Journal: Cancers

Article Title: Proteome-Wide Analysis Reveals TFEB Targets for Establishment of a Prognostic Signature to Predict Clinical Outcomes of Colorectal Cancer.

doi: 10.3390/cancers15030744

Figure Lengend Snippet: Figure 1. Mutations in S138 and S142 caused almost complete nuclear retention of TFEB. (A) Schematic of the human TFEB protein domains. S138 and S142 are localized in the proxim- ity of a NES. (B) S138 and S142 of TFEB are evolutionarily conserved in the indicated species. Alignment of the sequences near TFEB S138 and S142 is shown. (C) Western blots of endogenous and exogenous TFEB in CRC cells transfected with wild-type TFEB-GFP or TFEB-GFP mutant (S142A/S138A). Uncropped immunoblots are provided in the Figure S1. (D,E) Localization of TFEBWT and TFEBS142A/S138A in the cytosol and in the nucleus was detected by confocal microscopy. Representative images (D) and statistical results (E) are shown. Torin1 (250 nm), a positive control. Cell outlines in white dotted lines. DAPI was used to label the nucleus. Scale bars, 5 µm. Mean ± SD, n = 10 cells per condition, unpaired t-test. (F) Cell viability of indicated cells were analyzed using a CCK8 assay. Mean ± SEM, n = 3, unpaired t-test. ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies (against TFEB, GFP, LAMP2, NDRG1, SMPD1, and actin) and the HRP-conjugated secondary antibodies (goat anti-rabbit antibody) were supplied by Proteintech (Wuhan, China).

Techniques: Western Blot, Transfection, Mutagenesis, Confocal Microscopy, Positive Control, CCK-8 Assay

Journal: Molecular Systems Biology

Article Title: Clustering phenotype populations by genome-wide RNAi and multiparametric imaging

doi: 10.1038/msb.2010.25

Figure Lengend Snippet: Functional analysis of candidate genes for roles in cell-cycle progression and spindle organization. ( A ) Time-resolved cell-cycle analysis in HeLa cells transfected with indicated siRNAs at different time points. Box colours represent fractions of cells with DNA content corresponding to sub-G1, G1/G0, S and G2/M. HeLa cells transfected with siRNAs against DONSON showed a delay in S-phase progression. ( B ) Assessment of S-phase progression by BrdU incorporation; 48 h after siRNA transfection, U2OS cells were synchronized for 16 h with 1 mM hydroxyurea (HU) and released for 6 h in BrdU-containing medium. BrdU-positive cells were stained with anti-BrdU primary antibody (Calbiochem) and Alexa 488 secondary antibody. DNA was counterstained with propidium iodide. Imaging and quantification were performed with Acumen Explorer microplate reader. Values are shown as mean±s.d. of three biological replicates. ( C ) Cell-cycle-dependent protein expression of DONSON. U2OS cells were synchronized either in G1/S with HU or in G2/M with nocodazole (Noc). Cells were collected at different time points after release into cell cycle for western blot analysis. ( D ) DONSON depletion is associated with a 10-fold increase in multipolar spindles compared to control treatments. U2OS cells were transfected with a DONSON siRNA pool and immunostained for α- and γ-tubulin at indicated time points. U2OS cells transfected with Rluc siRNAs serving as negative control. Data represent mean±s.d. of three biological replicates. At least 200 metaphase spindles were counted in each experiment. Scale bar indicates 2.5 μm. ( E ) DONSON protein co-localizes with centrosomes. HeLa cells were transfected with HA-tagged DONSON for 48 h and immunostained with primary HA antibody and Alexa 488-conjugated secondary antibody. Arrows indicate the centrosomal staining of DONSON (green). DNA was counterstained with DAPI (blue). Scale bar indicates 2.5 μm. ( F ) DONSON co-localizes with centrin. U2OS cells were transfected with HA-tagged DONSON; 48 h after transfection, cells were immunostained with anti-centrin and anti-HA-tagged primary antibodies and Alexa 488-, Alexa 594-conjugated secondary antibodies, respectively. Scale bar indicates 2.5 μm. Source data is available for this figure at www.nature.com/msb .

Article Snippet: An HA-tagged version of human DONSON was generated by modification of a DONSON cDNA clone (SC111799, OriGene).

Techniques: Functional Assay, Cell Cycle Assay, Transfection, BrdU Incorporation Assay, Staining, Imaging, Expressing, Western Blot, Control, Negative Control

Journal: Molecular Systems Biology

Article Title: Clustering phenotype populations by genome-wide RNAi and multiparametric imaging

doi: 10.1038/msb.2010.25

Figure Lengend Snippet: Functional assays for candidate genes in maintenance of genomic integrity. ( A ) Depletion of RRM1, CLSPN, CD3EAP, CADM1, DONSON and SON induced γH2AX foci formation. U2OS cells were transfected with siRNA pools and immunostained with γH2AX antibody 72 h after transfection. Representative images of γH2AX foci formation are shown. Scale bar indicates 2.5 μm. ( B ) Quantification of γH2AX foci formation on depletion of candidate genes; 72 h after siRNA transfection, U2OS cells were fixed, immunostained for γH2AX and γH2AX-positive cells were quantified. Ratios of γH2AX-positive cells were normalized to the negative control Rluc siRNA treatment. Data are mean±s.d. of three biological replicates. ( C ) γH2AX accumulation in DONSON and SON-depleted cells is not caused by an accumulation of S-phase cells. U2OS cells were collected 72 h after siRNA transfection and cell lysates were analysed by western blotting using indicated antibodies. Source data is available for this figure at www.nature.com/msb .

Article Snippet: An HA-tagged version of human DONSON was generated by modification of a DONSON cDNA clone (SC111799, OriGene).

Techniques: Functional Assay, Transfection, Negative Control, Western Blot

Journal: Molecular Systems Biology

Article Title: Clustering phenotype populations by genome-wide RNAi and multiparametric imaging

doi: 10.1038/msb.2010.25

Figure Lengend Snippet: SON, DONSON and CD3EAP are required for the DNA damage response. ( A ) SON, DONSON and CD3EAP depletion leads to a decreased phosphorylation of CHEK1 on γ irradiation, similar to ATR; 48 h after siRNA transfection, cells were γ irradiated and collected 1 or 2 h later for immunoblot analysis, probing with indicated antibodies. ( B ) SON and DONSON depletion leads to attenuated phosphorylation of CHEK1 on UV exposure, similar to ATR depletion. U2OS cells were UVC irradiated (20 J/m 2 ) 48 h after siRNA transfection. Cell lysates were collected for immunoblotting 2 h later and probed with indicated antibodies. ( C ) Knock down of DONSON and SON impairs RPA2 recruitment onto chromatin and the phosphorylation of ATR substrates. U2OS cells were transfected with siRNAs and UVC irradiated (20 J/m 2 ). Subsequently, the chromatin-associated insoluble fraction was extracted 2 h after UV exposure, and the fractions were analysed for the indicated proteins by immunoblot.

Article Snippet: An HA-tagged version of human DONSON was generated by modification of a DONSON cDNA clone (SC111799, OriGene).

Techniques: Phospho-proteomics, Irradiation, Transfection, Western Blot, Knockdown

Journal: Molecular microbiology

Article Title: The Regulation of Antimicrobial Peptide Resistance in the Transition to Insect Symbiosis

doi: 10.1111/mmi.13598

Figure Lengend Snippet: Distribution and sequence coverage of Tn5 mutants in the S. praecaptivus genome are depicted as a histogram (A, Upper). Note that mutations affecting Sant_4061 are the most abundant in the library, following growth in LB media. The zoomed in regions of the histogram (A, Lower) reveal the distribution and abundance of Tn5 mutants in the genomic regions encoding phoPQ and Sant_4061 (in red), with the mutants recovered in the screen for polymyxin B sensitivity highlighted in orange, with the number of recovered mutants shown above each column. Growth curves for WT S. praecaptivus and Sant_4061 and phoP mutants grown in either LB or LB + Polymyxin B (PB) for 10 hours (B). Standard errors are shown above and below the average of three replicates at each time point. ΔSant_4061, ΔphoQ, and ΔphoP are unable to grow on LB supplemented with polymyxin B (C).

Article Snippet: Construction of phoP and phoQ knockouts A plasmid construct consisting of approximately 500 bp of homologous sequence up and downstream of phoP with an internal kanamycin cassette was commercially produced (Genscript).

Techniques: Sequencing

Journal: Molecular microbiology

Article Title: The Regulation of Antimicrobial Peptide Resistance in the Transition to Insect Symbiosis

doi: 10.1111/mmi.13598

Figure Lengend Snippet: Phylogeny of S. praecaptivus and related Sodalis-allied endosymbionts and free-living bacteria based on maximum likelihood analyses of the phoQ coding sequence (1.45 kbp) and 16S rRNA (1.46 kbp). Sequences below strain names on the phoQ phylogeny show the amino acids sequences of the Mg2+-binding site within PhoQ, with acidic residues highlighted in bold. The numbers adjacent to nodes indicate maximum likelihood bootstrap values shown for nodes with bootstrap support > 80%.

Article Snippet: Construction of phoP and phoQ knockouts A plasmid construct consisting of approximately 500 bp of homologous sequence up and downstream of phoP with an internal kanamycin cassette was commercially produced (Genscript).

Techniques: Bacteria, Sequencing, Binding Assay

Journal: Molecular microbiology

Article Title: The Regulation of Antimicrobial Peptide Resistance in the Transition to Insect Symbiosis

doi: 10.1111/mmi.13598

Figure Lengend Snippet: Bacterial strains used in this study

Article Snippet: Construction of phoP and phoQ knockouts A plasmid construct consisting of approximately 500 bp of homologous sequence up and downstream of phoP with an internal kanamycin cassette was commercially produced (Genscript).

Techniques:

Journal: Oncotarget

Article Title: MiR-302a sensitizes leukemia cells to etoposide by targeting Rad52

doi: 10.18632/oncotarget.17878

Figure Lengend Snippet: (A) Schematic representation of Rad52 3′-UTRs showing putative miR-302a target site. (B) the relative luciferase activity of miR-302a mimics or mutant with pGL3-Rad52 constructs in HL-60 cell lines. (C) the relative luciferase activity of Rad52 indicated constructs in HL-60 cell lines. (D) qRT-PCR was performed to detect the expression of Rad52 in HL-60 transfected with miR-302a mimics or negative control. (E) Western blot analysis of Rad52 expression in HL-60 cells transfected with negative control or miR-302. Each bar represents the mean of three independent experiments.* P<0.05, ** P<0.01.

Article Snippet: SiRNAs of Rad52 and its negative control were synthesized by Genscript (Nanjing, China).

Techniques: Luciferase, Activity Assay, Mutagenesis, Construct, Quantitative RT-PCR, Expressing, Transfection, Negative Control, Western Blot

Journal: Oncotarget

Article Title: MiR-302a sensitizes leukemia cells to etoposide by targeting Rad52

doi: 10.18632/oncotarget.17878

Figure Lengend Snippet: (A,B) downregulation of Rad52 in HL-60 transfected with Rad52 siRNA, control sequence served as loading control. qRT-PCR and Western blot were conducted to measure the Rad52 expression level. β-actin served as loading control. (C) qRT-PCR analysis of Rad52 mRNA expression in MNCs from healthy people, AML patients and AML cell line HL-60. (D) downregulation of Rad52 enhance the sensitivity to VP16 in HL-60 cell. (E,F) HL-60 cells were stained with PI and FITC-Annexin V, the percentage of apoptosis cell measured by flow cytometry. Each result represents the mean of three independent experiments.* P<0.05, ** P<0.01.

Article Snippet: SiRNAs of Rad52 and its negative control were synthesized by Genscript (Nanjing, China).

Techniques: Transfection, Control, Sequencing, Quantitative RT-PCR, Western Blot, Expressing, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: MiR-302a sensitizes leukemia cells to etoposide by targeting Rad52

doi: 10.18632/oncotarget.17878

Figure Lengend Snippet: (A) HL-60 cell lines were transfected with miR-302a or miR-302a and Rad52 overexpression vector respectively. The expression of Rad52, p-AKT, AKT, p-GSK3β, β-catenin were detected by Western blot. (B) Western blot analysis of Rad52, p-AKT, AKT, p-GSK3β, β-catenin expression in HL-60 cells transfected with negative control or Rad52 siRNA. (C) HL-60 cell lines treated with VP-16 or co-transfected with miR-302a mimics. The expression of Rad52, p-AKT, AKT, p-GSK3β, β-catenin were measured by Western blot. β-actin treated as loading control.

Article Snippet: SiRNAs of Rad52 and its negative control were synthesized by Genscript (Nanjing, China).

Techniques: Transfection, Over Expression, Plasmid Preparation, Expressing, Western Blot, Negative Control, Control