Journal: bioRxiv

Article Title: Non-redundant dimethyl sulfoxide reductases influence Salmonella enterica serotype Typhimurium anaerobic growth and virulence

doi: 10.1101/2022.11.29.517730

Figure Lengend Snippet: (A, D) Overnight cultures of the indicated bacterial strains bearing a P prgH - lacZY transcriptional fusion were diluted into LB and grown aerobically at 37°C. Where indicated, LB was supplemented with 1% (v/v) DMSO or DMS after 1.5 hours growth and incubation continued for another 1.5 hours. The activity of the prgH promoter was estimated by measuring β-galactosidase activity, which was converted into Miller Units. (B) Bacteria were prepared as in (A) and invasion efficiency into HCT116 epithelial cell monolayers was determined by gentamicin protection assay at 1 hour post-infection. Invasion efficiency was normalized to the untreated WT strain. (B) HCT116 cells were infected with the indicated bacterial strains (constitutively expressing mCherry ) bearing a P prgH -GFP[LVA] transcriptional fusion. Cells were fixed at the indicated timepoints, and the proportion of GFP-positive bacteria quantified by fluorescence microscopy. Bars represent the mean +/- SEM of 3 (A, C, D) or 4 (B) independent experiments. Asterisks (*) indicate significant difference from media alone as detected by Student’s t-test (p<0.05).

Article Snippet: HCT116 (human colonic epithelial) cells and J774A.1 (mouse macrophage-like) cells were purchased from ATCC and maintained in McCoy’s 5A media (Corning) containing 10% heat-inactivated fetal calf serum (FCS, Invitrogen) or Dulbecco’s Modified Eagle’s medium (Corning) containing 10% heat-inactivated FCS, respectively.

Techniques: Incubation, Activity Assay, Bacteria, Infection, Expressing, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases

doi: 10.1371/journal.pone.0108839

Figure Lengend Snippet: A. HCT116 cells were cultured in the presence of 1 µM b-AP15 or 100 nM bortezomib and harvested at the indicated time points. Lysates were subjected to immunoblotting for K48-linked polyubiquitin, HSP-70B', p21, PARP or β-actin (loading control). B. HCT116 cells or hTERT-RPE1 cells were exposed to different concentrations of b-AP15 for 1 hour, followed by washing and incubation for 16 hours in drug-free medium. Lysates were subjected to immunoblotting for K48-linked polyubiquitin, PARP and β-actin (loading control).

Article Snippet: HCT116 colon carcinoma cells (ATCC) were maintained in McCoy's 5A modified medium/10% fetal calf serum, hTERT-RPE1 cells in DMEM:F12 medium/10% fetal calf serum, human diploid fibroblasts and HeLa cells (European Collection of Cell Cultures) were maintained in DMEM medium/10% fetal calf serum and MelJuSo-Ub G76V -YFP cells in Iscove's DMEM medium/10% fetal calf serum.

Techniques: Cell Culture, Western Blot, Control, Incubation

Journal: PLoS ONE

Article Title: Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases

doi: 10.1371/journal.pone.0108839

Figure Lengend Snippet: A. and B. HCT116 cells were treated for 1 h with DMSO, tunicamycin (10 µg/ml) or CAM741 (10 µM) before pulse-labelling for 10 min with [ 35 S] Met/Cys. Endogenous prosaposin (pSAP) was recovered by immunoprecipitation and newly synthesised pSAP species were visualised by phosphorimaging. In DMSO-treated cells, pSAP was fully glycosylated (pSAP-5), whereas Endo H digestion or tunicamycin treatment yielded non-glycosylated pSAP (pSAP-0). Inhibition of protein translocation into the ER by CAM741 resulted in the appearance of a pSAP species that migrated more slowly than the non-glycosylated protein and was Endo H-resistant. This species may represent signal sequence-containing preprosaposin (prepSAP-0) that has failed to translocate across the ER membrane. C. Distinct forms of endogenous pSAP in HCT116 cells treated with DMSO, b-AP15 (1 µM), bortezomib (20 nM), cpdA (10 µM) or tunicamycin (10 µg/ml) were recovered by immunoprecipitation and visualised by phosphorimaging. Treatment with cpdA specifically inhibits the co-translational translocation of pSAP into the ER as judged by the appearance of prepSAP-0 species.

Article Snippet: HCT116 colon carcinoma cells (ATCC) were maintained in McCoy's 5A modified medium/10% fetal calf serum, hTERT-RPE1 cells in DMEM:F12 medium/10% fetal calf serum, human diploid fibroblasts and HeLa cells (European Collection of Cell Cultures) were maintained in DMEM medium/10% fetal calf serum and MelJuSo-Ub G76V -YFP cells in Iscove's DMEM medium/10% fetal calf serum.

Techniques: Immunoprecipitation, Inhibition, Translocation Assay, Sequencing, Membrane

Journal: PLoS ONE

Article Title: Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases

doi: 10.1371/journal.pone.0108839

Figure Lengend Snippet: A. HCT116 cells were pre-treated with or without (control) 10 µM cpdA for 16 hours, exposed to the indicated concentration of b-AP15 for 1 hour, then incubated for a further 16 hours in drug-free medium. Lysates were subjected to immunoblotting for K48-linked polyubiquitin, PARP and β-actin (loading control). B. HCT116 cells were treated as above. Following treatment, the number of dead cells was measured by Trypan-blue staining. Bar chart shows mean +/− SD of three independent experiments. Statistical significance was calculated using the Student's t-test. P values * = 0.05 and ** = 0.01. C. and D. hTERT-RPE1 cells were treated and analyzed as above. E. and F. Human diploid fibroblasts were treated and analyzed as above, and the number of dead cells was determined 36 hours after drug treatment.

Article Snippet: HCT116 colon carcinoma cells (ATCC) were maintained in McCoy's 5A modified medium/10% fetal calf serum, hTERT-RPE1 cells in DMEM:F12 medium/10% fetal calf serum, human diploid fibroblasts and HeLa cells (European Collection of Cell Cultures) were maintained in DMEM medium/10% fetal calf serum and MelJuSo-Ub G76V -YFP cells in Iscove's DMEM medium/10% fetal calf serum.

Techniques: Control, Concentration Assay, Incubation, Western Blot, Staining

Journal: PLoS ONE

Article Title: Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases

doi: 10.1371/journal.pone.0108839

Figure Lengend Snippet: A. HCT116 cells were exposed to 1 µM b-AP15 or 100 nM bortezomib (BZ) for 8 hours or 16 hours, in the presence or absence of 1 mM cysteine as indicated. Cell lysates were subjected to immunoblotting for LC3-I/II and β-actin (loading control). B. HCT116 cells were exposed to 1 µM b-AP15 for 16 hours in the presence or absence of 1 mM cysteine. Where indicated, cells were pre-treated with 10 µM cpdA for 16 hours prior to b-AP15 treatment. Apoptosis was determined by the appearance of caspase-cleaved K18. Bar chart shows mean values ± SD of of three independent experiments.

Article Snippet: HCT116 colon carcinoma cells (ATCC) were maintained in McCoy's 5A modified medium/10% fetal calf serum, hTERT-RPE1 cells in DMEM:F12 medium/10% fetal calf serum, human diploid fibroblasts and HeLa cells (European Collection of Cell Cultures) were maintained in DMEM medium/10% fetal calf serum and MelJuSo-Ub G76V -YFP cells in Iscove's DMEM medium/10% fetal calf serum.

Techniques: Western Blot, Control

Journal: PLoS ONE

Article Title: Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases

doi: 10.1371/journal.pone.0108839

Figure Lengend Snippet: Treatment of HCT116 cells with b-AP15 and cpdA does not deplete cellular cysteine levels.

Article Snippet: HCT116 colon carcinoma cells (ATCC) were maintained in McCoy's 5A modified medium/10% fetal calf serum, hTERT-RPE1 cells in DMEM:F12 medium/10% fetal calf serum, human diploid fibroblasts and HeLa cells (European Collection of Cell Cultures) were maintained in DMEM medium/10% fetal calf serum and MelJuSo-Ub G76V -YFP cells in Iscove's DMEM medium/10% fetal calf serum.

Techniques: