Journal: Oncotarget

Article Title: Interference with mutagenic aflatoxin B1-induced checkpoints through antagonistic action of ochratoxin A in intestinal cancer cells: a molecular explanation on potential risk of crosstalk between carcinogens

doi: 10.18632/oncotarget.8914

Figure Lengend Snippet: A–B. HCT-8 cells stably-transfected withempty vector (con) or plasmid for CYP3A4/5 shRNA (shCYP3A5/4) were treated with DMSO or 10 mM AFB 1 for 24 h. Cells were analyzed for cell cycle according to PI staining followed by FACS analysis. Figures in the box indicate CYP3A5 expression in the stable cell lines. C. HCT-8 cells stably-transfected withempty vector or shCYP3A5 were treated with DMSO or 10 mM AFB 1 for 72 h. AFB 1 DNA adduct (6A10) were detected using immunodot-blot assay. DNA adduct measured by multi gauge software (bottom panel), respectively. D. HCT-8 cells stably-transfected withempty vector or shCYP3A5 were treated with DMSO or 10 mM AFB 1 for 24 h. Total cell lysates were subjected to Western blot analysis. An asterisk (*) indicates a significant difference compared to AFB 1 -treated, an empty vector-transfected HCT-116 cells ( p < 0.05).

Article Snippet: To assess the formation of AFB 1 -induced DNA adducts, mycotoxin-treated cells were subjected to an immunodot-blot assay using mouse monoclonal anti-AFB 1 (6A10) antibody (Novus Biologicals, Inc., Littleton, CO, USA) raised against the open imidazole ring persistent form of the major N7 guanine adduct of AFB 1 .

Techniques: Stable Transfection, Transfection, Plasmid Preparation, shRNA, Staining, Expressing, Software, Western Blot

Journal: Nature cell biology

Article Title: Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity

doi: 10.1038/ncb2332

Figure Lengend Snippet: Autophagy controls intracellular MBd levels. (a) Single-plane confocal images of MBds within LC3-positive autophagosomes in MEFs expressing GFP-LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MBd markers: Cep55, MKLP1, or mgcRACGAP. Autophagosomes: GFP-LC3 or LC3. Note that MKLP1 (blue) and mgcRACGAP (red) are co-localized (magenta) in the autophagosome (green), suggesting that MBds are sorted into autophagosomes. Bars, 2 μm. (b) Decreasing autophagy levels by deletion of Atg5 gene (left, MEFs) or depletion of Atg7 by siRNA (right, HeLa) significantly increases the percent of MBd+ cells (p=0.0019 and p=0.021, respectively, n=3). Immunoblots confirm loss of the Atg5-Atg12 conjugation in mutant cells and depletion of Atg7 (asterisk). (c) Rapamycin (Rapa) and lithium chloride (LiCl) co-treatment induces autophagy and decreases the percent of MBd+ cells (left, HeLa; p=0.0056, n=3). Immunoblots showing increased LC3-II levels confirm autophagy induction. Induction of autophagy by over-expression of Flag-tagged BECN1 reduces the percent of MBd+ cells (right, MCF-7; p=0.0008, n=4) (d) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d/PepA. U, uninhibited. I, inhibited. Below, the average of the percent change in LC3-II levels after lysosomal inhibition from 3 experiments. α-tubulin, loading control. (e) Quantification of autophagic flux from 3 experiments in different cell lines. Normal dividing cells (MBd-poor) typically have high autophagic flux, whereas stem and cancer cells (MBd-rich) have low autophagic flux. The data are presented as mean ± s.d. (b-e).

Article Snippet: Atg5 (1:2000, Cosmo Bio, CAC-TMD-PH-ATG); Atg7 (1:1000, ProSci, 3617); Actin (1:300, Sigma, AC-40); Aurora B (1:100, BD Trans Lab, 611082); CD13 (1:50, BioLegend, 301707); CD133 (1:200, eBioscience, 14-1331); Cep55 (1:50, 1:100 and 1:1000 for immunofluorescence, Abnova #H00055165-B01, Abnova #H00055165-A01, and the gift from K. Kurtche, respectively; 1:500 for immunoblotting, Genetax #GTX112190); hCenexin1 (1:100, a gift from K.S.

Techniques: Expressing, Staining, Western Blot, Conjugation Assay, Mutagenesis, Over Expression, Activity Assay, Inhibition, Control

Journal: Nature cell biology

Article Title: Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity

doi: 10.1038/ncb2332

Figure Lengend Snippet: NBR1 is a receptor for targeting MBds to the autophagy pathway. (a) Single-plane confocal images showing co-localization of the MBd and the autophagic receptor, NBR1, in U2OS cells and p62-deleted MEFs. MBd markers: MKLP1 or Cep55. Bar, 2 μm. (b) The percent of MBd+ cells is significantly increased following the depletion of NBR1 (p=0.022, n=3), but not another autophagic receptor, p62. Co-depletion of NBR1 and p62 does not further increase MBd levels over NBR1 depletion alone. (c) Deletion of the p62 gene does not affect the percent of MBd+ cells. For (b) and (c), immunoblots verify protein loss. (d) Co-immunoprecipitation reveals Cep55 and NBR1 form a complex. Precipitated proteins and 5% of the input material (Input) were analyzed by immunoblotting with antibodies against NBR1 or Cep55. (e-g) Over-expression of CEP55-EGFP increases the percent of MBd+ cells (e; p=0.0007, n=3) and the percent of NBR1-negative MBds (f; p=0.0568, n=3), presumably by sequestering NBR1 (red) away from MBds in cells expressing CEP55-EGFP (green) as shown in (g), and consequently preventing MBd degradation. The dotted box in (g) is enlarged (top right panel), and the labeling of NBR1 and CEP55-EGFP (middle and bottom right panel) are also presented. DAPI, DNA (blue). Bar, 5 μm. The data are presented as mean ± s.d. (b, c, e, and f).

Article Snippet: Atg5 (1:2000, Cosmo Bio, CAC-TMD-PH-ATG); Atg7 (1:1000, ProSci, 3617); Actin (1:300, Sigma, AC-40); Aurora B (1:100, BD Trans Lab, 611082); CD13 (1:50, BioLegend, 301707); CD133 (1:200, eBioscience, 14-1331); Cep55 (1:50, 1:100 and 1:1000 for immunofluorescence, Abnova #H00055165-B01, Abnova #H00055165-A01, and the gift from K. Kurtche, respectively; 1:500 for immunoblotting, Genetax #GTX112190); hCenexin1 (1:100, a gift from K.S.

Techniques: Western Blot, Immunoprecipitation, Over Expression, Expressing, Labeling

Journal: Nature cell biology

Article Title: Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity

doi: 10.1038/ncb2332

Figure Lengend Snippet: MBd enrichment increases reprogramming efficiency and enhances in vitro tumorigenicity. (a-c) Reprogramming is more efficient after MBd enrichment. Differentiated cells (dH1f) and embryonic fibroblasts (IMR90) are reprogrammed after stable expression of either NBR1-specific shRNA (shNBR1) or non-targeting shRNA (shNT). Emerging iPSC colonies are scored based on Tra-1-60 expression37. (a, b) Cells depleted of NBR1 to increase MBd levels show an increase in iPSC colony formation (a, dH1f: 3.1±0.5-fold, n=15, p=0.00035; IMR90: 3.4±0.8-fold, n=3, p=0.02; data are mean ± s.e.m.) but insignificant changes in autophagic activity (c) over shNT control. (b) Representative plates with Tra-1-60-immunostained iPSC colonies. Immunoblot (c, top) and densitometry (c, bottom; percent of autophagic flux) show representative result (n=3); α-tubulin, loading control. (d) MCF-7 side-population (SP) cells have a significantly higher percentage of MBd+ cells over the non-SP population (MP; p=0.0015, n=3; data are mean ± s.d.). (e, f) MBd enrichment in cancer cells leads to increased anchorage-independent growth. MKLP1-GFP-expressing HeLa cells are separated into “MBd high” and “MBd low” subpopulations. An increase in the “MBd high” over “MBd low” ratio is associated with an increase in soft-agar colony formation (e). No significant difference was observed when the enrichment of MBd high subpopulation was less than 3-fold. More soft-agar colonies are formed when MBds are enriched by NBR1-depletion (shNBR1) in HeLa (f, left; p=0.0012, n=3) and mouse 134-4 cells (f, right; p=0.0086, n=3); control, shNT. Data are mean ± s.d., and the colony number (e, f) is the sum of INT-violet-stained colonies from 10 random fields. (g) Model for MBd fate in cells. The newly-formed MBd is preferentially inherited by the daughter cell with the older centrosome (top panel). The inherited MBd (black ring) is recognized by binding of the NBR1 autophagic receptor (grey circle) with the MB protein Cep55 (magenta). The MBd is then encapsulated by the autophagosome (yellow circle), and degraded after fusion of autophagosome and lysosome (red circle) in differentiated cells. This pathway prevents MBd-accumulation. In contrast, stem cells efficiently accumulate MBds through successive divisions and evasion of NBR1-mediated autophagy. Additionally, differentiated and stem cells possess overall high and low autophagic activity, respectively.

Article Snippet: Atg5 (1:2000, Cosmo Bio, CAC-TMD-PH-ATG); Atg7 (1:1000, ProSci, 3617); Actin (1:300, Sigma, AC-40); Aurora B (1:100, BD Trans Lab, 611082); CD13 (1:50, BioLegend, 301707); CD133 (1:200, eBioscience, 14-1331); Cep55 (1:50, 1:100 and 1:1000 for immunofluorescence, Abnova #H00055165-B01, Abnova #H00055165-A01, and the gift from K. Kurtche, respectively; 1:500 for immunoblotting, Genetax #GTX112190); hCenexin1 (1:100, a gift from K.S.

Techniques: In Vitro, Expressing, shRNA, Activity Assay, Control, Western Blot, Staining, Binding Assay