Figure 6.
Figure 6. SHC1 knockdown sensitizes early infected cells to apoptosis. (A) Western blots showing TNF-induced PARP cleavage (black arrowheads) in HeLa cells with Luciferase or SHC1 knockdown after C. trachomatis infection (MOI 50) for 6 h and an additional induction of apoptosis for 4 h (25 ng/ml TNF and 10 µg/ml cycloheximide). Actin and Hsp60 were used as loading control (white arrowheads). Infection with C. trachomatis L2 blocked PARP cleavage in infected Luciferase-transfected HeLa cells, whereas SHC1 knockdown sensitizes infected cells to apoptosis as indicated by PARP cleavage. (B) Quantification of PARP cleavage in C. trachomatis and C. pneumoniae infection from chemiluminescence imager recorded blots (data from A and not depicted, n = 3, error bars indicate SE). Band signals from PARP and cleaved PARP were measured using AIDA software, and the ratio was calculated. The ratio of apoptosis inhibition in uninfected TNF-stimulated versus infected TNF-stimulated cells is depicted. C. trachomatis–infected control cells show a high degree of apoptosis resistance, which is significantly reduced upon SHC1 knockdown. In contrast, C. pneumoniae–infected cells exhibit a diminished apoptosis resistance, independent of SHC1. (C and E) Immunostainings showing TNF-induced apoptosis in HeLa cells with Luciferase or SHC1 knockdown after infection with C. trachomatis and apoptosis induction (identical conditions to A). SHC1 knockdown sensitizes infected cells to apoptosis, whereas apoptosis is blocked in infected control cells. Apoptosis induction was assessed by cytokeratin 18 cleavage (red) and TUNEL assay (green), respectively. Nuclei are stained blue with Hoechst 33342. Image area: 450 µm × 450 µm. (D and F) Quantification of cells positive for cytokeratin 18 cleavage (data from B and not shown; n = 2, error bars indicate SE) and TUNEL (data from D and not depicted; n = 2, error bars indicate SE), respectively. A mean of 10,000 cells per condition were analyzed. Apoptosis inhibition is depicted as in B. C. trachomatis infection leads to apoptosis inhibition in control cells, but not in SHC1 knockdown cells. In contrast, C. pneumoniae fails to block apoptosis in both control and SHC1 knockdown cells.

SHC1 knockdown sensitizes early infected cells to apoptosis. (A) Western blots showing TNF-induced PARP cleavage (black arrowheads) in HeLa cells with Luciferase or SHC1 knockdown after C. trachomatis infection (MOI 50) for 6 h and an additional induction of apoptosis for 4 h (25 ng/ml TNF and 10 µg/ml cycloheximide). Actin and Hsp60 were used as loading control (white arrowheads). Infection with C. trachomatis L2 blocked PARP cleavage in infected Luciferase-transfected HeLa cells, whereas SHC1 knockdown sensitizes infected cells to apoptosis as indicated by PARP cleavage. (B) Quantification of PARP cleavage in C. trachomatis and C. pneumoniae infection from chemiluminescence imager recorded blots (data from A and not depicted, n = 3, error bars indicate SE). Band signals from PARP and cleaved PARP were measured using AIDA software, and the ratio was calculated. The ratio of apoptosis inhibition in uninfected TNF-stimulated versus infected TNF-stimulated cells is depicted. C. trachomatis–infected control cells show a high degree of apoptosis resistance, which is significantly reduced upon SHC1 knockdown. In contrast, C. pneumoniae–infected cells exhibit a diminished apoptosis resistance, independent of SHC1. (C and E) Immunostainings showing TNF-induced apoptosis in HeLa cells with Luciferase or SHC1 knockdown after infection with C. trachomatis and apoptosis induction (identical conditions to A). SHC1 knockdown sensitizes infected cells to apoptosis, whereas apoptosis is blocked in infected control cells. Apoptosis induction was assessed by cytokeratin 18 cleavage (red) and TUNEL assay (green), respectively. Nuclei are stained blue with Hoechst 33342. Image area: 450 µm × 450 µm. (D and F) Quantification of cells positive for cytokeratin 18 cleavage (data from B and not shown; n = 2, error bars indicate SE) and TUNEL (data from D and not depicted; n = 2, error bars indicate SE), respectively. A mean of 10,000 cells per condition were analyzed. Apoptosis inhibition is depicted as in B. C. trachomatis infection leads to apoptosis inhibition in control cells, but not in SHC1 knockdown cells. In contrast, C. pneumoniae fails to block apoptosis in both control and SHC1 knockdown cells.

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