Journal: Molecular and Cellular Biology

Article Title: Formation of tRNA Wobble Inosine in Humans Is Disrupted by a Millennia-Old Mutation Causing Intellectual Disability

doi: 10.1128/mcb.00203-19

Figure Lengend Snippet: Figure 8. ADAT3-V144M is bound by the HSP60 and TRiC chaperonins. (A) Silver stain of 1017

Article Snippet: Antibodies were against the following proteins : FLAG epitope tag 553 (A2220, Sigma), 6xHis tag (MA1-21315, Thermo Fisher), GFP (sc-9996, Santa Cruz 554 Biotechnology), Strep-tag II-tag (NC9261069, Thermo Fisher), ADAT3 (Abcam, ab192987), 555 ADAT3 (H00113179-B01P, Abnova), ADAT2 (ab135429, Abcam), HSP60 (A302-845A, 556 Bethyl Labs), CCT1 (sc-53454, Santa Cruz Biotechnologies), CCT7 (A304-730A-M, Bethyl 557 Labs) and actin (MAB1501, EMD Millipore).

Techniques: Silver Staining

Journal: PLOS Pathogens

Article Title: Chlamydial protease-like activity factor targets SLC7A11 for degradation to induce ferroptosis and facilitate progeny releases

doi: 10.1371/journal.ppat.1013060

Figure Lengend Snippet: ( A ) A schematic diagram illustrates a series of reported pharmaceuticals that block ferroptosis by inhibiting lipid ROS accumulation. ( B ) The release of LDH and the levels of lipid ROS in Chlamydia trachomatis serovar D (MOI 10)-infected HeLa-229 cells were assessed following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) for 72 hours. Statistical analysis was conducted using a one-way ANOVA with Bonferroni’s multiple comparisons (n=3). ( C ) Immunoblot analysis of chlamydial MOMP from cell supernatant and GAPDH from cell lysate were conducted following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) for 72 hours. ( D ) The copy number of the chlamydial cryptic plasmid in the cell supernatant of Chlamydia trachomatis serovar D (MOI 10)-infected cells was determined following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) for 72 hours. Statistical analysis was conducted using a one-way ANOVA with Bonferroni’s multiple comparisons (n=3). ( E ) The copy number of ompA in the total culture (cell supernatant and monolayer) of Chlamydia trachomatis serovar D (MOI 10)-infected cells was determined following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) over a time course. Statistical analysis was conducted using a two-way ANOVA test (n=3). ( F , G ) The release of LDH, lipid ROS levels, and the copy number of the chlamydial cryptic plasmid of cell supernatant in Chlamydia trachomatis serovars L1 (MOI 3)- ( F ) and A (MOI 3)- ( G ) infected cells were measured following treatment with liproxstatin-1 (1 μM) for 72 hours. The Student’s t - test was used for statistical analysis of ( F ) and ( G ) (n=3). ( H ) The release of LDH and the level of lipid ROS in Chlamydia muridarum (CM) (MOI 2)-infected McCoy cells were measured following treatment with trolox (3.2 mM) for 48 hours. The Student’s t - test was used for statistical analysis (n=3). ( I ) Immunoblot analysis of chlamydial HSP60 in the cell supernatant and β-actin in the cell lysate was performed following treatment with trolox (3.2 mM) for 48 hours. ( J ) The copy number of the Nigg II plasmid in the cell supernatant and total culture (cell supernatant and monolayer) of CM (MOI 2)-infected McCoy cells was quantified over a time-course following treatment with trolox (3.2 mM). The Student’s t-test was used for statistical analysis of the cell supernatant data (left panel), while a two-way ANOVA was applied to the data from the total culture (cell supernatant and monolayer) over the time course (right panel). Data are presented as the mean ± SD (n=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: For monitor the growth dynamic of CT, a mouse anti-chlamydial Hsp60 antibody conjugated to Alexa Fluor 488 (Santa Cruz; sc-57840 AF488) was used.

Techniques: Blocking Assay, Infection, Western Blot, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: Chlamydial protease-like activity factor targets SLC7A11 for degradation to induce ferroptosis and facilitate progeny releases

doi: 10.1371/journal.ppat.1013060

Figure Lengend Snippet: (A) Immunoblot analysis of multiple ferroptosis-associated proteins and chlamydial HSP60 was performed in cells infected with various MOIs of Chlamydia trachomatis serovar D (CT-D) at 72 h.p.i., compared to mock-infected cells. (B) Immunoblot analysis of SLC7A11, GPx4, GAPDH, and chlamydial HSP60 was conducted in cells infected with Chlamydia trachomatis serovar L1 (CT-L1) (MOI 3) and Chlamydia muridarum (CM) (MOI 2), compared to mock-infected cells. (C) A schematic representation of the SLC7A11-GSH-GPx4 pathway in the regulation of ferroptosis. (D) Intracellular glutathione (GSH) levels were measured in CT-D (MOI 5)-infected cells at 72 h.p.i by flow cytometry, compared to mock-infected cells. The fraction of cells with high intracellular GSH was calculated. The Student’s t-test was used for statistical analysis. Data are presented as the mean ± SD (n=3). **, P < 0.01.

Article Snippet: For monitor the growth dynamic of CT, a mouse anti-chlamydial Hsp60 antibody conjugated to Alexa Fluor 488 (Santa Cruz; sc-57840 AF488) was used.

Techniques: Western Blot, Infection, Flow Cytometry