goat secondary antibody (1:500, Zymed Laboratories, San Francisco, USA).

Techniques:

Number of ca-PEDV syncytia (mono- and mixed infections [ C. trachomatis S45]) at 24 h pi (A) and at 48 h pi (B). Statistically significant differences between mono- and dual infections are indicated (*): (A) p = 0.031 and (B) p 1 = 0.035, p 2 = 0.02; chi-square test.

Journal: Veterinary Microbiology

Article Title: Mixed infections in vitro with different Chlamydiaceae strains and a cell culture adapted porcine epidemic diarrhea virus

doi: 10.1016/j.vetmic.2004.10.023

Figure Lengend Snippet: Number of ca-PEDV syncytia (mono- and mixed infections [ C. trachomatis S45]) at 24 h pi (A) and at 48 h pi (B). Statistically significant differences between mono- and dual infections are indicated (*): (A) p = 0.031 and (B) p 1 = 0.035, p 2 = 0.02; chi-square test.

Techniques:

Immunohistochemical labeling of chlamydial inclusions (A, C), ca-PEDV single and syncytial cells (B) as well as viral syncytial cell with chlamydial inclusion (D). Vero cells were dually infected with C. trachomatis S45 and ca-PEDV and pelleted. Paraffin sections. Immunoperoxidase stain. (A) Chlamydial inclusions; red (AEC). (B) Ca-PEDV syncytial cell with apoptosis (bottom) and single cells (top); brown (DAB). (C) Chlamydial inclusions (brown, DAB+) and syncytial cells (left one showing apoptosis; pink, Fast Red). (D) Syncytial cell (pink) with chlamydial inclusion (brown). Magnifications: 40× (A, B, C), 100× (D). Bars 20 μm.

Journal: Veterinary Microbiology

Article Title: Mixed infections in vitro with different Chlamydiaceae strains and a cell culture adapted porcine epidemic diarrhea virus

doi: 10.1016/j.vetmic.2004.10.023

Figure Lengend Snippet: Immunohistochemical labeling of chlamydial inclusions (A, C), ca-PEDV single and syncytial cells (B) as well as viral syncytial cell with chlamydial inclusion (D). Vero cells were dually infected with C. trachomatis S45 and ca-PEDV and pelleted. Paraffin sections. Immunoperoxidase stain. (A) Chlamydial inclusions; red (AEC). (B) Ca-PEDV syncytial cell with apoptosis (bottom) and single cells (top); brown (DAB). (C) Chlamydial inclusions (brown, DAB+) and syncytial cells (left one showing apoptosis; pink, Fast Red). (D) Syncytial cell (pink) with chlamydial inclusion (brown). Magnifications: 40× (A, B, C), 100× (D). Bars 20 μm.

Techniques: Immunohistochemical staining, Labeling, Infection, Staining

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