Journal: EBioMedicine

Article Title: Chlamydia trachomatis Prevents Apoptosis Via Activation of PDPK1-MYC and Enhanced Mitochondrial Binding of Hexokinase II

doi: 10.1016/j.ebiom.2017.08.005

Figure Lengend Snippet: CTL2 induces PDPK1 phosphorylation downstream of PI3K. a) Western blot showing an increase of PDPK1 phosphorylated at Ser-241 compared to total PDPK1 at 24 and 48 h p.i. in CTL2-infected (MOI 1) whole cell lysates. β-actin served as loading control. b) The band densities from a) were quantified and normalized to corresponding band densities of the β-actin loading control. Alterations in expression levels compared to non-infected controls are represented as mean fold change ± SEM, * p < 0.05, t -test, n = 3. c) CTL2 infection increases levels of PDPK1 phosphorylated at Ser-241, which is recruited as a rim-like structure at the inclusions in HeLa cells. PDPK1 phosphorylated at Ser-241, IncA and nuclei were labelled with Cy3-conjugated phosphorylated PDPK1 and IncA antibodies and DAPI. Scale bar: 30 μm. Asterisks are CTL2 inclusions. d) Monolayers of fallopian tube mesenchymal stem cells, infected with CTL2 for 48 h p.i. (MOI 0.5) were labelled with antibodies against Chlamydia trachomatis , PDPK1 phosphorylated at Ser-241 and DAPI. Results were similar to c). Scale bar: 30 μm. e) Human primary fallopian tube epithelial organoids, infected with CTL2 for 48 h p.i. were labelled with antibodies against Chlamydia trachomatis , PDPK1 phosphorylated at Ser-241 and E -Cadherin. Results were similar to c). Scale bar: 30 μm.

Article Snippet: The following antibodies were used: rabbit monoclonal antibodies against total MYC (D84C12, 5605), total HKII (C64G5, 2867) and total PDHK1 (C47H1, 3820), rabbit polyclonal antibodies against total PDPK1 (3062) and anti-P-PDPK1 (Ser-241, 3061) (all Cell Signaling); rabbit polyclonal anti-P-MYC (T58, ab28842), mouse monoclonal anti-P-MYC (S62, 33A12E10, ab78318, both from Abcam); mouse monoclonal anti-VDAC1 (B-6, sc-390996), Rho A (24C4, sc-418) and anti-MYC (9E10, sc-40 all from Santa Cruz Biotechnology Inc.); mouse monoclonal anti- Chlamydia trachomatis MAb species-specific KK-12 IgG2a (supplied by David Grayston, University of Washington, Seattle); rabbit polyclonal anti- Chlamydia genus-specific antibody (AG, 3-090, Milan Analytica), mouse monoclonal anti- Chlamydia trachomatis Hsp60 (ALX-804-072-R100, Enzo Life Sciences), rabbit anti-INCA antibody ( ); goat anti-chlamydia (1990-0404, AbD Serotec); rabbit monoclonal cleaved PARP (Asp214) and rabbit polyclonal anti-cleaved caspase 3 (Asp175, 9661, both from Cell Signaling).

Techniques: Phospho-proteomics, Western Blot, Infection, Control, Expressing