Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: H19 long noncoding RNA controls the mRNA decay promoting function of KSRP

doi: 10.1073/pnas.1415098111

Figure Lengend Snippet: The interaction with H19 favors the decay-promoting function of KSRP. (A) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-KSRP (flag-KSRP), or pTAG2B-H19 (H19) plus pCDNA3-Flag-KSRP (flag-KSRP). Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. (B) HEK-293 cells were transiently transfected with GST-fused MS2 binding protein (GST-MS2BP) together with pTAG2B-myog 3′UTR and pTAG2B-MS2-12XH19 (in which murine H19 was tagged with MS2 RNA hairpins; MS2-H19) or pTAG2B-MS2-12X (empty vector; MS2). Total cell extracts were prepared 48 h after transfection and precipitated by glutathione–Sepharose beads. RNA was purified and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. (C) HEK-293 cells were transiently cotransfected with pCMV-TAG2B-KSRP (expressing Flag-tagged WT KSRP) or pCMV-TAG2B expressing the indicated Flag-tagged KSRP mutants (KH1GDDG, KH2GDDG, KH3GDDG, KH4GDDG) together with pTAG2B-myog 3′UTR and pTAG2B-H19. Total cell extracts were prepared 48 h after transfection and immunoprecipitated by anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect myogenin 3′UTR (m.Myog 3′UTR) or transfected murine H19 (m.H19). (D) In vitro RNA degradation assays using S100 extracts from shKSRP C2C12 cells preincubated (for 90 min at 4 °C) with anti-Flag immunoprecipitates from HEK-293 cells transiently transfected with Flag-tagged WT KSRP (KSRP) or Flag-tagged KH1GDDG mutant (KSRP[KH1GDDG]) together with the E3 sequence or murine H19 cloned in expression vectors. Internally 32P-labeled and capped RNA substrates were incubated with the aforementioned reaction mixtures, and their decay was monitored for the indicated times. RNA was analyzed by denaturing polyacrylamide gel electrophoresis followed by autoradiography. E3 is a stable transcript used to detect background decay. Representative autoradiograms are displayed. (E) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5, or pTAG2B-H19 (H19) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5. Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. The values of RT-qPCR experiments shown are averages (±SEM) of three independent experiments performed in triplicate (*P < 0.01 and **P < 0.001, Student t test.)

Article Snippet: To generate the pTAG2B-MS2-12X vector, a fragment encompassing 12 MS2 binding sites was excised from the pSLMS2-12X (Addgene) and cloned in the EcoRI/EcoRV sites of pCMV-TAG2B.

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Purification, Quantitative RT-PCR, Binding Assay, Expressing, In Vitro, Mutagenesis, Sequencing, Clone Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

Journal: Bioengineered

Article Title: LncRNA DDX11 antisense RNA 1 promotes EMT process of esophageal squamous cell carcinoma by sponging miR-30d-5p to regulate SNAI1/ZEB2 expression and Wnt/β-catenin pathway

doi: 10.1080/21655979.2021.2008759

Figure Lengend Snippet: DDX11-AS1 was up-regulated in ESCC tissues and linked to poor prognosis of ESCC patients. (a) DDX11-AS1 expression was notably elevated in ESCC tissues as opposed to normal tissues, *P < 0.05. (b) DDX11-AS1 expression in tumor and matched contiguous normal tissue of each ESCC patient. (c) The fold change of DDX11-AS1 expression in tumor tissues contrasted with paired adjacent normal tissues. (d) Relative DDX11-AS1 expression level in five human esophageal cancer cell lines identified by RT-qPCR method. Pools: average expression in 10 normal tissues was utilized as normal control. (e) Correlation of the expression level of DDX11-AS1 with clinicopathologic features of ESCC patients. (f-j) Kaplan–Meier univariate survival analysis of DDX11-AS1 and TNM stage, UGIC family history in ESCC cases

Article Snippet: Subcloning of the MS2-12X fragment from pSL-MS2-12X plasmid (Addgene, USA), gave pcDNA3.1, pcDNA3.1-DDX11-AS1, pcDNA3.1-DDX11-AS1-MUT (miR-30d-5p) to build the pcDNA3.1-MS2, pcDNA3.1-MS2-DDX11-AS1, and pcDNA3.1-MS2-DDX11-AS1-MUT plasmids, in that order.

Techniques: Expressing, Quantitative RT-PCR

Journal: Bioengineered

Article Title: LncRNA DDX11 antisense RNA 1 promotes EMT process of esophageal squamous cell carcinoma by sponging miR-30d-5p to regulate SNAI1/ZEB2 expression and Wnt/β-catenin pathway

doi: 10.1080/21655979.2021.2008759

Figure Lengend Snippet: DDX11-AS1 promotes esophageal cancer cells proliferation, migration, and invasion. (a, b) The overexpression efficiency or knockdown of DDX11-AS1 transfect in esophageal cancer cells was identified by the qRT-PCR method. NC, negative control. (c) The Eca109 and Kyse150 cell growth abilities were identified by MTS. *P < 0.05, contrasted with NC cohort. (d, e) The impact of DDX11-AS1 on cell migration and invasiveness ability of Eca109 and Kyse150 cells were identified by transwell invasion assay and wound-healing experiment. *P < 0.05, contrasted with NC cohort. (f-h) The rescue experiments were performed to evaluate the function of DDX11-AS1 in TE13 cells. *P < 0.05, contrasted with NC cohort. #P < 0.05, contrasted with pcDNA3.1-DDX11-AS1 group

Article Snippet: Subcloning of the MS2-12X fragment from pSL-MS2-12X plasmid (Addgene, USA), gave pcDNA3.1, pcDNA3.1-DDX11-AS1, pcDNA3.1-DDX11-AS1-MUT (miR-30d-5p) to build the pcDNA3.1-MS2, pcDNA3.1-MS2-DDX11-AS1, and pcDNA3.1-MS2-DDX11-AS1-MUT plasmids, in that order.

Techniques: Migration, Over Expression, Quantitative RT-PCR, Negative Control, Transwell Invasion Assay

Journal: Bioengineered

Article Title: LncRNA DDX11 antisense RNA 1 promotes EMT process of esophageal squamous cell carcinoma by sponging miR-30d-5p to regulate SNAI1/ZEB2 expression and Wnt/β-catenin pathway

doi: 10.1080/21655979.2021.2008759

Figure Lengend Snippet: DDX11-AS1 promotes the TGF-β induced EMT process. (a) Cell morphology of Eca109 cells treated or untreated with TGF‑β. (b) Relative expression of EMT-related markers was detected in Eca109 cells with or without TGF‑β treatment. *P < 0.05, contrasted with untreated cohort. (c) Relative expression of DDX11-AS1 was identified in esophageal cancer cells with or without TGF‑β treatment. *P < 0.05, contrasted with the untreated cohort. (d, e) The effect of DDX11-AS1 on the expression of EMT-related markers. *P < 0.05, compared with NC cohort

Article Snippet: Subcloning of the MS2-12X fragment from pSL-MS2-12X plasmid (Addgene, USA), gave pcDNA3.1, pcDNA3.1-DDX11-AS1, pcDNA3.1-DDX11-AS1-MUT (miR-30d-5p) to build the pcDNA3.1-MS2, pcDNA3.1-MS2-DDX11-AS1, and pcDNA3.1-MS2-DDX11-AS1-MUT plasmids, in that order.

Techniques: Expressing

Journal: Bioengineered

Article Title: LncRNA DDX11 antisense RNA 1 promotes EMT process of esophageal squamous cell carcinoma by sponging miR-30d-5p to regulate SNAI1/ZEB2 expression and Wnt/β-catenin pathway

doi: 10.1080/21655979.2021.2008759

Figure Lengend Snippet: DDX11-AS1 served as a molecular sponge of miR-30d-5p. (a) The subcellular localization of DDX11-AS1 was forecasted by the lncLocator online tool. (b) The subcellular localization of DDX11-AS1 in ESCC cells was identified by the RT-qPCR method. (c) The probable binding sites of DDX11-AS1 to miR-30d-5p were forecasted by LncBase. (d) The miR-30d-5p expression was considerably reduced in ESCC tissues contrasted with normal tissues, *P < 0.05. (e) The relationship between DDX11-AS1 and miR-30d-5p expression was assessed in ESCC tissues. (f) Relative expression of miR-30d-5p in various subgroups. (g) The regulation of DDX11-AS1 on miR-30d-5p expression was identified by the qRT-PCR method. (h) MiR-30d-5p mimic or inhibitor did not affect DDX11-AS1 expression in ESCC cells. (i) The effect of miR-30d-5p mimic on the activity of DDX11-AS1 conveyed by dual-luciferase reporter assay. (j) RNA immunoprecipitation (RIP) assay illustrating the greater enrichment of miR-30d-5p to DDX11-AS1. *P < 0.05, contrasted with NC cohort

Article Snippet: Subcloning of the MS2-12X fragment from pSL-MS2-12X plasmid (Addgene, USA), gave pcDNA3.1, pcDNA3.1-DDX11-AS1, pcDNA3.1-DDX11-AS1-MUT (miR-30d-5p) to build the pcDNA3.1-MS2, pcDNA3.1-MS2-DDX11-AS1, and pcDNA3.1-MS2-DDX11-AS1-MUT plasmids, in that order.

Techniques: Quantitative RT-PCR, Binding Assay, Expressing, Activity Assay, Luciferase, Reporter Assay, Immunoprecipitation

Journal: Bioengineered

Article Title: LncRNA DDX11 antisense RNA 1 promotes EMT process of esophageal squamous cell carcinoma by sponging miR-30d-5p to regulate SNAI1/ZEB2 expression and Wnt/β-catenin pathway

doi: 10.1080/21655979.2021.2008759

Figure Lengend Snippet: SNAI1 and ZEB2 were the target gene of miR-30d-5p and regulated by DDX11-AS1. (a) The probable target genes of miR-30d-5p are forecasted by miRDB, miRWalk v3.0, and microT-CDS. (b) SNAI1 and ZEB2 expression were considerably elevated in ESCC tissues contrasted with normal tissues, which were measured by qRT-PCR. *P < 0.05. (c) The relationship between SNAI1/ZEB2 and miR-30d-5p expression was assessed in ESCC tissues. (d) The effect of miR-30d-5p mimic/inhibitor on the expression of SNAI1/ZEB2 was reported by qRT-PCR. *P < 0.05, contrasted with NC cohort. (e) The effect of miR-30d-5p mimic/inhibitor on luciferase activity of SNAI1/ZEB2 reported by dual-luciferase reporter assay. *P < 0.05, contrasted with NC cohort. (f) The expression of SNAI1/ZEB2 was upregulated by DDX11-AS1, whereas miR-30d-5p partially rescued the increased effect in ESCC cells. *P < 0.05. (g) MiR-30d-5p partially rescued the upregulation of luciferase activity caused by DDX11-AS1 overexpression. *P < 0.05. (h, i) Cell viability and invasive cell counts were rescued by cotransfection with miR-30d-5p mimics in DDX11-AS1 overexpression cells, which were performed by MTS and transwell invasion assays. *P < 0.05, contrasted with NC cohort, #P < 0.05, contrasted with pcDNA3.1-DDX11-AS1 cohort

Article Snippet: Subcloning of the MS2-12X fragment from pSL-MS2-12X plasmid (Addgene, USA), gave pcDNA3.1, pcDNA3.1-DDX11-AS1, pcDNA3.1-DDX11-AS1-MUT (miR-30d-5p) to build the pcDNA3.1-MS2, pcDNA3.1-MS2-DDX11-AS1, and pcDNA3.1-MS2-DDX11-AS1-MUT plasmids, in that order.

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Reporter Assay, Over Expression, Cotransfection

Journal: Bioengineered

Article Title: LncRNA DDX11 antisense RNA 1 promotes EMT process of esophageal squamous cell carcinoma by sponging miR-30d-5p to regulate SNAI1/ZEB2 expression and Wnt/β-catenin pathway

doi: 10.1080/21655979.2021.2008759

Figure Lengend Snippet: DDX11-AS1 activated the Wnt/β-catenin Signaling pathway via targeting miR-30d-5p. (a) The KEGG pathway analysis on the downstream target genes of miR-30d-5p using Starbase 3.0 online tool. (b, c) The effect of miR-30d-5p or DDX11-AS1 on the TOP/FOP Flash luciferase activity. *P < 0.05. (d) The effect of miR-30d-5p or DDX11-AS1 on downstream target genes of the Wnt pathway. *P < 0.05, contrasted with NC cohort

Article Snippet: Subcloning of the MS2-12X fragment from pSL-MS2-12X plasmid (Addgene, USA), gave pcDNA3.1, pcDNA3.1-DDX11-AS1, pcDNA3.1-DDX11-AS1-MUT (miR-30d-5p) to build the pcDNA3.1-MS2, pcDNA3.1-MS2-DDX11-AS1, and pcDNA3.1-MS2-DDX11-AS1-MUT plasmids, in that order.

Techniques: Luciferase, Activity Assay

Journal: Bioengineered

Article Title: LncRNA DDX11 antisense RNA 1 promotes EMT process of esophageal squamous cell carcinoma by sponging miR-30d-5p to regulate SNAI1/ZEB2 expression and Wnt/β-catenin pathway

doi: 10.1080/21655979.2021.2008759

Figure Lengend Snippet: Univariate analysis for OS in ESCC cases (Cox’s test)

Article Snippet: Subcloning of the MS2-12X fragment from pSL-MS2-12X plasmid (Addgene, USA), gave pcDNA3.1, pcDNA3.1-DDX11-AS1, pcDNA3.1-DDX11-AS1-MUT (miR-30d-5p) to build the pcDNA3.1-MS2, pcDNA3.1-MS2-DDX11-AS1, and pcDNA3.1-MS2-DDX11-AS1-MUT plasmids, in that order.

Techniques: Expressing

Journal: Bioengineered

Article Title: LncRNA DDX11 antisense RNA 1 promotes EMT process of esophageal squamous cell carcinoma by sponging miR-30d-5p to regulate SNAI1/ZEB2 expression and Wnt/β-catenin pathway

doi: 10.1080/21655979.2021.2008759

Figure Lengend Snippet: Multivariate analysis for OS in ESCC cases (Cox’s test)

Article Snippet: Subcloning of the MS2-12X fragment from pSL-MS2-12X plasmid (Addgene, USA), gave pcDNA3.1, pcDNA3.1-DDX11-AS1, pcDNA3.1-DDX11-AS1-MUT (miR-30d-5p) to build the pcDNA3.1-MS2, pcDNA3.1-MS2-DDX11-AS1, and pcDNA3.1-MS2-DDX11-AS1-MUT plasmids, in that order.

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: H19 long noncoding RNA controls the mRNA decay promoting function of KSRP

doi: 10.1073/pnas.1415098111

Figure Lengend Snippet: The interaction with H19 favors the decay-promoting function of KSRP. (A) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-KSRP (flag-KSRP), or pTAG2B-H19 (H19) plus pCDNA3-Flag-KSRP (flag-KSRP). Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. (B) HEK-293 cells were transiently transfected with GST-fused MS2 binding protein (GST-MS2BP) together with pTAG2B-myog 3′UTR and pTAG2B-MS2-12XH19 (in which murine H19 was tagged with MS2 RNA hairpins; MS2-H19) or pTAG2B-MS2-12X (empty vector; MS2). Total cell extracts were prepared 48 h after transfection and precipitated by glutathione–Sepharose beads. RNA was purified and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. (C) HEK-293 cells were transiently cotransfected with pCMV-TAG2B-KSRP (expressing Flag-tagged WT KSRP) or pCMV-TAG2B expressing the indicated Flag-tagged KSRP mutants (KH1GDDG, KH2GDDG, KH3GDDG, KH4GDDG) together with pTAG2B-myog 3′UTR and pTAG2B-H19. Total cell extracts were prepared 48 h after transfection and immunoprecipitated by anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect myogenin 3′UTR (m.Myog 3′UTR) or transfected murine H19 (m.H19). (D) In vitro RNA degradation assays using S100 extracts from shKSRP C2C12 cells preincubated (for 90 min at 4 °C) with anti-Flag immunoprecipitates from HEK-293 cells transiently transfected with Flag-tagged WT KSRP (KSRP) or Flag-tagged KH1GDDG mutant (KSRP[KH1GDDG]) together with the E3 sequence or murine H19 cloned in expression vectors. Internally 32P-labeled and capped RNA substrates were incubated with the aforementioned reaction mixtures, and their decay was monitored for the indicated times. RNA was analyzed by denaturing polyacrylamide gel electrophoresis followed by autoradiography. E3 is a stable transcript used to detect background decay. Representative autoradiograms are displayed. (E) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5, or pTAG2B-H19 (H19) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5. Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. The values of RT-qPCR experiments shown are averages (±SEM) of three independent experiments performed in triplicate (*P < 0.01 and **P < 0.001, Student t test.)

Article Snippet: To generate the pTAG2B-MS2-12X vector, a fragment encompassing 12 MS2 binding sites was excised from the pSLMS2-12X (Addgene) and cloned in the EcoRI/EcoRV sites of pCMV-TAG2B.

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Purification, Quantitative RT-PCR, Binding Assay, Expressing, In Vitro, Mutagenesis, Sequencing, Clone Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA CNALPTC1 promotes cell proliferation and migration of papillary thyroid cancer via sponging miR-30 family

doi:

Figure Lengend Snippet: The expression pattern of CNALPTC1 in PTC. A. Genomic copy number levels of CNALPTC1 were determined by qRT-PCR in 64 pairs of PTC tissues and adjacent noncancerous thyroid tissues. The horizontal lines in the box plots represent the medians, the boxes represent the interquartile range, and the whiskers represent the minimum and maximum values. ***P < 0.001 by Wilcoxon signed-rank test. B. CNALPTC1 RNA expression levels were determined by qRT-PCR in 64 pairs of PTC tissues and adjacent noncancerous thyroid tissues. The horizontal lines in the box plots represent the medians, the boxes represent the interquartile range, and the whiskers represent the minimum and maximum values. ***P < 0.001 by Wilcoxon signed-rank test. C. Correlation between CNALPTC1 RNA expression levels and CNALPTC1 genomic copy number levels in PTC tissues (n = 64). x, the relative CNALPTC1 genomic copy number levels. y, the relative CNALPTC1 RNA expression levels. P < 0.001, r = 0.759 by Pearson correlation analysis. PTC, papillary thyroid cancer; qRT-PCR, quantitative real-time PCR.

Article Snippet: pSL-MS2-12X (Addgene, Cambridge, MA, USA) was double digested using EcoR I and Not I, and the MS2-12X fragment was inserted into pcDNA3.1, pcDNA3.1-CNALPTC1, or pcDNA3.1-CNALPTC1-mut, named as pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2, respectively. pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2 was co-transfected with pMS2-GFP (Addgene) into TPC-1 cells.

Techniques: Expressing, Quantitative RT-PCR, RNA Expression, Real-time Polymerase Chain Reaction

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA CNALPTC1 promotes cell proliferation and migration of papillary thyroid cancer via sponging miR-30 family

doi:

Figure Lengend Snippet: Association between CNALPTC1 expression and clinicopathological characteristics of PTC patients

Article Snippet: pSL-MS2-12X (Addgene, Cambridge, MA, USA) was double digested using EcoR I and Not I, and the MS2-12X fragment was inserted into pcDNA3.1, pcDNA3.1-CNALPTC1, or pcDNA3.1-CNALPTC1-mut, named as pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2, respectively. pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2 was co-transfected with pMS2-GFP (Addgene) into TPC-1 cells.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA CNALPTC1 promotes cell proliferation and migration of papillary thyroid cancer via sponging miR-30 family

doi:

Figure Lengend Snippet: Depletion of CNALPTC1 inhibits proliferation of PTC cells. A. CNALPTC1 RNA expression levels were determined by qRT-PCR in CNALPTC1 stably depleted and control TPC-1 cells. B. Cell proliferation of CNALPTC1 stably depleted and control TPC-1 cells were determined by CCK-8 assays. C. Cell proliferation of CNALPTC1 stably depleted and control TPC-1 cells were determined by EdU incorporation assays. The red colour indicates EdU-positive nuclei. Scale bars = 100 µm. D. CNALPTC1 RNA expression levels were determined by qRT-PCR in CNALPTC1 stably depleted and control IHH-4 cells. E. Cell proliferation of CNALPTC1 stably depleted and control IHH-4 cells were determined by CCK-8 assays. F. Cell proliferation of CNALPTC1 stably depleted and control IHH-4 cells were determined by EdU incorporation assays. The red colour indicates EdU-positive nuclei. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. **P < 0.01, ***P < 0.001 by Student’s t-test. PTC, papillary thyroid cancer; qRT-PCR, quantitative real-time PCR; CCK-8, Cell Counting Kit-8; EdU, Ethynyl deoxyuridine.

Article Snippet: pSL-MS2-12X (Addgene, Cambridge, MA, USA) was double digested using EcoR I and Not I, and the MS2-12X fragment was inserted into pcDNA3.1, pcDNA3.1-CNALPTC1, or pcDNA3.1-CNALPTC1-mut, named as pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2, respectively. pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2 was co-transfected with pMS2-GFP (Addgene) into TPC-1 cells.

Techniques: RNA Expression, Quantitative RT-PCR, Stable Transfection, CCK-8 Assay, Real-time Polymerase Chain Reaction, Cell Counting

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA CNALPTC1 promotes cell proliferation and migration of papillary thyroid cancer via sponging miR-30 family

doi:

Figure Lengend Snippet: Depletion of CNALPTC1 promotes apoptosis of PTC cells. A. Apoptosis of CNALPTC1 stably depleted and control TPC-1 cells were determined by TUNEL assays. The green colour indicates TUNEL-positive and apoptotic cells. Scale bars = 100 µm. B. Apoptosis of CNALPTC1 stably depleted and control IHH-4 cells were determined by TUNEL assays. The green colour indicates TUNEL-positive and apoptotic cells. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. **P < 0.01 by Student’s t-test. PTC, papillary thyroid cancer; TUNEL, TdT-mediated dUTP nick end labeling.

Article Snippet: pSL-MS2-12X (Addgene, Cambridge, MA, USA) was double digested using EcoR I and Not I, and the MS2-12X fragment was inserted into pcDNA3.1, pcDNA3.1-CNALPTC1, or pcDNA3.1-CNALPTC1-mut, named as pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2, respectively. pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2 was co-transfected with pMS2-GFP (Addgene) into TPC-1 cells.

Techniques: Stable Transfection, TUNEL Assay, End Labeling

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA CNALPTC1 promotes cell proliferation and migration of papillary thyroid cancer via sponging miR-30 family

doi:

Figure Lengend Snippet: Depletion of CNALPTC1 represses migration of PTC cells. A. Migration of CNALPTC1 stably depleted and control TPC-1 cells were determined by transwell assays. Represent images are shown. Scale bars = 100 µm. B. Migration of CNALPTC1 stably depleted and control IHH-4 cells were determined by transwell assays. Represent images are shown. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. ***P < 0.001 by Student’s t-test. PTC, papillary thyroid cancer.

Article Snippet: pSL-MS2-12X (Addgene, Cambridge, MA, USA) was double digested using EcoR I and Not I, and the MS2-12X fragment was inserted into pcDNA3.1, pcDNA3.1-CNALPTC1, or pcDNA3.1-CNALPTC1-mut, named as pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2, respectively. pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2 was co-transfected with pMS2-GFP (Addgene) into TPC-1 cells.

Techniques: Migration, Stable Transfection

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA CNALPTC1 promotes cell proliferation and migration of papillary thyroid cancer via sponging miR-30 family

doi:

Figure Lengend Snippet: CNALPTC1 sponges and down-regulates miR-30 family. A. Schematic outlining the predicted binding site of miR-30 family on CNALPTC1. B. The specific bindings of miR-30 family to CNALPTC1 or miR-30 binding site mutated CNALPTC1 (CNALPTC1-mut) were determined by MS2 based RIP assays, followed by qRT-PCR. C. TPC-1 cell lysates were incubated with biotin-labeled CNALPTC1 or CNALPTC1-mut; after pull-down, microRNAs were extracted and determined by qRT-PCR. D. CNALPTC1 or miR-30 binding site mutated CNALPTC1 (CNALPTC1-mut) overexpression plasmids were transfected into TPC-1 cells. Forty-eight hours later, miR-30 family expression levels were determined by qRT-PCR. E. Expression levels of miR-30 family were determined by qRT-PCR in CNALPTC1 stably depleted and control TPC-1 cells. Results are shown as mean ± s.d. of 3 independent experiments. **P < 0.01, ***P < 0.001 by Student’s t-test. RIP, RNA Immunoprecipitation; qRT-PCR, quantitative real-time PCR.

Article Snippet: pSL-MS2-12X (Addgene, Cambridge, MA, USA) was double digested using EcoR I and Not I, and the MS2-12X fragment was inserted into pcDNA3.1, pcDNA3.1-CNALPTC1, or pcDNA3.1-CNALPTC1-mut, named as pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2, respectively. pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2 was co-transfected with pMS2-GFP (Addgene) into TPC-1 cells.

Techniques: Binding Assay, Quantitative RT-PCR, Incubation, Labeling, Over Expression, Transfection, Expressing, Stable Transfection, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA CNALPTC1 promotes cell proliferation and migration of papillary thyroid cancer via sponging miR-30 family

doi:

Figure Lengend Snippet: CNALPTC1 up-regulates miR-30 targets BCL9, SNAI1, and VIM expression. A. Luciferase activities of pmirGLO, pmirGLO-BCL9, pmirGLO-SNAI1, or pmirGLO-VIM upon transfection of CNALPTC1 or miR-30 binding site mutated CNALPTC1 (CNALPTC1-mut) overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix. Results are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. B. Luciferase activities of pmirGLO, pmirGLO-BCL9, pmirGLO-SNAI1, or pmirGLO-VIM in CNALPTC1 stably depleted and control TPC-1 cells. Results are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. C. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, BCL9, SNAI1, and VIM RNA expression levels were determined by qRT-PCR. D. BCL9, SNAI1, and VIM RNA expression levels were determined by qRT-PCR in CNALPTC1 stably depleted and control TPC-1 cells. E. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, BCL9, SNAI1, and VIM protein expression levels were determined by western blot. F. BCL9, SNAI1, and VIM protein expression levels were determined by western blot in CNALPTC1 stably depleted and control TPC-1 cells. Results are shown as mean ± s.d. of 3 independent experiments. **P < 0.01, ***P < 0.001 by Student’s t-test. qRT-PCR, quantitative real-time PCR.

Article Snippet: pSL-MS2-12X (Addgene, Cambridge, MA, USA) was double digested using EcoR I and Not I, and the MS2-12X fragment was inserted into pcDNA3.1, pcDNA3.1-CNALPTC1, or pcDNA3.1-CNALPTC1-mut, named as pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2, respectively. pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2 was co-transfected with pMS2-GFP (Addgene) into TPC-1 cells.

Techniques: Expressing, Luciferase, Transfection, Binding Assay, Over Expression, Cotransfection, Activity Assay, Stable Transfection, RNA Expression, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA CNALPTC1 promotes cell proliferation and migration of papillary thyroid cancer via sponging miR-30 family

doi:

Figure Lengend Snippet: CNALPTC1 exerts oncogenic activity in PTC via sponging miR-30 family. A. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell proliferation was determined by CCK-8 assays. B. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell proliferation was determined by EdU incorporation assays. The red colour indicates EdU-positive nuclei. Scale bars = 100 µm. C. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell apoptosis was determined by TUNEL assays. The green colour indicates TUNEL-positive or apoptotic cells. Scale bars = 100 µm. D. After transfection of CNALPTC1 or CNALPTC1-mut overexpression plasmids, or co-transfection of CNALPTC1 overexpression plasmids and miR-30 mimics mix into TPC-1 cells, cell migration was determined by transwell assays. Represent images are shown. Scale bars = 100 µm. Results are shown as mean ± s.d. of 3 independent experiments. *P < 0.05, **P < 0.01, ns, not significant, by Student’s t-test. PTC, papillary thyroid cancer; CCK-8, Cell Counting Kit-8; EdU, Ethynyl deoxyuridine; TUNEL, TdT-mediated dUTP nick end labeling.

Article Snippet: pSL-MS2-12X (Addgene, Cambridge, MA, USA) was double digested using EcoR I and Not I, and the MS2-12X fragment was inserted into pcDNA3.1, pcDNA3.1-CNALPTC1, or pcDNA3.1-CNALPTC1-mut, named as pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2, respectively. pcDNA3.1-MS2, pcDNA3.1-CNALPTC1-MS2, or pcDNA3.1-CNALPTC1-mut-MS2 was co-transfected with pMS2-GFP (Addgene) into TPC-1 cells.

Techniques: Activity Assay, Transfection, Over Expression, Cotransfection, CCK-8 Assay, TUNEL Assay, Migration, Cell Counting, End Labeling

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) Heatmap illustration of mRNA expression levels in TCGA LUAD and LUSC cohorts ( n = 1,016 tumors). An EMT score calculated for each tumor, as described previously , was correlated with each PI4K family member or, as a comparison, with the EMT-activating transcription factor using Pearson’s coefficient ( r value). ( B ) qPCR analysis of PI4K2A and PI4KB mRNA levels in human lung cancer cell lines classified as epithelial (E) or mesenchymal (M). ( C and D ) WB analysis of PI4K2A, PI4KB, and ZEB1 levels in epithelial ( C ) or mesenchymal ( D ) cells subjected to ZEB1 gain or loss of function, respectively. Relative densitometric values are shown under the gel lanes. α-Tubulin was used as a loading control. Empty vector (Vec), scrambled control (siCTL), and ZEB1 (siZEB1) siRNAs were used. ( E ) WB analysis of PI4K2A in cells transfected with miR mimics. ( F ) PI4K2A 3′-UTR reporter assays. H1299 cells were cotransfected with miR mimics and reporters containing WT or miR-182/-183 binding site mutant 3′-UTRs ( n = 4 replicates per condition). ( G – I ) PI4P ELISA in siRNA-transfected H1299 ( G ), H441 ( H ), and HCC827 ( I ) cells. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed Student’s t test for 2-group comparisons ( B ); 1-way ANOVA test for multiple comparisons ( F – I ). miR-NC, negative control mimic.

Article Snippet: To generate the TurboID-PI4K2A construct, PI4K2A coding sequences (a gift from Feng-Qian Li and Ken-Ichi Takemaru, Addgene plasmid 74223) were inserted into the Flag-TurboID-pcDNA3 vector using XhoI and XbaI. pSL-MS2-12X was double-digested with EcoRI and XhoI, and the MS2-12X fragment was subcloned into the pcDNA3.1 to make the pcDNA-MS2 (12X) vector.

Techniques: Expressing, Comparison, Control, Plasmid Preparation, Transfection, Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) qPCR analysis of ACBD3 mRNA levels in the cell lines (dots) described in B. ( B and C ) WB analysis of ACBD3 protein levels in epithelial ( B ) and mesenchymal ( C ) cells subjected to ZEB1 gain or loss of function, respectively. ( D and E ) PI4P ELISA in mesenchymal ( D ) and epithelial ( E ) cells subjected to ACBD3 gain or loss of function, respectively. ( F ) Schema showing constructs containing MS2 binding sites (12X) fused downstream of a WT or mutant (MT) ACBD3 3′-UTR lacking the miR-34a–binding site (BS). ( G ) MS2-based RIP. MS2-UTR–associated miR-34a was quantified as fold enrichment values relative to MS2. miR-200b was included as a negative control. ( H and I ) WB analysis of ACBD3 and ZFP36L1 levels in cells transfected with miR mimics ( H ) or siRNAs ( I ). ( J and K ) WB analysis of ZFP36L1 levels in cell lysates (input), an MS2-based RIP complex (GFP), or a negative control IP (IgG). ( L ) Schema of the working model. ZEB1 executes a PI4KB-to-PI4K2A dependency switch by silencing miR-34a and miR-182/-183. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. P values were determined by 2-tailed Student’s t test for 2-group comparisons ( A , D , and E ); 1-way ANOVA test for multiple comparisons.

Article Snippet: To generate the TurboID-PI4K2A construct, PI4K2A coding sequences (a gift from Feng-Qian Li and Ken-Ichi Takemaru, Addgene plasmid 74223) were inserted into the Flag-TurboID-pcDNA3 vector using XhoI and XbaI. pSL-MS2-12X was double-digested with EcoRI and XhoI, and the MS2-12X fragment was subcloned into the pcDNA3.1 to make the pcDNA-MS2 (12X) vector.

Techniques: Enzyme-linked Immunosorbent Assay, Construct, Binding Assay, Mutagenesis, Negative Control, Transfection

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) WB analysis confirming target gene deletion in PI4K2A-KO H1299 cells. ( B ) Orthotopic lung tumor size (left plot) and mediastinal and contralateral lung metastasis numbers (right plot) generated in nude mice (dots) by the intrathoracic injection of cells described in A . ( C ) WB analysis confirming target gene depletion in shRNA-transfected 344SQ cells. ( D ) Tumor weights (left plot) and lung metastasis numbers (right plot) generated in syngeneic, immunocompetent mice (dots) by subcutaneous injection of the cells described in C . ( E ) Daily subcutaneous tumor volume measurements (dots) in nude mice treated with PI-273 or vehicle (DMSO). ( F and G ) Tumor tissues removed at necropsy in E were imaged ( F ) and weighed ( G ). ( H ) Orthotopic lung tumor size (left plot) and metastasis numbers (right plot) in nude mice treated with PI-273 or vehicle. ( I ) Kaplan-Meier survival analysis of mice bearing orthotopic lung tumors treated with PI-273 or DMSO. ( J ) WB analysis demonstrating reconstitution of shPI4K2A-transfected H1299 cells (shUTR) with WT or enzyme-dead mutant (D308A) PI4K2A. Empty vector (Vec). ( K ) Orthotopic lung tumors (arrows) generated in nude mice by the cells in J . Scale bars: 5 mm. ( L ) Orthotopic lung tumor size (left plot) and metastasis numbers (right plot). ( M ) Annexin V/propidium iodide flow cytometric analysis of the apoptotic fraction in siRNA-transfected cells. ( N ) Colonies formed in soft agar by siRNA-transfected cells. Values are expressed relative to siCTL. ( O ) Boyden chamber migration and invasion assays on siRNA-transfected cells. Values are expressed relative to siCTL. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. P values were determined by 2-tailed Student’s t test for 2-group comparisons ( E – H ); 1-way ANOVA test for multiple comparisons ( B , D , and L – O ; and log-rank test ( I ). shCTL, control shRNA.

Article Snippet: To generate the TurboID-PI4K2A construct, PI4K2A coding sequences (a gift from Feng-Qian Li and Ken-Ichi Takemaru, Addgene plasmid 74223) were inserted into the Flag-TurboID-pcDNA3 vector using XhoI and XbaI. pSL-MS2-12X was double-digested with EcoRI and XhoI, and the MS2-12X fragment was subcloned into the pcDNA3.1 to make the pcDNA-MS2 (12X) vector.

Techniques: Generated, Injection, shRNA, Transfection, Mutagenesis, Plasmid Preparation, Migration, Control

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) Relative soft agar colony numbers. siRNA-transfected H1299 cells were treated with CM samples from siRNA-transfected H1299 cells. ( B ) Apoptosis assays on siRNA-transfected H1299 cells treated with CM samples. WB analysis of cleaved PARP1 (C-PARP1) and PI4K2A (gel). ( C ) Volcano plot illustration of proteins (dots) identified by LC-MS analysis of CM samples. P value ( y axis) and fold change ( x axis) are shown. PI4K2A-upregulated secreted proteins (pink quadrant) of interest are labeled. ( D ) Gene Ontology analysis of the pink quadrant in C . ( E ) Subcutaneous tumors (dots) were weighed (left plot) and subjected to flow cytometry to quantify CD31 + cells (middle plot) and cleaved-caspase 3 + (CC3 + ) cells (right plot). ( F ) Heatmap illustration of the correlation between mRNAs and EMT scores (Byers or Creighton) in TCGA LUAD cohort. r values were determined by Pearson’s correlation. ( G ) Kaplan-Meier survival analysis of TCGA LUAD and LUSC cohorts based on 6-gene signatures of secreted proteins. Tumors were scored as being above (high) or below (low) each cohort’s median values. ( H – J ) Apoptosis assays and WB analysis of cleaved PARP1 (gel) ( H ), soft agar colony formation assays ( I ), and Boyden chamber migration and invasion assays ( J ) were carried out on siRNA-transfected H1299 cells. ( K ) HUVEC migration in Boyden chambers. CM samples from siRNA-transfected H1299 cells were loaded into the lower wells. Scale bars: 200 μm. ( L ) HUVEC spheroid invasion assay. HUVEC spheroids were seeded in 3D collagen and treated with CM samples. Scale bar: 100 μm. ( M ) HUVEC tube formation assay in 3D Matrigel following treatment with CM samples. Scale bars: 100 μm. ( N ) HUVEC migration in Boyden chambers. CM from siRNA-transfected H1299 cells was loaded into the lower chambers. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed Student’s t test for 2-group comparisons ( E ); 1-way ANOVA test for multiple comparisons ( A , B , and H – N ).

Article Snippet: To generate the TurboID-PI4K2A construct, PI4K2A coding sequences (a gift from Feng-Qian Li and Ken-Ichi Takemaru, Addgene plasmid 74223) were inserted into the Flag-TurboID-pcDNA3 vector using XhoI and XbaI. pSL-MS2-12X was double-digested with EcoRI and XhoI, and the MS2-12X fragment was subcloned into the pcDNA3.1 to make the pcDNA-MS2 (12X) vector.

Techniques: Transfection, Liquid Chromatography with Mass Spectroscopy, Labeling, Flow Cytometry, Migration, Invasion Assay, HUVEC Tube Formation Assay

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) Single-channel and merged confocal micrographs of total and surface VSV-G in H1299 cells cotransfected with siRNAs and EGFP–VSV-G and imaged 30 minutes after transfer to the permissive temperature. Plot shows the ratio of surface VSV-G to total VSV-G in each cell (dot) 30 or 60 minutes after transfer to 32°C. Scale bar: 20 μm. ( B ) BRET measurement of PI4P in RAB6A + vesicles in siRNA-transfected H1299 cells. Results are expressed as a ratio of the values from GSK-A1–treated and vehicle-treated (DMSO) cells at each time point ( n = 5 replicates per group). ( C ) Confocal micrographs of RAB6A + vesicles (blue arrows) and unfissioned RAB6A + tubules (red arrows) emerging from the Golgi. Scale bar: 20 μm. Dotted lines indicate the cell boundaries. Results were quantified per cell (dot plots). ( D ) Venn diagram of PI42KA-interacting proteins identified by TurboID and IP approaches. Overlapping proteins are listed on the right. Reported PI4K2A-interacting proteins ( , ) are shown in bold. ( E ) Schematic illustration of full-length and truncated PI4K2A constructs. WB assays on whole-cell lysates (WCLs) (input) or IP proteins isolated from H1299 cells transfected with MYC-tagged PI4K2A constructs (gel). Full-length (1–479) and truncated constructs are indicated under the gels. IgG was used as the control IP. ( F ) WB analysis of WCLs (WCL) and Golgi-enriched fractions (Golgi) from siRNA-transfected H1299 cells. ( G ) Confocal micrographs of SPP1 + vesicles (arrows) in siRNA-transfected H1299 cells costained with anti-SPP1 and anti–Golgin 97 antibodies. Scale bars: 50 μm. Dot plot shows the vesicle numbers per cell. ( H ) WB analysis of WCLs or enriched subcellular fractions from siRNA-transfected H1299 cells. Densitometric values were normalized to siCTL (graph). Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. *** P < 0.001, by 2-tailed Student’s t test for 2-group comparisons ( B ); 1-way ANOVA test for multiple comparisons ( A , C , G , and H ).

Article Snippet: To generate the TurboID-PI4K2A construct, PI4K2A coding sequences (a gift from Feng-Qian Li and Ken-Ichi Takemaru, Addgene plasmid 74223) were inserted into the Flag-TurboID-pcDNA3 vector using XhoI and XbaI. pSL-MS2-12X was double-digested with EcoRI and XhoI, and the MS2-12X fragment was subcloned into the pcDNA3.1 to make the pcDNA-MS2 (12X) vector.

Techniques: Transfection, Construct, Isolation, Control

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) Intracellular levels of biotinylated Tfn in siRNA-transfected H1299 cells were quantified at the indicated time points after a 30-minute pulse. The percentage of the internalized Tfn pool was calculated relative to the initial loading ( n = 4 samples per condition). ( B ) WB analysis of CD44, ITGB1, and AXL protein levels in WCLs (input) or streptavidin bead–enriched protein samples from H1299 cells that were transfected with PI4K2A/TurboID construct and treated with biotin. Controls included the PI4KB/TurboID construct (PI4KB) and Turbo alone (CTL). ( C ) WB analysis of WCLs (input) or anti-HA immunoprecipitates from H1299 cells transfected with HA-tagged PI4K2A. IgG was used as the control IP. ( D ) SPP1 protein-protein interaction network (STRING-db.org). ( E ) WB analysis of proteins isolated by streptavidin bead–based pulldowns carried out on H1299 cells treated with biotin-labeled recombinant SPP1. ( F ) Confocal micrographs of plasma membrane–bound ITGB1 (arrows, upper panels) and CD44 (arrows, lower panels) in nonpermeabilized siRNA-transfected H1299 cells stained with antibodies against endogenous ITGB1 or CD44. Scale bars: 10 μm. ( G ) WB analysis of cell membrane–enriched fractions (Mem.) and WCL. Densitometric values are shown under the gels. ( H ) WB analysis of cleaved PARP1 (gel) and flow cytometric analysis of annexin V/PI–stained cells (graph) to quantify apoptosis in siRNA-transfected H1299 cells. ( I ) Boyden chamber migration and invasion assays on siRNA-transfected cells. ( J ) Schematic illustration of the working model. PI4K2A coordinates exocytic and endocytic vesicular trafficking to activate an SPP1-dependent autocrine loop. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. P values were determined by 1-way ANOVA test for multiple comparisons ( A , H , and I ).

Article Snippet: To generate the TurboID-PI4K2A construct, PI4K2A coding sequences (a gift from Feng-Qian Li and Ken-Ichi Takemaru, Addgene plasmid 74223) were inserted into the Flag-TurboID-pcDNA3 vector using XhoI and XbaI. pSL-MS2-12X was double-digested with EcoRI and XhoI, and the MS2-12X fragment was subcloned into the pcDNA3.1 to make the pcDNA-MS2 (12X) vector.

Techniques: Transfection, Construct, Control, Isolation, Labeling, Recombinant, Clinical Proteomics, Membrane, Staining, Migration

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) WB analysis of parental and PI4K2A-KO H1299 cells. Densitometric values are shown under the gels. Positive (EGFR) and negative (ITGB1) controls. ( B ) Correlation between AXL and PI4K2A protein levels in cell lines (dots). ( C ) WB analysis of parental and PI4K2A-KO H1299 cells treated with the AXL ligand Gas6. pAKT, phosphorylated AKT. Graphs show densitometric analysis of pAXL and pAKT levels normalized to t = 0 minutes. ( D ) Boyden chamber migration assays on parental and PI4K2A-KO H1299 cells treated with (+) or without (–) Gas6. ( E ) WB analysis of parental and PI4K2A-KO H1299 cells stably transfected with empty vector or AXL. ( F ) Boyden chamber migration assays on cells in E . ( G ) WB analysis of parental and PI4K2A-KO H1299 cells treated with cycloheximide (CHX). Graphs show the densitometric values. ( H ) Single-channel and merged confocal micrographs of parental and PI4K2A-KO cells costained with anti-AXL and anti-LAMP1 antibodies. Lysosomal AXL (inset, arrows) was quantified as the percentage of total AXL that colocalized with LAMP1 per field ( n = 10 fields per condition). Scale bar: 10 μm. Original magnification, ×2.5 (enlarged insets). ( I ) WB analysis of PI4K2A-KO cells treated with proteasomal (MG132) or lysosomal (leupeptin or monensin) inhibitors. DMSO was used as the vehicle. l.e., long exposure duration; s.e., short exposure duration. ( J ) WB analysis of siRNA-transfected H1299 cells. Densitometric values are shown under the gel. ( K ) WB analysis of WCLs (input) and anti-HA immunoprecipitates from H1299 cells transfected with HA-tagged PI4K2A. IgG was used as the negative control IP. ( L ) WB analysis of WCLs (input) and anti-HSP90 immunoprecipitates from parental and PI4K2A-KO H1299 cells. IgG was used as the negative control IP. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. ** P < 0.01 and *** P < 0.001, by 2-tailed Student’s t test for 2-group comparisons ( C , D , G , and H ); 1-way ANOVA test for multiple comparisons ( F ).

Article Snippet: To generate the TurboID-PI4K2A construct, PI4K2A coding sequences (a gift from Feng-Qian Li and Ken-Ichi Takemaru, Addgene plasmid 74223) were inserted into the Flag-TurboID-pcDNA3 vector using XhoI and XbaI. pSL-MS2-12X was double-digested with EcoRI and XhoI, and the MS2-12X fragment was subcloned into the pcDNA3.1 to make the pcDNA-MS2 (12X) vector.

Techniques: Migration, Stable Transfection, Transfection, Plasmid Preparation, Negative Control