tusc3 short hairpin rna  (Santa Cruz Biotechnology)

Journal: Nature communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC.

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: Fig. 1 miR-224 and -520c are responsible for TUSC3 deficiency in metastasized lung cancer patient tissues. a Expression analyses of TUSC3 by immunohistochemistry (IHC) showing suppressed expression in matched metastatic samples. 50 primary and their matched lymph node metastasized lung tumor tissues were analyzed. The summarized expressional scores are shown in Supplementary Fig. 1b. P-value was obtained by Pearson’s chi- squared test. b PrognoScan based Kaplan Meier plot showing decreased patient survivals associated with TUSC3 deficiency (GSE31210). c Luciferase reporter assays with TUSC3 3’UTR in miR-224 or -520c-overexpressing HEK293 cells. The pGL3-TUSC3-3’UTR plasmid or the mutants harboring deleted target sequences for miR-224 or miR-520c were co-transfected with miR-224 (left panels) or miR-520c (right panels). The relative values were obtained by normalizing to Renilla luciferase values. Bars represent means ± SD (n = 4) and the p-values were determined by two-tailed student t-test (*p < 0.001). d Decreased TUSC3 mRNA expression in miR-224 or miR-520c overexpressing NSCLC. Bars indicate means ±SD (n = 3) and p-values were addressed by two-tailed student t-test (*p < 0.01, **p < 0.05). e Western blot analyses showing that miR-224 and miR-520c suppressed the endogenous TUSC3 protein in A549 (left panel) and H460 (right panel) cells. Pre-miR-224/-520C (left) or anti-miR-224/-520c (right) were ectopically expressed in A549 (left) or H460 (right) cells. f In situ hybridization showing the enhanced miR-224 and miR-520c expression in lymph node metastasized lung tissues compared to their corresponding primary tumors. Total numbers of cases were summarized in Supplementary Fig. 1e. g, h Co-expression analyses of TUSC3 and miR- 224 (g) and miR-520c (h). The images in the miR-224 and miR-520c sections show a lung adenocarcinoma after co-expression of TUSC3 (fluorescence red and RGB brown) with miR-224 (g, fluorescence blue and RGB blue) or miR-520c (h, fluorescence blue and RGB blue). The chi-squared test statistic was generated and the null hypotheses that the expression of TUSC-3, miR-224, and miR-520 was equal in primary versus the metastatic tumors or that the expression of TUSC3 and a given miRNA was equal in the primary and metastatic tumors tested using 2 degrees of freedom. The scale bars indicate 150 µm (a), 100 µm (b), and 200 µm (g, h), respectively

Article Snippet: The CRISPR knock out (KO) constructs for TUSC3 gene and HRD1 gene were purchased from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Immunohistochemistry, Luciferase, Plasmid Preparation, Transfection, Two Tailed Test, Western Blot, In Situ Hybridization, Generated

Journal: Nature communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC.

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: Fig. 4 TUSC3 deficiency enhances HRD1-dependent pro-metastatic property by enhancing ERAD activity. a Enhanced A1AT-NHK-ddVenus GFP accumulation in A549 TUSC3KO cells. The A1AT-NHK-ddVenus stable A549 control (Ctrl) or TUSC3 KO cells were generated by transduction with pLL-Lenti-A1AT-NHK-ddVenus virus. The protein accumulation was monitored after incubating the cells with MG132 (20 uM) for 16 h. The accumulation was quantified by FACS analyses (lower panel) or Western blot analyses using anti-GFP antibody (upper panel). b Decreased accumulation of A1AT- NHK-ddVenus by miR-224 and -520c suppression. Bars indicate means ±SD (n = 3) and p-values were obtained by two-tailed student t-test (a and b, *p < 0.001, **p < 0.02). c Co-immunoprecipitation assays with TUSC3 and HRD1 proteins. pcDNA-TUSC3-V5-His or its mutants (TUSC3-CCSS and Del- TUSC3) was co-transfected with pCMV6-HRD1-Flag in HEK293 cells. d The changed affinity of HRD1 protein to its substrates by TUSC3. The endogenous HRD1 protein was precipitated with anti-HRD1 antibody in H460 control, TUSC3 KO or TUSC3-reconstituting TUSC3 KO cells. e In vitro competition assay with p53 and TUSC3 proteins in HEK293 cells. f In vivo ubiquitination assay of p53 protein showing enhanced p53 ubiquitination in response to TUSC3 downmodulation. g Decreased p53 protein in H460 TUSC3 KO cell and rescued in HRD1/TUSC3 DKO cells. The indicated cells were exposed by 5.0 ug/ml of TM for 1 h and subject to Western blot analysis. h Rescued p53 protein in TUSC3 or TUSC3-CCSS mutant expressing H460 TUSC3 KO cells. i Re-suppressed colonization effect of TUSC3KO cells upon TP53 reconstitution. Tail-vein injection was performed using the indicated cells, and subsequently the lung tissues were harvested after 4 weeks. p-values were obtained by student t-test (*p = 0.00031, **p < 0.038). j Co-expression analyses between TUSC3 and p53 (upper panels) or IRE1α (lower panels) in lung cancer patient samples. The p53 and IRE1α are shown in fluorescence green whereas TUSC3 is shown in fluorescence red. Scale bar indicates 150 µm. k GSEA plots indicating gene sets suppressed by TP53 were enriched in TUSC3KO cells either DMSO or TM treatment compared to the controls

Article Snippet: The CRISPR knock out (KO) constructs for TUSC3 gene and HRD1 gene were purchased from Santa Cruz Biotechnology, Inc.

Techniques: Activity Assay, Control, Generated, Transduction, Virus, Western Blot, Two Tailed Test, Immunoprecipitation, Transfection, In Vitro, Competitive Binding Assay, In Vivo, Ubiquitin Proteomics, Mutagenesis, Expressing, Injection

Journal: Nature communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC.

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: Fig. 5 Metastatic suppressor, NM23H1/2 is regulated by TUSC3 through p53 regulation. a The reduction of NM23H1/2 protein in TUSC3 KO cells in response to ER stress induction. The cells were incubated by 5.0 ug of TM for 9 h and subsequently harvested for Western blot analysis. b Decreased NM23H1 protein in p53 deficient cells. The H460 cells were transfected with siTP53 siRNAs for 48 h, and the expression of p53 and NM23H1/2 proteins was measured by Western blot analysis. c Restored NM23H1/2 proteins by p53 protein accumulation in A549 TUSC3 KO cells. The cells were incubated with Ntulin3a for 24 h, and Western blot analysis was performed to measure the expression of NM23H1/2. d HRD1-regulated NM23H1/2 protein in A549 TUSC3 KD cells. The A549 TUSC3 KD cells were transfected with scrambled or siHRD1 siRNAs. After 48 h, the cells were exposed to TM (5 ug/ml) for 6 h followed by Western blot analysis using indicated antibodies. e Accumulated NM23H1/2 proteins upon miR-224/-520c downregulation. The miRNA KD cells were transfected by scrambled or siTUSC3 siRNAs for 48 h, and the NM23H1/2 protein was measured by Western blot analysis. f The reduced migration and invasion of A549 TUSC3 KD cells in response to NM23H1 reconstitution. H460 TUSC3 KD cells were transfected by pCMV6-NM23H1 plasmid for 24 h. Subsequently, the cells were harvested and subject to the chambers of migration or invasion assays. Error bars indicate means ± SD (n = 3) and the p-values were calculated by two-tailed student t-test (*p < 0.03). g Restored expression of NM23H1 decreased colonization ability of H460 TUSC3KO cells. The cells overexpressing NM23H1 generated by the transduction of lent-NM23H1 virus. 1 × 106 of NM23H1 overexpressing TUSC3KO cells or control KO cells was intravenously injected into four NSG mice. The tumor area was obtained by Image J software by measuring the field of view in lung occupied by cancer. The region for the cancer was expressed as percentage. p-value was calculated by unpaired student t-test (*p < 0.001)

Article Snippet: The CRISPR knock out (KO) constructs for TUSC3 gene and HRD1 gene were purchased from Santa Cruz Biotechnology, Inc.

Techniques: Incubation, Western Blot, Transfection, Expressing, Migration, Plasmid Preparation, Two Tailed Test, Generated, Transduction, Virus, Control, Injection, Software

Journal: Nature communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC.

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: Fig. 6 The schematic diagram showing the working hypothesis by which miR-224/-520c-induced TUSC3 suppression enhanced metastatic potential of NSCLC through the alteration of UPRs and HRD1-dependent ERAD

Article Snippet: The CRISPR knock out (KO) constructs for TUSC3 gene and HRD1 gene were purchased from Santa Cruz Biotechnology, Inc.

Techniques:

Journal: Nature communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC.

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: Fig. 1 miR-224 and -520c are responsible for TUSC3 deficiency in metastasized lung cancer patient tissues. a Expression analyses of TUSC3 by immunohistochemistry (IHC) showing suppressed expression in matched metastatic samples. 50 primary and their matched lymph node metastasized lung tumor tissues were analyzed. The summarized expressional scores are shown in Supplementary Fig. 1b. P-value was obtained by Pearson’s chi- squared test. b PrognoScan based Kaplan Meier plot showing decreased patient survivals associated with TUSC3 deficiency (GSE31210). c Luciferase reporter assays with TUSC3 3’UTR in miR-224 or -520c-overexpressing HEK293 cells. The pGL3-TUSC3-3’UTR plasmid or the mutants harboring deleted target sequences for miR-224 or miR-520c were co-transfected with miR-224 (left panels) or miR-520c (right panels). The relative values were obtained by normalizing to Renilla luciferase values. Bars represent means ± SD (n = 4) and the p-values were determined by two-tailed student t-test (*p < 0.001). d Decreased TUSC3 mRNA expression in miR-224 or miR-520c overexpressing NSCLC. Bars indicate means ±SD (n = 3) and p-values were addressed by two-tailed student t-test (*p < 0.01, **p < 0.05). e Western blot analyses showing that miR-224 and miR-520c suppressed the endogenous TUSC3 protein in A549 (left panel) and H460 (right panel) cells. Pre-miR-224/-520C (left) or anti-miR-224/-520c (right) were ectopically expressed in A549 (left) or H460 (right) cells. f In situ hybridization showing the enhanced miR-224 and miR-520c expression in lymph node metastasized lung tissues compared to their corresponding primary tumors. Total numbers of cases were summarized in Supplementary Fig. 1e. g, h Co-expression analyses of TUSC3 and miR- 224 (g) and miR-520c (h). The images in the miR-224 and miR-520c sections show a lung adenocarcinoma after co-expression of TUSC3 (fluorescence red and RGB brown) with miR-224 (g, fluorescence blue and RGB blue) or miR-520c (h, fluorescence blue and RGB blue). The chi-squared test statistic was generated and the null hypotheses that the expression of TUSC-3, miR-224, and miR-520 was equal in primary versus the metastatic tumors or that the expression of TUSC3 and a given miRNA was equal in the primary and metastatic tumors tested using 2 degrees of freedom. The scale bars indicate 150 µm (a), 100 µm (b), and 200 µm (g, h), respectively

Article Snippet: TUSC3 shRNAs and recombinant lentivirus containing a pool of three target-specific shRNAs of TUSC3 were purchased from Santa Cruz Biotechnology, Inc. For the generation of TUSC3 knock down (KD) cells, H460 or A549 cells were transfected with TUSC3 shRNA plasmids.

Techniques: Expressing, Immunohistochemistry, Luciferase, Plasmid Preparation, Transfection, Two Tailed Test, Western Blot, In Situ Hybridization, Generated

Journal: Nature communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC.

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: Fig. 4 TUSC3 deficiency enhances HRD1-dependent pro-metastatic property by enhancing ERAD activity. a Enhanced A1AT-NHK-ddVenus GFP accumulation in A549 TUSC3KO cells. The A1AT-NHK-ddVenus stable A549 control (Ctrl) or TUSC3 KO cells were generated by transduction with pLL-Lenti-A1AT-NHK-ddVenus virus. The protein accumulation was monitored after incubating the cells with MG132 (20 uM) for 16 h. The accumulation was quantified by FACS analyses (lower panel) or Western blot analyses using anti-GFP antibody (upper panel). b Decreased accumulation of A1AT- NHK-ddVenus by miR-224 and -520c suppression. Bars indicate means ±SD (n = 3) and p-values were obtained by two-tailed student t-test (a and b, *p < 0.001, **p < 0.02). c Co-immunoprecipitation assays with TUSC3 and HRD1 proteins. pcDNA-TUSC3-V5-His or its mutants (TUSC3-CCSS and Del- TUSC3) was co-transfected with pCMV6-HRD1-Flag in HEK293 cells. d The changed affinity of HRD1 protein to its substrates by TUSC3. The endogenous HRD1 protein was precipitated with anti-HRD1 antibody in H460 control, TUSC3 KO or TUSC3-reconstituting TUSC3 KO cells. e In vitro competition assay with p53 and TUSC3 proteins in HEK293 cells. f In vivo ubiquitination assay of p53 protein showing enhanced p53 ubiquitination in response to TUSC3 downmodulation. g Decreased p53 protein in H460 TUSC3 KO cell and rescued in HRD1/TUSC3 DKO cells. The indicated cells were exposed by 5.0 ug/ml of TM for 1 h and subject to Western blot analysis. h Rescued p53 protein in TUSC3 or TUSC3-CCSS mutant expressing H460 TUSC3 KO cells. i Re-suppressed colonization effect of TUSC3KO cells upon TP53 reconstitution. Tail-vein injection was performed using the indicated cells, and subsequently the lung tissues were harvested after 4 weeks. p-values were obtained by student t-test (*p = 0.00031, **p < 0.038). j Co-expression analyses between TUSC3 and p53 (upper panels) or IRE1α (lower panels) in lung cancer patient samples. The p53 and IRE1α are shown in fluorescence green whereas TUSC3 is shown in fluorescence red. Scale bar indicates 150 µm. k GSEA plots indicating gene sets suppressed by TP53 were enriched in TUSC3KO cells either DMSO or TM treatment compared to the controls

Article Snippet: TUSC3 shRNAs and recombinant lentivirus containing a pool of three target-specific shRNAs of TUSC3 were purchased from Santa Cruz Biotechnology, Inc. For the generation of TUSC3 knock down (KD) cells, H460 or A549 cells were transfected with TUSC3 shRNA plasmids.

Techniques: Activity Assay, Control, Generated, Transduction, Virus, Western Blot, Two Tailed Test, Immunoprecipitation, Transfection, In Vitro, Competitive Binding Assay, In Vivo, Ubiquitin Proteomics, Mutagenesis, Expressing, Injection

Journal: Nature communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC.

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: Fig. 5 Metastatic suppressor, NM23H1/2 is regulated by TUSC3 through p53 regulation. a The reduction of NM23H1/2 protein in TUSC3 KO cells in response to ER stress induction. The cells were incubated by 5.0 ug of TM for 9 h and subsequently harvested for Western blot analysis. b Decreased NM23H1 protein in p53 deficient cells. The H460 cells were transfected with siTP53 siRNAs for 48 h, and the expression of p53 and NM23H1/2 proteins was measured by Western blot analysis. c Restored NM23H1/2 proteins by p53 protein accumulation in A549 TUSC3 KO cells. The cells were incubated with Ntulin3a for 24 h, and Western blot analysis was performed to measure the expression of NM23H1/2. d HRD1-regulated NM23H1/2 protein in A549 TUSC3 KD cells. The A549 TUSC3 KD cells were transfected with scrambled or siHRD1 siRNAs. After 48 h, the cells were exposed to TM (5 ug/ml) for 6 h followed by Western blot analysis using indicated antibodies. e Accumulated NM23H1/2 proteins upon miR-224/-520c downregulation. The miRNA KD cells were transfected by scrambled or siTUSC3 siRNAs for 48 h, and the NM23H1/2 protein was measured by Western blot analysis. f The reduced migration and invasion of A549 TUSC3 KD cells in response to NM23H1 reconstitution. H460 TUSC3 KD cells were transfected by pCMV6-NM23H1 plasmid for 24 h. Subsequently, the cells were harvested and subject to the chambers of migration or invasion assays. Error bars indicate means ± SD (n = 3) and the p-values were calculated by two-tailed student t-test (*p < 0.03). g Restored expression of NM23H1 decreased colonization ability of H460 TUSC3KO cells. The cells overexpressing NM23H1 generated by the transduction of lent-NM23H1 virus. 1 × 106 of NM23H1 overexpressing TUSC3KO cells or control KO cells was intravenously injected into four NSG mice. The tumor area was obtained by Image J software by measuring the field of view in lung occupied by cancer. The region for the cancer was expressed as percentage. p-value was calculated by unpaired student t-test (*p < 0.001)

Article Snippet: TUSC3 shRNAs and recombinant lentivirus containing a pool of three target-specific shRNAs of TUSC3 were purchased from Santa Cruz Biotechnology, Inc. For the generation of TUSC3 knock down (KD) cells, H460 or A549 cells were transfected with TUSC3 shRNA plasmids.

Techniques: Incubation, Western Blot, Transfection, Expressing, Migration, Plasmid Preparation, Two Tailed Test, Generated, Transduction, Virus, Control, Injection, Software

Journal: Nature communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC.

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: Fig. 6 The schematic diagram showing the working hypothesis by which miR-224/-520c-induced TUSC3 suppression enhanced metastatic potential of NSCLC through the alteration of UPRs and HRD1-dependent ERAD

Article Snippet: TUSC3 shRNAs and recombinant lentivirus containing a pool of three target-specific shRNAs of TUSC3 were purchased from Santa Cruz Biotechnology, Inc. For the generation of TUSC3 knock down (KD) cells, H460 or A549 cells were transfected with TUSC3 shRNA plasmids.

Techniques:

Journal: Nature Communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: miR-224 and -520c are responsible for TUSC3 deficiency in metastasized lung cancer patient tissues. a Expression analyses of TUSC3 by immunohistochemistry (IHC) showing suppressed expression in matched metastatic samples. 50 primary and their matched lymph node metastasized lung tumor tissues were analyzed. The summarized expressional scores are shown in Supplementary Fig. . P -value was obtained by Pearson’s chi-squared test. b PrognoScan based Kaplan Meier plot showing decreased patient survivals associated with TUSC3 deficiency (GSE31210). c Luciferase reporter assays with TUSC3 3’UTR in miR-224 or -520c-overexpressing HEK293 cells. The pGL3-TUSC3-3’UTR plasmid or the mutants harboring deleted target sequences for miR-224 or miR-520c were co-transfected with miR-224 (left panels) or miR-520c (right panels). The relative values were obtained by normalizing to Renilla luciferase values. Bars represent means ± SD ( n = 4) and the p -values were determined by two-tailed student t -test (* p < 0.001). d Decreased TUSC3 mRNA expression in miR-224 or miR-520c overexpressing NSCLC. Bars indicate means ±SD ( n = 3) and p -values were addressed by two-tailed student t -test (* p < 0.01, ** p < 0.05). e Western blot analyses showing that miR-224 and miR-520c suppressed the endogenous TUSC3 protein in A549 (left panel) and H460 (right panel) cells. Pre-miR-224/-520C (left) or anti-miR-224/-520c (right) were ectopically expressed in A549 (left) or H460 (right) cells. f In situ hybridization showing the enhanced miR-224 and miR-520c expression in lymph node metastasized lung tissues compared to their corresponding primary tumors. Total numbers of cases were summarized in Supplementary Fig. . g , h Co-expression analyses of TUSC3 and miR-224 ( g ) and miR-520c ( h ). The images in the miR-224 and miR-520c sections show a lung adenocarcinoma after co-expression of TUSC3 (fluorescence red and RGB brown) with miR-224 ( g , fluorescence blue and RGB blue) or miR-520c ( h , fluorescence blue and RGB blue). The chi-squared test statistic was generated and the null hypotheses that the expression of TUSC-3, miR-224, and miR-520 was equal in primary versus the metastatic tumors or that the expression of TUSC3 and a given miRNA was equal in the primary and metastatic tumors tested using 2 degrees of freedom. The scale bars indicate 150 µm ( a ), 100 µm ( b ), and 200 µm ( g , h ), respectively

Article Snippet: TUSC3 shRNAs and recombinant lentivirus containing a pool of three target-specific shRNAs of TUSC3 were purchased from Santa Cruz Biotechnology, Inc. For the generation of TUSC3 knock down (KD) cells, H460 or A549 cells were transfected with TUSC3 shRNA plasmids.

Techniques: Expressing, Immunohistochemistry, Luciferase, Plasmid Preparation, Transfection, Two Tailed Test, Western Blot, In Situ Hybridization, Fluorescence, Generated

Journal: Nature Communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: TUSC3 deficiency enhances metastatic potential of NCLC in vitro and in vivo. a Increased migration and invasion of TUSC3 knockdown (KD) cells. Two different TUSC3 KD cells (sh#2A1 and sh#2C1) were transfected with empty or mutant TUSC3 (smTUSC3) harboring three silence mutations corresponding to shTUSC3 shRNA target sequences. After 48 h, the cells were introduced into migration (left panels) and invasion (right panels) chambers. Bars represent means ± SD ( n = 3) and p -value were calculated by student t -test (* p < 0.05 and ** p < 0.01). b Differential expression of 84 known metastasis-related genes in H460 TUSC3KD cells (sh#2C1). The lists of genes showing differential expression is shown in Supplementary Dataset . c Gene set enrichment analysis (GSEA) for metastasis, migration and cell adhesion-related gene sets in A549 control or TUSC3KO cells with DMSO (left panels) or Tunicamycin (TM, 3ug/ml; right panels) for 16 h. The lists of gene sets are shown in Supplementary Dataset ( d ) IVIS in vivo images showing enhanced colonization ability of the A549 TUSC3 KD (sh#3C1). The cells (1 × 10 6 ) were intravenously injected into nude mice and the bioluminescence was obtained in weekly basis from 3 weeks of injection. Bars indicate means ± SD ( n = 3) and the p -values were addressed by two-tailed student t -test. e Anchorage independent cell growth is modulated by miRNA-dependent TUSC3 downregulation. Anti-miR-224 and -miR-520c were co-transfected with either scrambled or TUSC3 siRNAs for 48 h. After that, the cells were placed into an Agar Matrix Layer for 7 days. The numbers of colonies were quantified with a standard MTS assay. The changed expression of endogenous TUSC3 was analyzed by Western blot analysis (upper panel) or qRT-PCR analysis (Supplementary Fig. ). Bars indicate means ± SD ( n = 4) and p -value were calculated by two-tailed student t -test (* p < 0.005 and ** p < 0.001). f Lung metastasis of subcutaneously xenografted mice showing enhanced lung metastasis. The 5 × 10 5 of the indicated cells were subcutaneously injected into 4 nude mice, respectively. After 4 weeks, the lung tissues were harvested and dissected in 50-micron depth followed by H&E stain. Tumor areas were measured by Image J software. p -value were calculated by two tailed student t -test (* p < 0.001, ** p < 0.005)

Article Snippet: TUSC3 shRNAs and recombinant lentivirus containing a pool of three target-specific shRNAs of TUSC3 were purchased from Santa Cruz Biotechnology, Inc. For the generation of TUSC3 knock down (KD) cells, H460 or A549 cells were transfected with TUSC3 shRNA plasmids.

Techniques: In Vitro, In Vivo, Migration, Knockdown, Transfection, Mutagenesis, shRNA, Quantitative Proteomics, Control, Injection, Two Tailed Test, MTS Assay, Expressing, Western Blot, Quantitative RT-PCR, Staining, Software

Journal: Nature Communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: TUSC3 deficiency selectively modulates Unfolded Protein Responses. a GSEA plots showing upregulation of the genes involved in Unfolded Protein Reponses. b Western blot analyses showing weakened IRE1α-XBP1 and PERK-EIF2α pathways in A549 TUSC3 KO cells whereas enhanced in TUSC3/HRD1 DKO cells in response to ER stress induction. c Subcellular fractionation assay showing enhanced chromatin-bound ATF6α in A549 TUSC3 KO and HRD1/TUSC3 DKO cells. The indicated cells were stimulated by 1.0 uM of TG for 6 or 9 h, respectively. Anti-Calnexin or anti-PARP1 antibody was used for ER/Golgi or Nuclear fraction markers, respectively. d Increased ATF6α-dependent ER heat-shock proteins in TUSC3 deficient cells. The cells were transfected by scrambled or siATF6α siRNAs for 48 h followed by exposing to DMSO or TM (3 ug/ml, 16 h). Western blot analysis shows elevated expression of GRP78 and GRP94 in A549 TUSC3 KO cells. A mitochondrial heat-shock protein, GRP75 was used for negative control. e Schematic diagram showing primary structure of TUSC3 protein and its mutants (TUSC3 CCSS). CSVC indicates the amino acids in C-X-X-C motif. f Rescued IRE1α and PERK expression by reconstitution of TUSC3 or its CCSS mutant. g Restored nuclear localization of the ATF6α protein by the reconstitution of TUSC3 but not by CCSS mutant in A549 TUSC3 KO cells. h Rescued the colonization ability of H460 TUSC3KO cells by suppressing ATF6α expression. 1 × 10 6 of the control cells or ATF6α knock-downed TUSC3 KO ells was intravenously injected into four NOD scid gamma mice. p -value was calculated by unpaired student t -test (* p = 0.002). The data for the tumor area from the control cells-injected mice are shared with Fig. g. i Co-expression analysis of ATF6α with TUSC3 protein showing inverse correlation between TUSC3 and ATF6α activation in lung cancer patient samples. p -value was obtained by Chi square analysis. The scale bar is shown as 150 µm. j Rescued colonization ability of TUSC3/HRD1 DKO cells. The tumor area was calculated as the total area of lung occupied by cancer is field of view using Image J software. The region for the cancer was expressed as percentage . p -value was calculated by unpaired student t -test (* p < 0.001)

Article Snippet: TUSC3 shRNAs and recombinant lentivirus containing a pool of three target-specific shRNAs of TUSC3 were purchased from Santa Cruz Biotechnology, Inc. For the generation of TUSC3 knock down (KD) cells, H460 or A549 cells were transfected with TUSC3 shRNA plasmids.

Techniques: Western Blot, Fractionation, Transfection, Expressing, Negative Control, Mutagenesis, Control, Injection, Activation Assay, Software

Journal: Nature Communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: Metastatic suppressor, NM23H1/2 is regulated by TUSC3 through p53 regulation. a The reduction of NM23H1/2 protein in TUSC3 KO cells in response to ER stress induction. The cells were incubated by 5.0 ug of TM for 9 h and subsequently harvested for Western blot analysis. b Decreased NM23H1 protein in p53 deficient cells. The H460 cells were transfected with siTP53 siRNAs for 48 h, and the expression of p53 and NM23H1/2 proteins was measured by Western blot analysis. c Restored NM23H1/2 proteins by p53 protein accumulation in A549 TUSC3 KO cells. The cells were incubated with Ntulin3a for 24 h, and Western blot analysis was performed to measure the expression of NM23H1/2. d HRD1-regulated NM23H1/2 protein in A549 TUSC3 KD cells. The A549 TUSC3 KD cells were transfected with scrambled or siHRD1 siRNAs. After 48 h, the cells were exposed to TM (5 ug/ml) for 6 h followed by Western blot analysis using indicated antibodies. e Accumulated NM23H1/2 proteins upon miR-224/-520c downregulation. The miRNA KD cells were transfected by scrambled or siTUSC3 siRNAs for 48 h, and the NM23H1/2 protein was measured by Western blot analysis. f The reduced migration and invasion of A549 TUSC3 KD cells in response to NM23H1 reconstitution. H460 TUSC3 KD cells were transfected by pCMV6-NM23H1 plasmid for 24 h. Subsequently, the cells were harvested and subject to the chambers of migration or invasion assays. Error bars indicate means ± SD ( n = 3) and the p -values were calculated by two-tailed student t-test (* p < 0.03). g Restored expression of NM23H1 decreased colonization ability of H460 TUSC3KO cells. The cells overexpressing NM23H1 generated by the transduction of lent-NM23H1 virus. 1 × 10 6 of NM23H1 overexpressing TUSC3KO cells or control KO cells was intravenously injected into four NSG mice. The tumor area was obtained by Image J software by measuring the field of view in lung occupied by cancer. The region for the cancer was expressed as percentage. p -value was calculated by unpaired student t -test (* p < 0.001)

Article Snippet: TUSC3 shRNAs and recombinant lentivirus containing a pool of three target-specific shRNAs of TUSC3 were purchased from Santa Cruz Biotechnology, Inc. For the generation of TUSC3 knock down (KD) cells, H460 or A549 cells were transfected with TUSC3 shRNA plasmids.

Techniques: Incubation, Western Blot, Transfection, Expressing, Migration, Plasmid Preparation, Two Tailed Test, Generated, Transduction, Virus, Control, Injection, Software

Journal: Nature Communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: TUSC3 deficiency enhances HRD1-dependent pro-metastatic property by enhancing ERAD activity. a Enhanced A1AT-NHK-ddVenus GFP accumulation in A549 TUSC3KO cells. The A1AT-NHK-ddVenus stable A549 control (Ctrl) or TUSC3 KO cells were generated by transduction with pLL-Lenti-A1AT-NHK-ddVenus virus. The protein accumulation was monitored after incubating the cells with MG132 (20 uM) for 16 h. The accumulation was quantified by FACS analyses (lower panel) or Western blot analyses using anti-GFP antibody (upper panel). b Decreased accumulation of A1AT-NHK-ddVenus by miR-224 and -520c suppression. Bars indicate means ±SD ( n = 3) and p -values were obtained by two-tailed student t -test ( a and b , * p < 0.001, ** p < 0.02). c Co-immunoprecipitation assays with TUSC3 and HRD1 proteins. pcDNA-TUSC3-V5-His or its mutants (TUSC3-CCSS and Del-TUSC3) was co-transfected with pCMV6-HRD1-Flag in HEK293 cells. d The changed affinity of HRD1 protein to its substrates by TUSC3. The endogenous HRD1 protein was precipitated with anti-HRD1 antibody in H460 control, TUSC3 KO or TUSC3-reconstituting TUSC3 KO cells. e In vitro competition assay with p53 and TUSC3 proteins in HEK293 cells. f In vivo ubiquitination assay of p53 protein showing enhanced p53 ubiquitination in response to TUSC3 downmodulation. g Decreased p53 protein in H460 TUSC3 KO cell and rescued in HRD1/TUSC3 DKO cells. The indicated cells were exposed by 5.0 ug/ml of TM for 1 h and subject to Western blot analysis. h Rescued p53 protein in TUSC3 or TUSC3-CCSS mutant expressing H460 TUSC3 KO cells. i Re-suppressed colonization effect of TUSC3KO cells upon TP53 reconstitution. Tail-vein injection was performed using the indicated cells, and subsequently the lung tissues were harvested after 4 weeks. p -values were obtained by student t-test (* p = 0.00031, ** p < 0.038). j Co-expression analyses between TUSC3 and p53 (upper panels) or IRE1α (lower panels) in lung cancer patient samples. The p53 and IRE1α are shown in fluorescence green whereas TUSC3 is shown in fluorescence red. Scale bar indicates 150 µm. k GSEA plots indicating gene sets suppressed by TP53 were enriched in TUSC3KO cells either DMSO or TM treatment compared to the controls

Article Snippet: TUSC3 shRNAs and recombinant lentivirus containing a pool of three target-specific shRNAs of TUSC3 were purchased from Santa Cruz Biotechnology, Inc. For the generation of TUSC3 knock down (KD) cells, H460 or A549 cells were transfected with TUSC3 shRNA plasmids.

Techniques: Activity Assay, Control, Generated, Transduction, Virus, Western Blot, Two Tailed Test, Immunoprecipitation, Transfection, In Vitro, Competitive Binding Assay, In Vivo, Ubiquitin Proteomics, Mutagenesis, Expressing, Injection, Fluorescence

Journal: Nature Communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: The schematic diagram showing the working hypothesis by which miR-224/-520c-induced TUSC3 suppression enhanced metastatic potential of NSCLC through the alteration of UPRs and HRD1-dependent ERAD

Article Snippet: TUSC3 shRNAs and recombinant lentivirus containing a pool of three target-specific shRNAs of TUSC3 were purchased from Santa Cruz Biotechnology, Inc. For the generation of TUSC3 knock down (KD) cells, H460 or A549 cells were transfected with TUSC3 shRNA plasmids.

Techniques:

Journal: Nature Communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: Metastatic suppressor, NM23H1/2 is regulated by TUSC3 through p53 regulation. a The reduction of NM23H1/2 protein in TUSC3 KO cells in response to ER stress induction. The cells were incubated by 5.0 ug of TM for 9 h and subsequently harvested for Western blot analysis. b Decreased NM23H1 protein in p53 deficient cells. The H460 cells were transfected with siTP53 siRNAs for 48 h, and the expression of p53 and NM23H1/2 proteins was measured by Western blot analysis. c Restored NM23H1/2 proteins by p53 protein accumulation in A549 TUSC3 KO cells. The cells were incubated with Ntulin3a for 24 h, and Western blot analysis was performed to measure the expression of NM23H1/2. d HRD1-regulated NM23H1/2 protein in A549 TUSC3 KD cells. The A549 TUSC3 KD cells were transfected with scrambled or siHRD1 siRNAs. After 48 h, the cells were exposed to TM (5 ug/ml) for 6 h followed by Western blot analysis using indicated antibodies. e Accumulated NM23H1/2 proteins upon miR-224/-520c downregulation. The miRNA KD cells were transfected by scrambled or siTUSC3 siRNAs for 48 h, and the NM23H1/2 protein was measured by Western blot analysis. f The reduced migration and invasion of A549 TUSC3 KD cells in response to NM23H1 reconstitution. H460 TUSC3 KD cells were transfected by pCMV6-NM23H1 plasmid for 24 h. Subsequently, the cells were harvested and subject to the chambers of migration or invasion assays. Error bars indicate means ± SD ( n = 3) and the p -values were calculated by two-tailed student t-test (* p < 0.03). g Restored expression of NM23H1 decreased colonization ability of H460 TUSC3KO cells. The cells overexpressing NM23H1 generated by the transduction of lent-NM23H1 virus. 1 × 10 6 of NM23H1 overexpressing TUSC3KO cells or control KO cells was intravenously injected into four NSG mice. The tumor area was obtained by Image J software by measuring the field of view in lung occupied by cancer. The region for the cancer was expressed as percentage. p -value was calculated by unpaired student t -test (* p < 0.001)

Article Snippet: pCMV6-TUSC3, pCMV6-NM23H1, and pCMV6-Synoviolin (HRD1)-Flag plasmids were purchased from Origene. pcDNA-IRE1α-GFP, pcDNA-TP53-GFP, pcDNA-Ubiquitin-HA plasmids were purchased from Addgene Company.

Techniques: Incubation, Western Blot, Transfection, Expressing, Migration, Plasmid Preparation, Two Tailed Test, Generated, Transduction, Injection, Software

Journal: Nature Communications

Article Title: miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC

doi: 10.1038/s41467-018-07561-8

Figure Lengend Snippet: TUSC3 deficiency enhances HRD1-dependent pro-metastatic property by enhancing ERAD activity. a Enhanced A1AT-NHK-ddVenus GFP accumulation in A549 TUSC3KO cells. The A1AT-NHK-ddVenus stable A549 control (Ctrl) or TUSC3 KO cells were generated by transduction with pLL-Lenti-A1AT-NHK-ddVenus virus. The protein accumulation was monitored after incubating the cells with MG132 (20 uM) for 16 h. The accumulation was quantified by FACS analyses (lower panel) or Western blot analyses using anti-GFP antibody (upper panel). b Decreased accumulation of A1AT-NHK-ddVenus by miR-224 and -520c suppression. Bars indicate means ±SD ( n = 3) and p -values were obtained by two-tailed student t -test ( a and b , * p < 0.001, ** p < 0.02). c Co-immunoprecipitation assays with TUSC3 and HRD1 proteins. pcDNA-TUSC3-V5-His or its mutants (TUSC3-CCSS and Del-TUSC3) was co-transfected with pCMV6-HRD1-Flag in HEK293 cells. d The changed affinity of HRD1 protein to its substrates by TUSC3. The endogenous HRD1 protein was precipitated with anti-HRD1 antibody in H460 control, TUSC3 KO or TUSC3-reconstituting TUSC3 KO cells. e In vitro competition assay with p53 and TUSC3 proteins in HEK293 cells. f In vivo ubiquitination assay of p53 protein showing enhanced p53 ubiquitination in response to TUSC3 downmodulation. g Decreased p53 protein in H460 TUSC3 KO cell and rescued in HRD1/TUSC3 DKO cells. The indicated cells were exposed by 5.0 ug/ml of TM for 1 h and subject to Western blot analysis. h Rescued p53 protein in TUSC3 or TUSC3-CCSS mutant expressing H460 TUSC3 KO cells. i Re-suppressed colonization effect of TUSC3KO cells upon TP53 reconstitution. Tail-vein injection was performed using the indicated cells, and subsequently the lung tissues were harvested after 4 weeks. p -values were obtained by student t-test (* p = 0.00031, ** p < 0.038). j Co-expression analyses between TUSC3 and p53 (upper panels) or IRE1α (lower panels) in lung cancer patient samples. The p53 and IRE1α are shown in fluorescence green whereas TUSC3 is shown in fluorescence red. Scale bar indicates 150 µm. k GSEA plots indicating gene sets suppressed by TP53 were enriched in TUSC3KO cells either DMSO or TM treatment compared to the controls

Article Snippet: pCMV6-TUSC3, pCMV6-NM23H1, and pCMV6-Synoviolin (HRD1)-Flag plasmids were purchased from Origene. pcDNA-IRE1α-GFP, pcDNA-TP53-GFP, pcDNA-Ubiquitin-HA plasmids were purchased from Addgene Company.

Techniques: Activity Assay, Generated, Transduction, Western Blot, Two Tailed Test, Immunoprecipitation, Transfection, In Vitro, Competitive Binding Assay, In Vivo, Ubiquitin Assay, Mutagenesis, Expressing, Injection, Fluorescence