egfp depdc5 pljm1 vector  (Addgene inc)

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: