Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (a) HepG2 with or without various treatments for 24 hours and stable HepG2 cells that expressed a control shRNA (sh-Ctrl) or the Atg5 shRNA (sh-Atg5) were subjected to flow cytometry analysis for CD133+ cells. Results represent the mean ± SEM of three independent experiments. None, no treatment. (B) HepG2 cells with the treatments shown in (A) were lysed for immunoblot analysis. LC3-I, non-lipidated LC3; LC3-II, lipidated LC3. The β-actin protein was also analyzed to serve as the loading control. (C) Sphere-formation assay of CD133+ and CD133− HepG2 cells. The panels shown to the left are representative results of spheres formed by CD133+ and CD133− HepG2 cells with and without stable ATG5 knockdown. Scale bar=200 μm. The histogram shown to the right indicated the number of spheres larger than 100 μm in diameter when 500 CD133+ cells were seeded. The results represent the mean ± SEM of three independent experiments. (D) HepG2 cells with various treatments for 24 hours were incubated with MicroBeads (Miltenyi Biotec) for the isolation of CD133+ cells, which were then analyzed for their sphere-forming ability. 500 cells were seeded for the assay. Also see Figure S1.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Control, shRNA, Flow Cytometry, Western Blot, Tube Formation Assay, Knockdown, Incubation, Isolation

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Hep3B and Huh7 cells with various treatments for 24 hours were subjected to flow cytometry analysis for CD133+ cells. (B) HepG2 and Huh7 cells were transfected with the p53-expressing plasmid for two days, treated with 3-MA or rapamycin for another 24 hours and then subjected to flow cytometry analysis for CD133+ cells. (C) HepG2 cells transfected with the control siRNA (si-Ctrl) or the p53 siRNA (si-p53) for two days or treated with PFTα or DMSO for one day were analyzed for their CD133+ cells by flow cytometry (top panel) or sphere-forming ability of their CD133+ cells (bottom panel). The results shown in (A), (B) and (C) represent the mean ± SEM of three independent experiments. (D) HepG2 cells treated with DMSO or PFTα for one day or with siRNA for two days were lysed for immunoblot analysis. (E) Stable HepG2 cells that expressed control shRNA (sh-Ctrl), sh-Atg5, or both sh-Atg5 and sh-p53 were lysed for immunoblot analysis. (F) Cells mentioned in (E) were used for the sphere-formation assay. Also see Figure S2.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Control, Western Blot, shRNA, Tube Formation Assay

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) HepG2 cells with various treatments for 24 hours or stably expressing the control shRNA or the Atg5 shRNA were lysed for immunoblot analysis. (B) Immunoblot analysis of HepG2 cells transfected with either the control vector or the expression vector for various p53 proteins. Cells were lysed two days after DNA transfection for immunoblot analysis. None, control cells with no DNA transfection. (C) The experiments were conducted the same way as in (B), with the exception that Hep3B cells were used for the expression studies. (D) Hep3B or HepG2 cells were transfected with various p53-expressing plasmids or the control vector as indicated for two days followed by flow cytometry analysis for CD133+ cells. (E) HepG2 cells were transfected with the p53-expressing plasmids for two days, and CD133+ cells were then isolated for the sphere-formation assay. (F) The experiments were conducted the same way as in (E), with the exception that Hep3B cells were used for the studies. The results in (D–F) represented the mean ± SEM of three independent experiments. Also see Figure S3.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Stable Transfection, Expressing, Control, shRNA, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry, Isolation, Tube Formation Assay

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Top panel, HepG2 cells were treated with DMSO, Mdivi-1 or CCCP for one day and then subjected to flow cytometry analysis for CD133+ cells; bottom panel, CD133+ HepG2 cells were isolated and treated with Mdivi-1 or CCCP for two days and then analyzed for their sphere-forming ability. (B) HepG2 cells without treatment or with the treatment of DMSO, CCCP or Mdivi-1 for one day were lysed for immunoblot analysis. Cells were also subjected to subcellular fractionation for the isolation of mitochondria, cytosol, and nuclei for immunoblot analysis. Tom20, β-actin and lamin B1 were used as the loading controls for mitochondria, cytosol and nucleus, respectively, to ensure equal amount of proteins were loaded on the gel. (C) Confocal microscopy for the analysis of the subcellular localization of p53(pS392) in HepG2 cells treated with DMSO, Mdivi-1 or CCCP. TOM20 was used as the marker for mitochondria. The areas boxed are enlarged at the bottom. Scale bar, 10 μm. (D) The results shown in (C) were quantified with a Leica TCS SP8 fluorescent confocal microscope. The results indicated the percentages of p53(pS392) that colocalized with TOM20. The results represent the mean ± SEM of at least 30 cells that were analyzed. See also Figure S5.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Flow Cytometry, Isolation, Western Blot, Fractionation, Confocal Microscopy, Marker, Microscopy

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Effects of PINK1 knockdown on CD133+ HepG2 cells (top panel), their sphere-forming ability (middle panel) and their effects on the Nanog promoter using the Nanog-luc1 reporter (bottom panel). HepG2 cells transfected with either the control siRNA or the PINK1 siRNA for two days were analyzed. In the bottom panel, HepG2 cells were also transfected with the Nanog-luc1 reporter (see Figure 3B) for the analysis of luciferase activity. The luciferase activity of cells without the transfection of siRNA was arbitrarily defined as 1. The results represented the mean ± SEM of three independent experiments. (B) Effects of PINK1 over-expression on CD133+ HepG2 cells (top panel), their sphere-forming ability (middle panel) and their effects on the Nanog promoter (bottom panel). The experiments were conducted the same way as in (A), except that instead of using siRNA, cells were transfected with either the control vector or the PINK1-expressing plasmid. (C) Immunoblot analysis of HepG2 cells with PINK1 knockdown (left panels) or PINK1 over-expression (right panels) were lysed for immunoblot analysis. Total cell lysates as well as the nuclear lysates (bottom two panels) were analyzed. (D) PINK1 in HepG2, Hep3B or Huh7 cells was immunoprecipitated with a control antibody (−) or the anti-PINK1 antibody (+) and then incubated with GST-p53 in the presence of ATP. The GST-p53 phosphorylated at S392 was analyzed using the anti-p53 antibody that recognized phosphoserine-392. GST-p53 added in the reaction and PINK1 immunoprecipitated were also analyzed by immunoblot (bottom two panels). Numbers to the left of the top panel indicate protein molecular weight markers. (E) GST-p53 was mixed with GST-PINK1 or GST and incubated in the presence of ATP. The phosphorylation of p53 at S392 was then analyzed with the antibody that recognized phosphoserine-392. GST-p53, GST-PINK1 and GST used for the reaction was also analyzed by anti-p53, anti-PINK1 and anti-GST antibodies, respectively (bottom three panels). Numbers to the left indicate protein molecular weight markers. (F) Co-immunoprecipitation of p53 and p53(pS392) with PINK1. HepG2 cells were lysed and immunoprecipitated using the anti-PINK1 antibody or the control antibody followed by immunoblot analysis for p53, p53(pS392) and PINK1. (G) Co-immunoprecipitation of p53 and p53(pS392) with PINK1 using the anti-PINK1 antibody in different subcellular fractions (top 3 panels). β-actin, lamin B1 and Tom20 were used as the markers for cytosolic (C), nuclear (N) and mitochondrial (M) fractions. Equal amounts of p53 were used for the co-immunoprecipitation experiment (bottom 2 panels). See also Figure S6.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Knockdown, Transfection, Control, Luciferase, Activity Assay, Over Expression, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Incubation, Molecular Weight, Phospho-proteomics

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Virus, Recombinant, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Mouse Assay, Mutagenesis, Software, Imaging, Extraction, Isolation, DNA Labeling

Journal: mBio

Article Title: Identification of Novel Protein Lysine Acetyltransferases in Escherichia coli

doi: 10.1128/mBio.01905-18

Figure Lengend Snippet: Inactivation of the two known acetylation mechanisms in E. coli eliminates the majority of acetylation. Wild-type (WT) E. coli (strain BW25113) and an isogenic Δ pta yfiQ acs cobB mutant (Gutted) were aerated in TB7 supplemented with 0.4% glucose for 10 h. Whole-cell lysates were analyzed (A) by Coomassie blue-stained SDS-polyacrylamide gel to ensure equivalent loading and (B) by anti-acetyllysine Western blotting.

Article Snippet: Acetylated peptides were enriched using 1/4 tube of the anti-acetyllysine antibody-bead-conjugated PTMScan Acetyl-Lysine Motif [Ac-K] kit (Cell Signaling Technologies) for each of the 1-mg protein lysate samples according to the manufacturer’s instructions.

Techniques: Mutagenesis, Staining, Western Blot

Journal: mBio

Article Title: Identification of Novel Protein Lysine Acetyltransferases in Escherichia coli

doi: 10.1128/mBio.01905-18

Figure Lengend Snippet: Overexpression of five GNAT family members results in altered lysine acetylation patterns by anti-acetyllysine Western blotting. The gutted strain (BW25113 Δ pta yfiQ acs cobB ) was transformed with the pCA24n vector control (negative [−] control) or pCA24n containing the indicated genes under an IPTG-inducible promoter . As a positive (+) control, an isogenic strain that retained the WT allele of acs (Δ pta yfiQ cobB ) was transformed with pCA24n containing YfiQ. The resulting strains were aerated in TB7 supplemented with 0.4% glucose, 50 μM IPTG, and 25 μg/ml chloramphenicol for 10 h. Whole-cell lysates were analyzed (right panels) by Coomassie blue-stained SDS-polyacrylamide gel electrophoresis to ensure equivalent loading and (left panels) by anti-acetyllysine Western blotting. Note that the band in RimJ was not reproducible. The positive control contains one additional YfiQ-dependent band around 72 kDa, which corresponds to Acs . YncA and AstA each produce an acetylated band that can be observed in the Coomassie blue-stained gel at the expected molecular weight of these proteins.

Article Snippet: Acetylated peptides were enriched using 1/4 tube of the anti-acetyllysine antibody-bead-conjugated PTMScan Acetyl-Lysine Motif [Ac-K] kit (Cell Signaling Technologies) for each of the 1-mg protein lysate samples according to the manufacturer’s instructions.

Techniques: Over Expression, Western Blot, Transformation Assay, Plasmid Preparation, Negative Control, Positive Control, Staining, Polyacrylamide Gel Electrophoresis, Molecular Weight

Journal: mBio

Article Title: Identification of Novel Protein Lysine Acetyltransferases in Escherichia coli

doi: 10.1128/mBio.01905-18

Figure Lengend Snippet: Mutation of conserved catalytic amino acids prevents RimI-, PhnO-, YjaB-, and YiaC-dependent acetylation. The gutted strain (BW25113 Δ pta yfiQ acs cobB ) was transformed with the pCA24n vector control, pCA24n carrying the wild-type allele for each putative KAT, or mutant alleles for each putative KAT with alanine substitutions of the indicated residues. The resulting strains were grown in TB7 supplemented with 0.4% glucose, 100 μM IPTG, and 25 μg/ml chloramphenicol for 8 h. Crude lysates harvested after 4 h were analyzed for expression of the KAT proteins. Whole-cell lysates harvested after 8 h were analyzed for acetylation. Coomassie blue-stained SDS-PAGE gels (A and C) served as loading controls for anti-His (B) and anti-acetyllysine (D) Western blot analysis.

Article Snippet: Acetylated peptides were enriched using 1/4 tube of the anti-acetyllysine antibody-bead-conjugated PTMScan Acetyl-Lysine Motif [Ac-K] kit (Cell Signaling Technologies) for each of the 1-mg protein lysate samples according to the manufacturer’s instructions.

Techniques: Mutagenesis, Transformation Assay, Plasmid Preparation, Expressing, Staining, SDS Page, Western Blot

Journal: bioRxiv

Article Title: AGO2 slicing of a domesticated retrotransposon is necessary for normal vasculature development

doi: 10.1101/2025.04.02.646793

Figure Lengend Snippet: (A) Relative placental weights of mutants relative to wild-type littermates at E18.5. (B) Relative embryo weights of mutants relative to wild-type littermates at E18.5. (C) Relative placental efficiency of mutants relative to wild-type littermates at E18.5. Each dot represents an animal. p-values we calculated using a two-tailed t-test. (D) Histone H3 phosphorylation as measured by western blot on protein lysates from whole E14.5, E16.6, and E18.5 placentas. (E) Flow cytometry analysis of E16.5 placentas using antibodies against the proliferative marker Ki67 and the endothelial cell marker CD31. Plots show overlay of two biological replicates per genotype, each highlighted with a different color. (F) Quantification of (E). Each dot represents a biological replicate. P-values were calculated using a two-tailed t-test. (G) Schematic representation of a mid-gestation embryo, highlighting which structures are trophectoderm (blue) versus epiblast (pink) derived. (H) Genotyping PCR from 4 embryos (E1-E4), showing examples of a Ago2 flx/+ ;Sox2-Cre − embryo (E1), a Ago2 flx/D598A ;Sox2-Cre − embryo (E2), a Ago2 flx/+ ;Sox2-Cre + embryo (E3), and a Ago2 flx/D598A ;Sox2-Cre + embryo (E4).

Article Snippet: The cell pellets were resuspended in 100 μl cold FACS buffer (1X PBS, 0.5% BSA, 2mM EDTA) and used to purify endothelial cells using an initial lineage cell depletion step (Miltenyi Biotec #130-090-858) followed by endothelial cell purification using anti-CD31-conjugated magnetic beads (mouse CD31, Miltenyi Biotec #130-097-418) according to the manufacturer’s protocol.

Techniques: Two Tailed Test, Phospho-proteomics, Western Blot, Flow Cytometry, Marker, Derivative Assay

Journal: bioRxiv

Article Title: AGO2 slicing of a domesticated retrotransposon is necessary for normal vasculature development

doi: 10.1101/2025.04.02.646793

Figure Lengend Snippet: (A) Volcano plot showing genes significantly dysregulated in Ago2 mutant placentas (FDR<-0.05 and [log 2 (fold-change)] > 1.5). (B) Enriched GO terms (left) and MGI Phenotypes (right) amongst upregulated genes. Terms related to vascular development are highlighted in red. Neonatal lethality is highlighted in pink. (C) Gene Set Enrichment Analysis (GSEA) plots showing a significant enrichment of gene signatures for vascular and lymphatic endothelium amongst the genes dysregulated in Ago2 catalytic mutants. (D) Left, immunofluorescence staining of mouse placentas with an antibody against the CD31 endothelial cell marker. Right, quantification of vessel area in wild-type and mutant placentas. p-values we calculated using a tow-tailed t-test. (E) Hematoxylin-Eosin-stained sagittal sections of E18.5 embryos, showing enlarged vessels in mutant (bottom, closed arrow heads) compared to wild-type (top, open arrow heads) animals. (F) Quantification of vessel area in wild-type and mutant hearts.

Article Snippet: The cell pellets were resuspended in 100 μl cold FACS buffer (1X PBS, 0.5% BSA, 2mM EDTA) and used to purify endothelial cells using an initial lineage cell depletion step (Miltenyi Biotec #130-090-858) followed by endothelial cell purification using anti-CD31-conjugated magnetic beads (mouse CD31, Miltenyi Biotec #130-097-418) according to the manufacturer’s protocol.

Techniques: Mutagenesis, Immunofluorescence, Staining, Marker

Journal: bioRxiv

Article Title: AGO2 slicing of a domesticated retrotransposon is necessary for normal vasculature development

doi: 10.1101/2025.04.02.646793

Figure Lengend Snippet: (A) Enrichment analysis for gene signatures from 18 placental types in genes upregulated in Ago2 catalytic mutants. (B) Immunofluorescence staining of mouse placentas with an antibody against the CD31 endothelial cell marker. (C) Quantification of vessel area in wild-type and mutant placentas. p-values we calculated using a two-tailed t-test. (D) Representative E18.5 fetuses enveloped by the yolk-sac. Major vessels are highlighted. (E) Immunofluorescence staining of yolk-sacs with an antibody against the CD31 endothelial cell marker. Error bar, 100 μm.

Article Snippet: The cell pellets were resuspended in 100 μl cold FACS buffer (1X PBS, 0.5% BSA, 2mM EDTA) and used to purify endothelial cells using an initial lineage cell depletion step (Miltenyi Biotec #130-090-858) followed by endothelial cell purification using anti-CD31-conjugated magnetic beads (mouse CD31, Miltenyi Biotec #130-097-418) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Staining, Marker, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: AGO2 slicing of a domesticated retrotransposon is necessary for normal vasculature development

doi: 10.1101/2025.04.02.646793

Figure Lengend Snippet: (A) Relative Rtl1 expression in E14.5 placentas and embryos. All values are normalized to the average of the wild-type placenta. Each dot represents a biological replicate. P-values were calculated using a two-tailed t-test. (B) Relative Rtl1 expression from Ago2 flx/+ ;Sox2-Cre + and Ago2 flx/D598A ;Sox2-Cre + E18.5 placentas. Each dot represents a biological replicate. P-values were calculated using a two-tailed t-test. (C) Immunofluorescent staining of endothelial cell cultures of indicated genotypes infected with a control vector or a vector that knocks-down Rtl1 using the CRISPRi strategy. (D) Percentage of Ki67 positive cells in Ago2 +/D598A control (grey bars) and Ago2 D598A/D598A mutant (blue bars) endothelial cultures infected with the indicated constructs. Each dot represents a technical replicate. P-values were calculated using a two-tailed t-test. (E) Permeability assay performed on endothelial cells cultured in the presence of VEGF, a growth factor that increases vascular permeability, as a percentage of untreated control samples. (F) Average lumen area of tubes formed by the endothelial cells cultured in the presence of Matrigel. Each dot represents an independent replicate experiment. p-values we calculated using a two-tailed t-test.

Article Snippet: The cell pellets were resuspended in 100 μl cold FACS buffer (1X PBS, 0.5% BSA, 2mM EDTA) and used to purify endothelial cells using an initial lineage cell depletion step (Miltenyi Biotec #130-090-858) followed by endothelial cell purification using anti-CD31-conjugated magnetic beads (mouse CD31, Miltenyi Biotec #130-097-418) according to the manufacturer’s protocol.

Techniques: Expressing, Two Tailed Test, Staining, Infection, Control, Plasmid Preparation, Mutagenesis, Construct, Permeability, Cell Culture

Journal: bioRxiv

Article Title: AGO2 slicing of a domesticated retrotransposon is necessary for normal vasculature development

doi: 10.1101/2025.04.02.646793

Figure Lengend Snippet: (A) Schematic representation of the endothelial cell isolation strategy. (B, C) Rtl1 is expressed and upregulated in endothelial cells isolated from both placentas (B) and embryos (C). N.D., not detected. (D) Rtl1 expression in heterozygous control ( +/D598A ; grey) and homozygous mutant ( D598A/D598A ; blue) endothelial cell cultures stably infected with dCas9-KRAB control vector or a vector silencing Rtl1 expression. (E) Permeability assay performed on endothelial cells cultured in the absence of VEGF (F) Top, schematic representation of features quantified during the tube formation assay. Bottom, representative bright field image of tubes formed when indicated endothelial lines were cultured in Matrigel. (G-I) Number of junctions (G), meshes (H), and nodes (I) per field in indicated cell lines. Each dot represents a technical replicate. P-values were calculated using a two-tailed t-test.

Article Snippet: The cell pellets were resuspended in 100 μl cold FACS buffer (1X PBS, 0.5% BSA, 2mM EDTA) and used to purify endothelial cells using an initial lineage cell depletion step (Miltenyi Biotec #130-090-858) followed by endothelial cell purification using anti-CD31-conjugated magnetic beads (mouse CD31, Miltenyi Biotec #130-097-418) according to the manufacturer’s protocol.

Techniques: Cell Isolation, Isolation, Expressing, Control, Mutagenesis, Stable Transfection, Infection, Plasmid Preparation, Permeability, Cell Culture, Tube Formation Assay, Two Tailed Test