Journal: Molecular cell

Article Title: Translational control through differential ribosome pausing during amino acid limitation in mammalian cells

doi: 10.1016/j.molcel.2018.06.041

Figure Lengend Snippet: KEY RESOURCES TABLE :

Article Snippet: HEK293T cells were transfected in a 10 cm plate with donor expression plasmid pLJM1 containing the desired insert, psPAX2, from Didier Trono (Addgene # 12260), and pCMV-VSV-G, from Bob Weinberg (Addgene # 8454) in a 10:9:1 ratio (by weight).

Techniques: Recombinant, Staining, Western Blot, Stripping, Hybridization, SYBR Green Assay, Northern Blot, Homologous Recombination, Clone Assay, Sequencing, FLAG-tag, Software

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Establishment of the DEPDC5-KO HCC cells by using CRISPR/Cas9 system. ( a ) Schematics of the protein structure of DEPDC5. Grey and black bars show the position of amino acid substitutions induced by missense and stop-gain mutations in the ICGC public data. The arrow indicates the site that an sgRNA targets for knockout by using CRISPR/Cas9 technology in this study. ( b ) Sequence chromatograms of the DEPDC5-KO JHH5 and HLE cells around the sgRNA target site (grey background color). ( c ) Immunofluorescence analysis of the DEPDC5-WT and -KO JHH5 and HLE cells with DEPDC5 staining (red). Nuclei were counterstained with DAPI (blue). Magnification, ×200.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: CRISPR, Knock-Out, Sequencing, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Cellular response of the DEPDC5-KO cells to leucine deprivation involved in autophagy pathway. ( a ) Proliferation curves of the DEPDC5-WT and -KO JHH5 and HLE cells. The value of each sample was relative to that at Day 1. Error bars are the mean ± S.D. P values were calculated by Welch’s t -test. ( b ) Flow cytometric analysis with PI staining. The percentage of each phase is the mean ± S.D. P values were calculated by Welch’s t -test. ( c ) Immunoblots of LC3B and p62. The cells were exposed to leucine-free medium for the indicated time periods. GAPDH was used as a loading control. ( d ) Immunofluorescence analysis of the DEPDC5-WT and -KO JHH5 and HLE cells under leucine-depleted conditions with LC3 (green) and p62 (red) staining. Nuclei were counterstained with DAPI (blue). Magnification, ×200.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: Staining, Western Blot, Control, Immunofluorescence

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Reduction of cellular ROS levels in the DEPDC5-KO HCC cells. ( a ) Representative histogram images of cells with CellROX. The concentration of H2O2 was 100 μM in the right panels. The value of each mean relative fluorescence intensity (RFI) is the mean ± S.D. P values were calculated by Welch’s t -test. ( b ) Dose-response curves of the cell viability after H2O2 treatment. P values were calculated from the ANOVA table.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: Concentration Assay, Fluorescence

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Inhibition of cancer cell growth with elevated cellular ROS levels by DEPDC5 overexpression. ( a ) Immunofluorescence analysis of the doxycycline (DOX)-inducible DEPDC5-expressing HuH7 cells with DEPDC5 staining (red). Nuclei were counterstained with DAPI (blue). Magnification, ×200. ( b ) Quantification of colony-forming efficiency. Error bars are the mean ± S.D. P values were calculated by Welch’s t -test. ( c ) Immunoblots of p62. The cells were exposed to medium containing doxycycline for the indicated time periods. GAPDH was used as a loading control. ( d ) Representative histogram images of cells with CellROX. The value of each mean RFI is the mean ± S.D. P values were calculated by Welch’s t -test. ( e ) In vivo tumorigenicity of doxycycline-inducible DEPDC5-expressing HCC cells. The upper and lower panels show growth curves of transplanted tumors and representative photo images. Error bars are the mean ± S.E in the upper panel. P values were calculated by Welch’s t -test. The white scale bar is 1 cm in the lower panel.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: Inhibition, Over Expression, Immunofluorescence, Expressing, Staining, Western Blot, Control, In Vivo

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Relationship among DEPDC5 and p62 expression in HCC samples and patient prognosis. ( a ) Immunohistochemical analysis of DEPDC5 and p62 in a representative tissue sample including adjacent liver tissue (N) and cancer (T). Nuclei were counterstained with hematoxylin. In adjacent liver tissues of almost all cases, DEPDC5 was positive while p62 was negative. ( b ) Kaplan-Meier curves of the progression-free and overall survival in groups of HCC patients classified according to DEPDC5 and p62 expression patterns. P values were calculated by the log-rank test.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: Expressing, Immunohistochemical staining

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Relationship between DEPDC 5 expression and clinicopathological factors.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: Expressing

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Univariate and multivariate analysis of factors contributing to progression-free and overall survival.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques:

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pLJM1-CDKN2A (plasmid) , Twist Bioscience , , Lentiviral vector expressing codon-optimized CDKN2A.

Techniques: Recombinant, Plasmid Preparation, Expressing, Sequencing, Mutagenesis, Modification, Transfection, Software, Control

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/elife.95347

Figure Lengend Snippet: Figure 2. Functional characterization of all possible CDKN2A missense variants. (A) Functional classifications for 3120 CDKN2A variants, including 2964 missense variants and 156 synonymous variants. Variants were classified as functionally deleterious, indeterminate function, or neutral based on p-value using gamma generalized linear model (GLM). 525 (17.7%) variants were classified as functionally deleterious. (B) Log2 p-value (gamma GLM) for 32 benchmark pathogenic variants, 6 benign variants, 31 variants of uncertain significance (VUSs) previously reported to have functionally deleterious

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) CDKN2A GenBank Gene ID: 1029 NM_000077.5 NP_000068.1 Cell line (H. sapiens) PANC- 1 American Type Culture Collection Cat. #: CRL- 1469 RRID:CVCL_0480 Cell line (H. sapiens) 293T American Type Culture Collection Cat. #: CRL- 3216 RRID:CVCL_0063 Recombinant DNA reagent pHAGE- CDKN2A (plasmid) Addgene RRID:Addgene_116726 Lentiviral vector expressing CDKN2A Recombinant DNA reagent pLentiV_ Blast (plasmid) Addgene RRID:Addgene_111887 Recombinant DNA reagent pLJM1- Empty (plasmid) Addgene RRID:Addgene_91980 Recombinant DNA reagent psPAX2 (plasmid) Addgene RRID:Addgene_12260 Recombinant DNA reagent pCMV- VSV- G (plasmid) Addgene RRID:Addgene_8454 Recombinant DNA reagent pLJM1- CDKN2A (plasmid) Twist Bioscience Lentiviral vector expressing codon- optimized CDKN2A Recombinant DNA reagent pLJM1- CDKN2A- Leu32Leu (plasmid) This paper Lentiviral vector expressing CDKN2A- Leu32Leu Recombinant DNA reagent pLJM1- CDKN2A- Leu32Pro (plasmid) This paper Lentiviral vector expressing CDKN2ALeu32Pro Recombinant DNA reagent pLJM1- CDKN2A- Gly101Gly (plasmid) This paper Lentiviral vector expressing CDKN2AGly101Gly Recombinant DNA reagent pLJM1- CDKN2A- Gly101Trp (plasmid) This paper Lentiviral vector expressing CDKN2AGly101Trp Recombinant DNA reagent pLJM1- CDKN2A- Val126Asp (plasmid) This paper Lentiviral vector expressing CDKN2AVal126Asp Recombinant DNA reagent pLJM1- CDKN2A- Val126Val (plasmid) This paper Lentiviral vector expressing CDKN2AVal126Val Kimura et al. eLife 2024;13:RP95347.

Techniques: Functional Assay