cry2 mcherry nls  (Addgene inc)

Journal: Cell Death & Disease

Article Title: IGF1-mediated HOXA13 overexpression promotes colorectal cancer metastasis through upregulating ACLY and IGF1R

doi: 10.1038/s41419-021-03833-2

Figure Lengend Snippet: A (Left) Real-time PCR analysis of HOXA13 expression in 120 pairs of CRC and adjacent nontumor specimens, and normal colorectal epithelial specimens ( n = 20). (Middle) The mRNA expression of HOXA13 in CRC specimens from patients with recurrence ( n = 52) or without recurrence ( n = 68). (Right) The mRNA expression of HOXA13 in CRC specimens from patients with metastasis ( n = 56) or without metastasis ( n = 64). B The protein and mRNA expression of HOXA13 were detected by IHC staining (left) and real-time PCR (right) analyses in 20 pairs of normal colorectal epithelial tissues, primary colon cancer, and paired metastatic CRC tissues. The scale bars represent 250 μm (low magnification) and 50 μm (high magnification). C (Left)The representative image of IHC staining of HOXA13 in CRC and adjacent nontumor tissues microarray. The scale bars represent 250 μm (low magnification) and 50 μm (high magnification). (Right) The IHC score of HOXA13 in CRC and adjacent nontumor samples in two independent cohorts. D Kaplan–Meier analysis of the relationships of HOXA13 expression and overall survival times or the recurrence rates in two independent CRC cohorts. E Real-time PCR (upper) and western blotting (lower) analyses of the mRNA and protein expression of HOXA13 in CRC cell lines. F Western blotting (lower) analysis of HOXA13 protein expression in SW480 and SW620 cells after lentivirus transfection. G Cell migratory and invasive capabilities in the indicated CRC cell lines by Transwell assay. H In vivo liver metastatic assays. Four stable cell lines were injected into the spleens of nude mice ( n = 10 mice per group). Representative bioluminescent imaging, intensity of bioluminescence signals, number of metastatic liver nodules, overall survival times, liver tissues’ HE staining in each group ( n = 10), and the incidence of liver metastasis were shown. The scale bars represent 1 mm (low magnification) and 100 μm (high magnification). I In vivo lung metastatic assays. Four stable cell lines were injected into the tail veins of nude mice ( n = 10 mice per group). Representative bioluminescent imaging, intensity of bioluminescence signals, number of metastatic lung nodules, overall survival times, lung tissues’ HE staining in each group and the incidence of lung metastasis were shown. The scale bars represent 1 mm (low magnification) and 100 μm (high magnification). * P < 0.05 ** P < 0.01. Data are displayed as the mean ± s.d.

Article Snippet: Lentiviral vectors encoding the human HOXA13, ACLY, and IGF1R genes were constructed in pLV-puro or pLV-neo (Addgene) and designated as LV-HOXA13, LV-ACLY, and LV-IGF1R.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunohistochemistry, Microarray, Western Blot, Transfection, Transwell Assay, In Vivo, Stable Transfection, Injection, Imaging, Staining

Journal: Cell Death & Disease

Article Title: IGF1-mediated HOXA13 overexpression promotes colorectal cancer metastasis through upregulating ACLY and IGF1R

doi: 10.1038/s41419-021-03833-2

Figure Lengend Snippet: Correlation between HOXA13 expression and clinicopathological characteristics of CRC in two independent cohorts of human CRC tissues.

Article Snippet: Lentiviral vectors encoding the human HOXA13, ACLY, and IGF1R genes were constructed in pLV-puro or pLV-neo (Addgene) and designated as LV-HOXA13, LV-ACLY, and LV-IGF1R.

Techniques: Expressing, Significance Assay

Journal: Cell Death & Disease

Article Title: IGF1-mediated HOXA13 overexpression promotes colorectal cancer metastasis through upregulating ACLY and IGF1R

doi: 10.1038/s41419-021-03833-2

Figure Lengend Snippet: Univariate and multivariate analysis of factors associated with survival and recurrence in two independent cohorts of human CRC.

Article Snippet: Lentiviral vectors encoding the human HOXA13, ACLY, and IGF1R genes were constructed in pLV-puro or pLV-neo (Addgene) and designated as LV-HOXA13, LV-ACLY, and LV-IGF1R.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: IGF1-mediated HOXA13 overexpression promotes colorectal cancer metastasis through upregulating ACLY and IGF1R

doi: 10.1038/s41419-021-03833-2

Figure Lengend Snippet: A Venn diagram presenting the overlap of upregulated genes in both SW480-HOXA13 and DLD-1-HOXA13 cell lines (fold change >2.0). B (Left) Western blotting analysis of ACLY and IGF1R expression in the corresponding CRC cell lines after lentivirus transfection. (Right) Real-time PCR analysis of ACLY and IGF1R expression in the corresponding CRC cell lines after lentivirus transfection. C Luciferase reporter assay displayed the promoter activities of ACLY and IGF1R after SW480 cells were co-transfected with pCMV-HOXA13 and ACLY or IGF1R promoter constructs. D , E HOXA13-responsive regions in the ACLY and IGF1R promoter were analyzed by deletion and selective mutation. Serially truncated and mutated ACLY or IGF1R promoter constructs were co-transfected with pCMV-HOXA13, and relative luciferase activities were determined. Left-component was the schematic constructs, and right-component presented the relative luciferase activities. F , G ChIP assays revealed the direct binding of HOXA13 to the ACLY ( F ) or IGF1R ( G ) promoter in SW480-HOXA13 cells and the enriched binding sites of endogenous HOXA13 to the ACLY or IGF1R promoter in CRC tissues. The amounts of immunoprecipitated products were examined by real-time PCR. H Western blotting analysis presenting ACLY and IGF1R expression in SW480 and SW620 cells after lentivirus transfection. I Transwell assays showed that downregulation of ACLY and IGF1R reduced the migration and invasion abilities of SW480-HOXA13 cells, while upregulation of ACLY and IGF1R increased the migration and invasion abilities of SW620-shHOXA13 cells. J In vivo liver metastatic assays. Four stable cell lines were injected into the spleens of nude mice ( n = 10 mice per group). Representative bioluminescent imaging, incidence of liver metastasis, intensity of bioluminescence signals, overall survival times, liver tissues’ HE staining in each group, and the number of metastatic liver nodules were shown. The scale bars represent 1 mm (low magnification) and 100μm (high magnification). K In vivo lung metastatic assays. Four stable cell lines were injected into the tail veins of nude mice ( n = 10 mice per group). Representative bioluminescent imaging, incidence of lung metastasis, intensity of bioluminescence signals, overall survival times, lung tissues’ HE staining in each group and the number of metastatic lung nodules were shown. The scale bars represent 1 mm (low magnification) and 100μm (high magnification). * P < 0.05, ** P < 0.01. Data are displayed as the mean ± s.d.

Article Snippet: Lentiviral vectors encoding the human HOXA13, ACLY, and IGF1R genes were constructed in pLV-puro or pLV-neo (Addgene) and designated as LV-HOXA13, LV-ACLY, and LV-IGF1R.

Techniques: Western Blot, Expressing, Transfection, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Construct, Mutagenesis, Binding Assay, Immunoprecipitation, Migration, In Vivo, Stable Transfection, Injection, Imaging, Staining

Journal: Cell Death & Disease

Article Title: IGF1-mediated HOXA13 overexpression promotes colorectal cancer metastasis through upregulating ACLY and IGF1R

doi: 10.1038/s41419-021-03833-2

Figure Lengend Snippet: A SW480 and DLD-1 cells were administrated with gradient concentrations of IGF1 for 24 h, and then the mRNA and protein levels of HOXA13 and p-IGF1R were examined by real-time PCR and western blotting. B Relative luciferase activities were detected in SW480 cells and DLD-1 cells, which were transfected with plasmid constructs containing HOXA13 promoter after IGF1 treatment (50 ng/mL, 24 h). C Deletion and selective mutation analysis showed that two HIF1α binding regions within the HOXA13 promoter were responsible for IGF1-induced HOXA13 promoter transactivation. D Knockdown of HIF1α through HIF1α siRNA reduced IGF1-mediated HOXA13 expression, which were detected by luciferase reporter assay (upper), real-time PCR (upper), and western blotting analyses (lower). E HIF1α inhibitor YC-1 reduced IGF1-mediated HOXA13 expression. The promoter activity (left), mRNA expression (middle), and protein expression (right) of HOXA13 were examined when SW480 cells were treated with or without IGF1 (50 ng/mL, 24 h) in the presence of YC-1. F The protein expression of HOXA13, phosphorylated and total Akt and ERK1/2 were detected by western blotting when SW480 cells were pretreated with inhibitors of ERK1/2 (SCH772984) or PI3K (LY294002), and then were treated with IGF1 (50 ng/mL, 24 h) or not. G The ChIP assay showed the direct binding abilities of HIF1α to the HOXA13 promoter induced by IGF1, and PI3K inhibitor reduced the binding abilities of HIF1α to the HOXA13 promoter. H The expression of IGF1 and HOXA13 in CRC and adjacent nontumor tissues by IHC analysis. I The correlation analysis between IGF1 and HOXA13 in two independent cohorts. J Kaplan–Meier’s analysis showed the correlation between HOXA13/IGF1 expression and overall survival or recurrence in two independent cohorts. * P < 0.05, ** P < 0.01. Data are displayed as the mean ± s.d.

Article Snippet: Lentiviral vectors encoding the human HOXA13, ACLY, and IGF1R genes were constructed in pLV-puro or pLV-neo (Addgene) and designated as LV-HOXA13, LV-ACLY, and LV-IGF1R.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Transfection, Plasmid Preparation, Construct, Mutagenesis, Binding Assay, Knockdown, Expressing, Reporter Assay, Activity Assay

Journal: Cell Death & Disease

Article Title: IGF1-mediated HOXA13 overexpression promotes colorectal cancer metastasis through upregulating ACLY and IGF1R

doi: 10.1038/s41419-021-03833-2

Figure Lengend Snippet: A SW480 cells were transfected with LV-shcontrol or LV-shHOXA13 lentiviral vectors and then treated with or without IGF1 (50 ng/mL, 24 h). Next, the HOXA13 protein expression was analyzed by western blotting. B Transwell assays demonstrated that IGF1 treatment increased the migratory and invasive abilities of SW480 cells, while HOXA13 knockdown reduced these capabilities of SW480 cells. C SW480 cells were transfected with lentiviral vectors to construct IGF1-overexpressing SW480 cells (SW480-IGF1), and HOXA13 expression was further knockdown via lentiviral transfection in SW480-IGF1 cells. IGF1 and HOXA13 expression in the indicated cell lines was examined by western blotting. D Transwell assays revealed that overexpression of IGF1 enhanced the migratory and invasive capabilities of SW480 cells, while HOXA13 knockdown impaired the enhanced migratory and invasive abilities of SW480-IGF1 cells. E In vivo liver metastatic assays. Four stable cell lines were injected into the spleens of nude mice ( n = 10 mice per group). Representative bioluminescent imaging, intensity of bioluminescence signals, incidence of liver metastasis, liver tissues’ HE staining, number of metastatic liver nodules, and overall survival times in each group were shown. The scale bars represent 1 mm (low magnification) and 100 μm (high magnification). F In vivo lung metastatic assays. Four stable cell lines were injected into the tail veins of nude mice ( n = 10 mice per group). Representative bioluminescent imaging, intensity of bioluminescence signals, incidence of lung metastasis, lung tissues’ HE staining, number of metastatic lung nodules, and overall survival times in each group ( n = 10) were shown. The scale bars represent 1 mm (low magnification) and 100 μm (high magnification). * P < 0.05, ** P < 0.01. Data are displayed as the mean ± s.d.

Article Snippet: Lentiviral vectors encoding the human HOXA13, ACLY, and IGF1R genes were constructed in pLV-puro or pLV-neo (Addgene) and designated as LV-HOXA13, LV-ACLY, and LV-IGF1R.

Techniques: Transfection, Expressing, Western Blot, Knockdown, Construct, Over Expression, In Vivo, Stable Transfection, Injection, Imaging, Staining

Journal: Cell Death & Disease

Article Title: IGF1-mediated HOXA13 overexpression promotes colorectal cancer metastasis through upregulating ACLY and IGF1R

doi: 10.1038/s41419-021-03833-2

Figure Lengend Snippet: A Representative images of IHC staining of HOXA13, ACLY, and IGF1R expression in human CRC and adjacent nontumor samples were shown. The scale bars represent 250 μm (low magnification) and 50 μm (high magnification). B , C Correlation analysis of HOXA13 and ACLY or IGF1R expression in the CRC tissues in cohort I ( B ) and cohort II ( C ). D , E Kaplan–Meier’s curves generated with the data from the CRC patients with negative versus positive ACLY or IGF1R expression. The correlation between HOXA13 and ACLY (left) or IGF1R (right) expression and overall survival or recurrence in patients with CRC in cohort I ( D ) and cohort II ( E ). F Real-time PCR (upper) and IHC analyses (lower) of ACLY, IGF1R, and IGF1 expression in adjacent nontumor samples, primary CRC, and paired metastasis CRC samples. The scale bars represent 250 μm (low magnification) and 50 μm (high magnification). * P < 0.05, ** P < 0.01. Data are displayed as the mean ± s.d.

Article Snippet: Lentiviral vectors encoding the human HOXA13, ACLY, and IGF1R genes were constructed in pLV-puro or pLV-neo (Addgene) and designated as LV-HOXA13, LV-ACLY, and LV-IGF1R.

Techniques: Immunohistochemistry, Expressing, Generated, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: IGF1-mediated HOXA13 overexpression promotes colorectal cancer metastasis through upregulating ACLY and IGF1R

doi: 10.1038/s41419-021-03833-2

Figure Lengend Snippet: A The SW480-HOXA13 cells were respectively treated with ACLY inhibitor ETC-1002 (100 μM, 12 h), IGF1R inhibitor Linsitinib (10 μM, 6 h) or both two agents. The protein expression levels of p-AMPKα, total AMPKα, p-IGF1R, and total IGF1R were detected by western blotting. B Transwell assays showed the migratory and invasive capabilities of SW480-HOXA13 cells after treatment with ACLY inhibitor ETC-1002 (100 μM, 12 h), IGF1R inhibitor Linsitinib (10 μM, 6 h) or the combination of both two agents. C The diagram of agent treatment in four randomly assigned groups of nude mice. These mice were treated with vehicle, ETC-1002 (30 mg/kg), Linsitinib (50 mg/kg) or combined treatment 1 week after injection of SW480-HOXA13 cells. D In vivo liver metastatic assays revealed that combined administration of ETC-1002 and Linsitinib significantly suppress HOXA13-mediated CRC metastasis. Four stable cell lines were injected into the spleens of nude mice ( n = 10 mice per group). Representative bioluminescent imaging, intensity of bioluminescence signals, incidence of liver metastasis, liver tissues’ HE staining, number of metastatic liver nodules, and overall survival times in each group were shown. The scale bars represent 1 mm (low magnification) and 100 μm (high magnification). E In vivo lung metastatic assays revealed that combined administration of ETC-1002 and Linsitinib significantly suppress HOXA13-mediated CRC metastasis. Four stable cell lines were injected into the tail veins of nude mice ( n = 10 mice per group). Representative bioluminescent imaging, intensity of bioluminescence signals, number of metastatic lung nodules, overall survival times, lung tissues’ HE staining in each group, and the incidence of lung metastasis were shown. The scale bars represent 1 mm (low magnification) and 100 μm (high magnification). F A schematic diagram for the role of IGF1-HOXA13-IGF1R positive feedback loop in CRC metastasis. IGF1-IGF1R pathway upregulates HOXA13 via PI3K/AKT/HIF1α pathway. HOXA13 promotes CRC metastasis through upregulating ACLY and IGF1R expression. Combined administration of ACLY inhibitor (ETC-1002) and IGF1R inhibitor (Linsitinib) disrupts the IGF1-HOXA13-IGF1R loop and inhibits CRC metastasis. * P < 0.05, ** P < 0.01. Data are displayed as the mean ± s.d.

Article Snippet: Lentiviral vectors encoding the human HOXA13, ACLY, and IGF1R genes were constructed in pLV-puro or pLV-neo (Addgene) and designated as LV-HOXA13, LV-ACLY, and LV-IGF1R.

Techniques: Expressing, Western Blot, Injection, In Vivo, Stable Transfection, Imaging, Staining