Journal: Cell Death & Disease

Article Title: Bacterial type III effector protein HopQ inhibits melanoma motility through autophagic degradation of vimentin

doi: 10.1038/s41419-020-2427-y

Figure Lengend Snippet: a B16F10 cells were transfected with plasmids expressing EV or Myc-HopQ and whole-cell lysates were immunoprecipitated (IP) with anti-Myc antibody and immunoblot analysis with anti-14-3-3 or anti-pSerine antibodies. b B16F10 cells were transfected with EV, Myc-HopQ, or its mutants for 24 h. Whole-cell lysates were then harvested for IP with anti-Myc antibody and immunoblot analysis with the anti-14-3-3 antibody. Arrowheads indicate Myc-HopQ mutant bands. c Schematic depiction of the 14-3-3-binding motif of HopQ. d B16F10 cells were transfected with EV, Myc-HopQ WT, or Myc-HopQ S51A for 24 h. Whole-cell lysates were then harvested for IP with anti-Myc antibody and immunoblot analysis with anti-14-3-3. e B16F10 cells were transfected with Myc-HopQ WT or Myc-HopQ S51A for 24 h; cells were then fixed with 4% paraformaldehyde and subjected to confocal microscopy. Scale bar: 20 μm. f B16F10 cells transiently expressing EV, Myc-HopQ WT, or Myc-HopQ S51A for 12 h were then treated with 40 μg/ml mitomycin C for 2 h and scratch wound-healing assays were performed. Bar graphs show the percentage of the cell-covered area. (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). Scale bar: 250 μm. g , h EV-, Myc-HopQ-, or Myc-HopQ S51A-expressing B16F10 were seeded in transwell chambers and incubated for 24 h. The lower chamber containing 10% FBS was used as a chemoattractant. For the cell invasion assay, the membrane was coated with 10 mg/ml of Matrigel. Bar graphs show the percentage of migrating or invading cells (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). Scale bar: 200 μm.

Article Snippet: Anti-Ubiquitin (#3933), anti-14-3-3 eta (#9640), anti-14-3-3 tau (#9638), anti-phospho-FOXO1 (#9461), anti-FOXO1 (#2880), anti-p53 (#2524), anti-phospho-AKT (#9271), anti-AKT (#4685), anti-phospho-GSK3β (#9336), anti-GSK3β (#9315), anti-phospho-ERK1/2 (#4370), anti-ERK1/2 (#4695), anti-Snail (#3879), anti-β-Catenin (#8480), anti-Cyclin D1 (#2978), and anti-E-cadherin (#14472) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Binding Assay, Confocal Microscopy, Incubation, Invasion Assay

Journal: Cell Death & Disease

Article Title: Bacterial type III effector protein HopQ inhibits melanoma motility through autophagic degradation of vimentin

doi: 10.1038/s41419-020-2427-y

Figure Lengend Snippet: a B16F10 cells were transfected with Myc-HopQ WT or S51A for 16 h and treated with cycloheximide (CHX) 10 μM at the indicated time points. Cell lysates were then collected for immunoblot. b Quantification of the relative levels of vimentin after the treatments described in a . (** P ≤ 0.01). The amount of vimentin was normalized to the β-actin level. c B16F10 cells were transfected with EV, Myc-HopQ WT, or Myc-HopQ S51A for 24 h. Whole-cell lysates were then harvested for IP with anti-Vimentin antibody and immunoblot analysis with anti-Myc. d B16F10 cells were transfected with EV or Myc-HopQ for 12 h and treated with R18 10 μM and 25 μM for an additional 12 h. Whole-cell lysates were then harvested for IP with anti-Vimentin antibody and immunoblot analysis with anti-Myc or anti-14-3-3. Arrowheads indicate specific bands. e B16F10 cells were transfected with Myc-HopQ with increasing doses of N90 for 24 h, cell lysates were collected for immunoblot. f B16F10 cells were transfected with increasing doses of Myc-HopQ WT, ΔN or N90 mutant for 24 h and cell lysates were collected for immunoblot.

Article Snippet: Anti-Ubiquitin (#3933), anti-14-3-3 eta (#9640), anti-14-3-3 tau (#9638), anti-phospho-FOXO1 (#9461), anti-FOXO1 (#2880), anti-p53 (#2524), anti-phospho-AKT (#9271), anti-AKT (#4685), anti-phospho-GSK3β (#9336), anti-GSK3β (#9315), anti-phospho-ERK1/2 (#4370), anti-ERK1/2 (#4695), anti-Snail (#3879), anti-β-Catenin (#8480), anti-Cyclin D1 (#2978), and anti-E-cadherin (#14472) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Western Blot, Mutagenesis