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pclneomyc lats2 plasmid (Addgene inc)

Name: pclneomyc lats2 plasmid
Brand: Addgene inc
Rating: 93 (6 reviews)

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Addgene inc pclneomyc lats2 plasmid

<t>LATS2</t> is a target gene of miR-25 . (a) The predicted binding site of miR-25 in the 3′UTR of LATS2 from miRBase. The mutated LATS2 3′UTR-binding site is indicated. (b) LATS2 expression in 15 pairs of NSCLC tissues and adjacent normal tissues by qRT-PCR. (c) LATS2 expression in NSCLC cell lines and the noncancerous lung epithelial cell line BEAS-2B. (d) Kaplan-Meier curve for overall survival in lung cancer patients with high or low LATS2 expression. Data were taken from TCGA. (e, f) Luciferase reporter assays in HEK-293 cells. (g, h) Luciferase reporter assays in H1299 cells. HEK-293T, A549, and H1299 cells were cotransfected with pRL-TK carrying a wild-type or mutant 3′UTR of LATS2 and the miR-25 precursor (60 ng) or the miR-25 inhibitor (10 pmol), and the luciferase activity was measured 48 h posttransfection. (i) Western blot assays for LATS2 in A549, H1299, and BEAS-2B cells. (j) Western blot analysis of LATS2 in A549 and H1299 cells transfected with miR-25 precursor or the miR-25 inhibitor. Experiments in this section were performed using our documented protocols . Results were presented as the mean ± SD. ∗ P < 0.05 and ∗∗∗ P < 0.001.

Pclneomyc Lats2 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "miR-25 Promotes Cell Proliferation, Migration, and Invasion of Non-Small-Cell Lung Cancer by Targeting the LATS2/YAP Signaling Pathway"

Article Title: miR-25 Promotes Cell Proliferation, Migration, and Invasion of Non-Small-Cell Lung Cancer by Targeting the LATS2/YAP Signaling Pathway

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2019/9719723

LATS2 is a target gene of miR-25 . (a) The predicted binding site of miR-25 in the 3′UTR of LATS2 from miRBase. The mutated LATS2 3′UTR-binding site is indicated. (b) LATS2 expression in 15 pairs of NSCLC tissues and adjacent normal tissues by qRT-PCR. (c) LATS2 expression in NSCLC cell lines and the noncancerous lung epithelial cell line BEAS-2B. (d) Kaplan-Meier curve for overall survival in lung cancer patients with high or low LATS2 expression. Data were taken from TCGA. (e, f) Luciferase reporter assays in HEK-293 cells. (g, h) Luciferase reporter assays in H1299 cells. HEK-293T, A549, and H1299 cells were cotransfected with pRL-TK carrying a wild-type or mutant 3′UTR of LATS2 and the miR-25 precursor (60 ng) or the miR-25 inhibitor (10 pmol), and the luciferase activity was measured 48 h posttransfection. (i) Western blot assays for LATS2 in A549, H1299, and BEAS-2B cells. (j) Western blot analysis of LATS2 in A549 and H1299 cells transfected with miR-25 precursor or the miR-25 inhibitor. Experiments in this section were performed using our documented protocols . Results were presented as the mean ± SD. ∗ P < 0.05 and ∗∗∗ P < 0.001.

Figure Legend Snippet: LATS2 is a target gene of miR-25 . (a) The predicted binding site of miR-25 in the 3′UTR of LATS2 from miRBase. The mutated LATS2 3′UTR-binding site is indicated. (b) LATS2 expression in 15 pairs of NSCLC tissues and adjacent normal tissues by qRT-PCR. (c) LATS2 expression in NSCLC cell lines and the noncancerous lung epithelial cell line BEAS-2B. (d) Kaplan-Meier curve for overall survival in lung cancer patients with high or low LATS2 expression. Data were taken from TCGA. (e, f) Luciferase reporter assays in HEK-293 cells. (g, h) Luciferase reporter assays in H1299 cells. HEK-293T, A549, and H1299 cells were cotransfected with pRL-TK carrying a wild-type or mutant 3′UTR of LATS2 and the miR-25 precursor (60 ng) or the miR-25 inhibitor (10 pmol), and the luciferase activity was measured 48 h posttransfection. (i) Western blot assays for LATS2 in A549, H1299, and BEAS-2B cells. (j) Western blot analysis of LATS2 in A549 and H1299 cells transfected with miR-25 precursor or the miR-25 inhibitor. Experiments in this section were performed using our documented protocols . Results were presented as the mean ± SD. ∗ P < 0.05 and ∗∗∗ P < 0.001.

Techniques Used: Binding Assay, Expressing, Quantitative RT-PCR, Luciferase, Mutagenesis, Activity Assay, Western Blot, Transfection

The effects of LATS2 expression on A549 cell proliferation, migration, and invasion. (a) Colony formation of A549 cells transfected with LATS2, miR-25, or the combination of both miR-25 and LATS2 plasmids. (b, c) Wound healing and transwell assays for migration and invasion capacities of A549 cells transfected with miR-25 and/or LATS2. (d) Western blot assays for LATS2, downstream effectors of the Hippo pathway (phosphorylated YAP and YAP), and metastasis-related proteins (E-cadherin, Vimentin, and MMP9) in A549 cells transfected with miR-25 and/or LATS2. Results were presented as the mean ± SD. (e) Immunofluorescence assay for nuclear translocation of YAP protein. Representative immunofluorescent images of YAP staining were shown in the left panel (green: YAP; blue: nuclear; scale bar, 10 μ m). The nuclear localization of YAP is quantified in the right panel by ImageJ software. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Figure Legend Snippet: The effects of LATS2 expression on A549 cell proliferation, migration, and invasion. (a) Colony formation of A549 cells transfected with LATS2, miR-25, or the combination of both miR-25 and LATS2 plasmids. (b, c) Wound healing and transwell assays for migration and invasion capacities of A549 cells transfected with miR-25 and/or LATS2. (d) Western blot assays for LATS2, downstream effectors of the Hippo pathway (phosphorylated YAP and YAP), and metastasis-related proteins (E-cadherin, Vimentin, and MMP9) in A549 cells transfected with miR-25 and/or LATS2. Results were presented as the mean ± SD. (e) Immunofluorescence assay for nuclear translocation of YAP protein. Representative immunofluorescent images of YAP staining were shown in the left panel (green: YAP; blue: nuclear; scale bar, 10 μ m). The nuclear localization of YAP is quantified in the right panel by ImageJ software. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Techniques Used: Expressing, Migration, Transfection, Western Blot, Immunofluorescence, Translocation Assay, Staining, Software

miR-25 antagomir inhibits lung cancer growth and metastasis in mouse xenografts. (a) Time course of tumor volumes from immunodeficient mice treated with the miR-25 antagomir or a control. Results were presented as the mean ± SD. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (b, c) The image and the weight of xenograft tumors one month after the treatment. Mean ± SD was shown. ∗∗ P < 0.01. (d) Representative images of mouse lungs and livers. (e) Histological morphology of mouse lungs and livers by HE staining. The magnification of the upper row images was 40x, bar scale = 500 μ m. The magnification of the bottom row images was 400x, bar scale = 50 μ m. (f, h) The incidence of lung and liver metastasis in mice after tail intravenous injections with the miR-25 antagomir was shown in the table. (g) Quantification of the metastasis nodules in the lungs of each group ( n = 10). ∗ P < 0.05. (i) Representative images of immunohistochemical analysis of LATS2, E-cadherin, Vimentin, and MMP9 in lung metastasis (magnification, 400x. bar scale = 50 μ m). Brown represented the target protein in IHC, and blue represented nuclear staining.

Figure Legend Snippet: miR-25 antagomir inhibits lung cancer growth and metastasis in mouse xenografts. (a) Time course of tumor volumes from immunodeficient mice treated with the miR-25 antagomir or a control. Results were presented as the mean ± SD. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (b, c) The image and the weight of xenograft tumors one month after the treatment. Mean ± SD was shown. ∗∗ P < 0.01. (d) Representative images of mouse lungs and livers. (e) Histological morphology of mouse lungs and livers by HE staining. The magnification of the upper row images was 40x, bar scale = 500 μ m. The magnification of the bottom row images was 400x, bar scale = 50 μ m. (f, h) The incidence of lung and liver metastasis in mice after tail intravenous injections with the miR-25 antagomir was shown in the table. (g) Quantification of the metastasis nodules in the lungs of each group ( n = 10). ∗ P < 0.05. (i) Representative images of immunohistochemical analysis of LATS2, E-cadherin, Vimentin, and MMP9 in lung metastasis (magnification, 400x. bar scale = 50 μ m). Brown represented the target protein in IHC, and blue represented nuclear staining.

Techniques Used: Staining, Immunohistochemical staining

Figure Legend Snippet: Schematic presentation of a positive feedback loop involving miR-25/LATS2/YAP/TAZ/TEAD/ MCM7 /miR-25 in lung cancer.

Techniques Used:

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Journal: Oxidative Medicine and Cellular Longevity

Article Title: miR-25 Promotes Cell Proliferation, Migration, and Invasion of Non-Small-Cell Lung Cancer by Targeting the LATS2/YAP Signaling Pathway

doi: 10.1155/2019/9719723

Figure Lengend Snippet: LATS2 is a target gene of miR-25 . (a) The predicted binding site of miR-25 in the 3′UTR of LATS2 from miRBase. The mutated LATS2 3′UTR-binding site is indicated. (b) LATS2 expression in 15 pairs of NSCLC tissues and adjacent normal tissues by qRT-PCR. (c) LATS2 expression in NSCLC cell lines and the noncancerous lung epithelial cell line BEAS-2B. (d) Kaplan-Meier curve for overall survival in lung cancer patients with high or low LATS2 expression. Data were taken from TCGA. (e, f) Luciferase reporter assays in HEK-293 cells. (g, h) Luciferase reporter assays in H1299 cells. HEK-293T, A549, and H1299 cells were cotransfected with pRL-TK carrying a wild-type or mutant 3′UTR of LATS2 and the miR-25 precursor (60 ng) or the miR-25 inhibitor (10 pmol), and the luciferase activity was measured 48 h posttransfection. (i) Western blot assays for LATS2 in A549, H1299, and BEAS-2B cells. (j) Western blot analysis of LATS2 in A549 and H1299 cells transfected with miR-25 precursor or the miR-25 inhibitor. Experiments in this section were performed using our documented protocols . Results were presented as the mean ± SD. ∗ P < 0.05 and ∗∗∗ P < 0.001.

Article Snippet: The pClneoMyc-LATS2 plasmid was a gift from Yutaka Hata (Addgene plasmid), which expressed Myc-tagged LATS2 in mammalian cells [ ].

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Luciferase, Mutagenesis, Activity Assay, Western Blot, Transfection

Journal: Oxidative Medicine and Cellular Longevity

Article Title: miR-25 Promotes Cell Proliferation, Migration, and Invasion of Non-Small-Cell Lung Cancer by Targeting the LATS2/YAP Signaling Pathway

doi: 10.1155/2019/9719723

Figure Lengend Snippet: The effects of LATS2 expression on A549 cell proliferation, migration, and invasion. (a) Colony formation of A549 cells transfected with LATS2, miR-25, or the combination of both miR-25 and LATS2 plasmids. (b, c) Wound healing and transwell assays for migration and invasion capacities of A549 cells transfected with miR-25 and/or LATS2. (d) Western blot assays for LATS2, downstream effectors of the Hippo pathway (phosphorylated YAP and YAP), and metastasis-related proteins (E-cadherin, Vimentin, and MMP9) in A549 cells transfected with miR-25 and/or LATS2. Results were presented as the mean ± SD. (e) Immunofluorescence assay for nuclear translocation of YAP protein. Representative immunofluorescent images of YAP staining were shown in the left panel (green: YAP; blue: nuclear; scale bar, 10 μ m). The nuclear localization of YAP is quantified in the right panel by ImageJ software. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The pClneoMyc-LATS2 plasmid was a gift from Yutaka Hata (Addgene plasmid), which expressed Myc-tagged LATS2 in mammalian cells [ ].

Techniques: Expressing, Migration, Transfection, Western Blot, Immunofluorescence, Translocation Assay, Staining, Software

Journal: Oxidative Medicine and Cellular Longevity

Article Title: miR-25 Promotes Cell Proliferation, Migration, and Invasion of Non-Small-Cell Lung Cancer by Targeting the LATS2/YAP Signaling Pathway

doi: 10.1155/2019/9719723

Figure Lengend Snippet: miR-25 antagomir inhibits lung cancer growth and metastasis in mouse xenografts. (a) Time course of tumor volumes from immunodeficient mice treated with the miR-25 antagomir or a control. Results were presented as the mean ± SD. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (b, c) The image and the weight of xenograft tumors one month after the treatment. Mean ± SD was shown. ∗∗ P < 0.01. (d) Representative images of mouse lungs and livers. (e) Histological morphology of mouse lungs and livers by HE staining. The magnification of the upper row images was 40x, bar scale = 500 μ m. The magnification of the bottom row images was 400x, bar scale = 50 μ m. (f, h) The incidence of lung and liver metastasis in mice after tail intravenous injections with the miR-25 antagomir was shown in the table. (g) Quantification of the metastasis nodules in the lungs of each group ( n = 10). ∗ P < 0.05. (i) Representative images of immunohistochemical analysis of LATS2, E-cadherin, Vimentin, and MMP9 in lung metastasis (magnification, 400x. bar scale = 50 μ m). Brown represented the target protein in IHC, and blue represented nuclear staining.

Article Snippet: The pClneoMyc-LATS2 plasmid was a gift from Yutaka Hata (Addgene plasmid), which expressed Myc-tagged LATS2 in mammalian cells [ ].

Techniques: Staining, Immunohistochemical staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: miR-25 Promotes Cell Proliferation, Migration, and Invasion of Non-Small-Cell Lung Cancer by Targeting the LATS2/YAP Signaling Pathway

doi: 10.1155/2019/9719723

Figure Lengend Snippet: Schematic presentation of a positive feedback loop involving miR-25/LATS2/YAP/TAZ/TEAD/ MCM7 /miR-25 in lung cancer.

Article Snippet: The pClneoMyc-LATS2 plasmid was a gift from Yutaka Hata (Addgene plasmid), which expressed Myc-tagged LATS2 in mammalian cells [ ].

Techniques:

( A ) IB analysis of IFN-I signaling in HEK293T transfected with HA-LATS1 and treated with IFN-α (1000 IU/ml). ( B ) IB analysis of IRF9 in Lats1/2 +/+ and Lats1/2 −/− MEFs. ( C ) IB analysis of pY701-STAT1 in HEK293T transfected with shLATS1 and treated with IFN-ɑ (1000 IU/ml). ( D ) IB analysis of pY690-STAT2 (pSTAT2), STAT2, and IRF9 in Lats1/2 +/+ and Lats1/2 −/− MEFs treated with mIFNβ (1000 IU/ml). ( E and F ) IB analysis of pS727-STAT1 in HeLa cells transfected with shLATS1 (E) or in HEK293T transfected with shLATS1 (F) and then treated with IFN-ɑ (1000 IU/ml). ( G and H ) IB analysis of pS727-STAT1 in Lats1/2 +/+ or Lats1/2 −/− MEFs treated with mIFNβ (500 or 1000 IU/ml, 1 hour) (G) or transfected with Flag-LATS1 or Flag-LATS2 and treated with mIFNβ (1000 IU/ml, 1 hour) (H). ( I ) IB analysis of pS727-STAT1 in HEK293T transfected with Flag-LATS1 (WT or YF/YF) and treated with IFN-ɑ (1000 IU/ml, 1 hour). ( J ) RT-qPCR analysis of the representative ISG ( Ifit1 ) in STAT1-WT or STAT1-S727A-knockin 2fTGH cells transfected with shCON or shLATS1, followed by IFN-α treatment (1000 IU/ml, 4 hours) (right). S727A-knockin was confirmed by sequencing (left). ( K ) Median tissue culture infectious dose (TCID 50 ) assay to analyze VSV titers in supernatants from HEK293T transfected with shLATS1 and stimulated with IFN-ɑ (60 IU/ml), followed by VSV infection (multiplicity of infection = 0.1, 24 hours). Data are shown as means and SD of three biological replicates (J to K) or are representative of three independent experiments (A to I). N.S ( P > 0.05), * P < 0.05, ** P < 0.01, and *** P < 0.001 (two-tailed unpaired Student’s t test).

Journal: Science Advances

Article Title: LATS1 is a central signal transmitter for achieving full type-I interferon activity

doi: 10.1126/sciadv.abj3887

Figure Lengend Snippet: ( A ) IB analysis of IFN-I signaling in HEK293T transfected with HA-LATS1 and treated with IFN-α (1000 IU/ml). ( B ) IB analysis of IRF9 in Lats1/2 +/+ and Lats1/2 −/− MEFs. ( C ) IB analysis of pY701-STAT1 in HEK293T transfected with shLATS1 and treated with IFN-ɑ (1000 IU/ml). ( D ) IB analysis of pY690-STAT2 (pSTAT2), STAT2, and IRF9 in Lats1/2 +/+ and Lats1/2 −/− MEFs treated with mIFNβ (1000 IU/ml). ( E and F ) IB analysis of pS727-STAT1 in HeLa cells transfected with shLATS1 (E) or in HEK293T transfected with shLATS1 (F) and then treated with IFN-ɑ (1000 IU/ml). ( G and H ) IB analysis of pS727-STAT1 in Lats1/2 +/+ or Lats1/2 −/− MEFs treated with mIFNβ (500 or 1000 IU/ml, 1 hour) (G) or transfected with Flag-LATS1 or Flag-LATS2 and treated with mIFNβ (1000 IU/ml, 1 hour) (H). ( I ) IB analysis of pS727-STAT1 in HEK293T transfected with Flag-LATS1 (WT or YF/YF) and treated with IFN-ɑ (1000 IU/ml, 1 hour). ( J ) RT-qPCR analysis of the representative ISG ( Ifit1 ) in STAT1-WT or STAT1-S727A-knockin 2fTGH cells transfected with shCON or shLATS1, followed by IFN-α treatment (1000 IU/ml, 4 hours) (right). S727A-knockin was confirmed by sequencing (left). ( K ) Median tissue culture infectious dose (TCID 50 ) assay to analyze VSV titers in supernatants from HEK293T transfected with shLATS1 and stimulated with IFN-ɑ (60 IU/ml), followed by VSV infection (multiplicity of infection = 0.1, 24 hours). Data are shown as means and SD of three biological replicates (J to K) or are representative of three independent experiments (A to I). N.S ( P > 0.05), * P < 0.05, ** P < 0.01, and *** P < 0.001 (two-tailed unpaired Student’s t test).

Article Snippet: Myc-LATS2 was a gift from Y. Hata (Addgene, no. 66852).

Techniques: Transfection, Quantitative RT-PCR, Knock-In, Sequencing, Infection, Two Tailed Test

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1) Product Images from "miR-25 Promotes Cell Proliferation, Migration, and Invasion of Non-Small-Cell Lung Cancer by Targeting the LATS2/YAP Signaling Pathway"

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