guide rna lentiviral expression vector  (Addgene inc)

Journal: Thoracic Cancer

Article Title: CDCA4 interacts with IGF2BP1 to regulate lung adenocarcinoma proliferation via the PI3K / AKT pathway

doi: 10.1111/1759-7714.14800

Figure Lengend Snippet: The cell division cycle‐associated 4 (CDCA4) expression was upregulated in the lung adenocarcinomas (LUAD) tissues and cell lines. (a) The CDCA4 expression was higher in the 483 LUAD tissues than in the 347 normal lung tissues, with reference to the The Cancer Genome Atlas (TCGA) database ( p < 0.05). (b) Kaplan–Meier overall survival curves and disease‐free survival curves in 478 LUAD patients. (c) Comparison of the CDCA4 protein levels in the non‐small cell lung cancer (NSCLC) tissues and adjacent non‐tumor tissues. (d) The messenger (mRNA) and protein expression of CDCA4 in LUAD cell lines

Article Snippet: Lentiviral vectors expressing CDCA4‐Cas9/single guide RNA (sgRNA) plasmid and CDCA4 were obtained from Vigene Biosciences.

Techniques: Expressing, Comparison

Journal: Thoracic Cancer

Article Title: CDCA4 interacts with IGF2BP1 to regulate lung adenocarcinoma proliferation via the PI3K / AKT pathway

doi: 10.1111/1759-7714.14800

Figure Lengend Snippet: Knockout of cell division cycle‐associated 4 (CDCA4) inhibits lung adenocarcinomas (LUAD) cell proliferation. (a) Validation of the messenger (mRNA) and protein level efficiency of CDCA4 overexpression cells A549, H460, and CDCA4 knockdown cells H1299, PC9 (mean ± standard deviation [SD], Student's t ‐test, *** p < 0.005). (b) CDCA4 overexpression in A549 and H460 promotes the ability of colony formation and CDCA4 knockdown in H1299 and PC9 inhibits the cellular clonogenic ability (mean ± SD, Student's t ‐test, * p < 0.05, *** p < 0.005). (c) The knockdown of CDCA4 expression in H1299 and PC9 inhibited proliferation, whereas the CDCA4 overexpression in A549 and H460 promoted growth of cells (mean ± SD, Student's t ‐test, * p < 0.05, *** p < 0.005). (d) Differences in the cell proliferation capacity reflected by real‐time cell analysis (RTCA) results in A549 and H1299 (mean ± SD, Student's t ‐test, ** p < 0.01, *** p < 0.005). (e) The EdU assay was used to detect the changes in the proliferation ability after CDCA4 knockout and overexpression in LUAD cells (mean ± SD, Student's t ‐test, ** p < 0.01, *** p < 0.005)

Article Snippet: Lentiviral vectors expressing CDCA4‐Cas9/single guide RNA (sgRNA) plasmid and CDCA4 were obtained from Vigene Biosciences.

Techniques: Knock-Out, Biomarker Discovery, Over Expression, Knockdown, Standard Deviation, Expressing, Cell Analysis, EdU Assay

Journal: Thoracic Cancer

Article Title: CDCA4 interacts with IGF2BP1 to regulate lung adenocarcinoma proliferation via the PI3K / AKT pathway

doi: 10.1111/1759-7714.14800

Figure Lengend Snippet: Cell division cycle‐associated 4 (CDCA4) knockdown causes S‐phase arrest. The downregulation of CDCA4 in H1299 caused S‐phase arrest, whereas the proportion of S‐phase was decreased in A549 overexpressing CDCA4 when compared with the control cells (mean ± standard deviation [SD], Student's t ‐test, * p < 0.05, ** p < 0.01, #not significant)

Article Snippet: Lentiviral vectors expressing CDCA4‐Cas9/single guide RNA (sgRNA) plasmid and CDCA4 were obtained from Vigene Biosciences.

Techniques: Knockdown, Control, Standard Deviation

Journal: Thoracic Cancer

Article Title: CDCA4 interacts with IGF2BP1 to regulate lung adenocarcinoma proliferation via the PI3K / AKT pathway

doi: 10.1111/1759-7714.14800

Figure Lengend Snippet: Cell division cycle‐associated 4 (CDCA4) affects cell proliferation mediated by the phosphatidylinositol 3 kinase (PI3K)– protein kinase B (AKT) signaling pathway. Western blotting analysis indicates the expression of the PI3K–AKT signaling pathway‐related proteins in H1299 and A549 cells (mean ± standard deviation [SD], Student's t ‐test, ** p < 0.01, #not significant)

Article Snippet: Lentiviral vectors expressing CDCA4‐Cas9/single guide RNA (sgRNA) plasmid and CDCA4 were obtained from Vigene Biosciences.

Techniques: Western Blot, Expressing, Standard Deviation

Journal: Thoracic Cancer

Article Title: CDCA4 interacts with IGF2BP1 to regulate lung adenocarcinoma proliferation via the PI3K / AKT pathway

doi: 10.1111/1759-7714.14800

Figure Lengend Snippet: Cell division cycle‐associated 4 (CDCA4) interacts with IGF2BP1 and promotes the post‐transcriptional translation level of IGF2BP1. (a) The results of mass spectrometry analysis of differential fragments in A549 cells overexpressing CDCA4. (b) Reducing IGF2BP1 inhibits the clonogenic ability of A549 (mean ± SD, Student's t ‐test, * p < 0.05). (c) The downregulation of CDCA4 in A549 inhibits cell proliferation (mean ± SD, Student's t ‐test, *** p < 0.005). (d) The effect of decreasing IGF2BP1 expression on the phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway. (e) CO‐immunoprecipitation (IP) assay demonstrating the binding of CDCA4 and IGF2BP1 in A549 cells. (f) Western blotting analysis indicating changes in the IGF2BP1 protein expression in stable transgenic strains A549 and H1299 (mean ± standard deviation [SD], Student's t ‐test, * p < 0.05, ** p < 0.01). (g) The effect of upregulation versus inhibition of CDCA4 on the messenger RNA (mRNA) level of IGF2BP1. (h) Quantitative polymerase chain reaction (qPCR) analysis indicating the changes in the CDCA4 expression in A549 transfection with si‐IGF2BP1. (i) The effect of downregulation of IGF2BP1 in A549 on the expression of CDCA4 protein

Article Snippet: Lentiviral vectors expressing CDCA4‐Cas9/single guide RNA (sgRNA) plasmid and CDCA4 were obtained from Vigene Biosciences.

Techniques: Mass Spectrometry, Expressing, Immunoprecipitation, Binding Assay, Western Blot, Transgenic Assay, Standard Deviation, Inhibition, Real-time Polymerase Chain Reaction, Transfection

Journal: Thoracic Cancer

Article Title: CDCA4 interacts with IGF2BP1 to regulate lung adenocarcinoma proliferation via the PI3K / AKT pathway

doi: 10.1111/1759-7714.14800

Figure Lengend Snippet: IGF2BP1 knockdown can alter the function of cell division cycle‐associated 4 (CDCA4). (a) Western blotting analysis showing the expression of IGF2BP1 protein in H1299, A549 stable transfer strain transfection with si‐IGF2BP1. (b) Effect of downregulation of IGF2BP1 on the proliferation efficiency of CDCA4 knockout cells H1299 and CDCA4 overexpressing cells A549 (mean ± standard deviation [SD], Student's t ‐test, *** p < 0.005). (c) Differences in the cell proliferation capacity as reflected by the real‐time cell analysis (RTCA) results (mean ± SD, Student's t ‐test, * p < 0.05，** p < 0.01, *** p < 0.005). (d) The effect of the downregulation of IGF2BP1 on the phenotype of CDCA4 clone formation (mean ± SD, Student's t ‐test, * p < 0.05，** p < 0.01). (e) EdU assay indicated the effect of IGF2BP1 knockdown on the proliferation ability of lung adenocarcinomas (LUAD) cells with high and low expression of CDCA4 (mean ± SD, Student's t ‐test, * p < 0.05，** p < 0.01, *** p < 0.005)

Article Snippet: Lentiviral vectors expressing CDCA4‐Cas9/single guide RNA (sgRNA) plasmid and CDCA4 were obtained from Vigene Biosciences.

Techniques: Knockdown, Western Blot, Expressing, Transfection, Knock-Out, Standard Deviation, Cell Analysis, EdU Assay

Journal: Thoracic Cancer

Article Title: CDCA4 interacts with IGF2BP1 to regulate lung adenocarcinoma proliferation via the PI3K / AKT pathway

doi: 10.1111/1759-7714.14800

Figure Lengend Snippet: The knockdown of cell division cycle‐associated 4 (CDCA4) inhibits the ability of lung adenocarcinomas (LUAD) cells to proliferate in vivo. (a) Western blotting analysis indicating the expression of CDCA4 protein in A549 transfection with shCDCA4. (b,c) Bodyweight changes and tumor volume changes in nude mice after subcutaneous injection of tumor cells (mean ± SD, Student's t ‐test, * p < 0.05). (d,e) The downregulation of the CDCA4 expression significantly inhibits tumor growth in vivo (mean ± standard deviation [SD], Student's t ‐test, *** p < 0.005). (f) The results of KI67 staining of tumor sections

Article Snippet: Lentiviral vectors expressing CDCA4‐Cas9/single guide RNA (sgRNA) plasmid and CDCA4 were obtained from Vigene Biosciences.

Techniques: Knockdown, In Vivo, Western Blot, Expressing, Transfection, Injection, Standard Deviation, Staining

Journal: Blood

Article Title: Salt-inducible kinase inhibition suppresses acute myeloid leukemia progression in vivo

doi: 10.1182/blood.2019001576

Figure Lengend Snippet: SIK3 and MEF2C are selectively essential for the growth of AML and multiple myeloma cells. (A) SIK3 and MEF2C essentiality scores extracted from the DepMap database of cancer cell lines.35 Shown is a boxplot distribution of the copy number–adjusted essentiality score (CERES; a normalized metric of gene essentiality) of SIK3 and MEF2C across all 558 cell lines, 16 AML lines, 18 multiple myeloma lines, and 498 solid tumor cell lines. (B-C) Scatterplots of SIK3 and MEF2C essentiality scores in human AML cell lines in the DepMap (CERES) or from Wang et al52 (CRISPR scores). (D) Western blot analysis of MEF2C in RN2 and 3T3 whole-cell lysates. (E) Competition-based proliferation assays in which cells were infected with the indicated sgRNAs linked to GFP. Bar graphs represent the mean ± standard error of the mean (SEM; n = 3). (F) Bright-field images of methylcellulose-based colony-formation assays of normal myeloid progenitors or RN2 cells on day 7 after retroviral transduction with control or Sik3 shRNAs. (G) Quantification of the immature/blast colonies shown in panel F. Mean ± SEM (n = 4). (H) Western blot analysis of SIK3 performed on day 6 after infection of the indicated shRNAs. (I) Bioluminescence imaging of wild-type C57BL/6 mice receiving transplants of Cas9-expressing RN2 cells transduced with the indicated sgRNA. Representative images are shown on the indicated day after transplantation. (J) Quantification of bioluminescence from panel I. Values represent photons per second (p/s) of bioluminescent signal detection (mean ± SEM). The P value was calculated by unpaired Student t test (n = 5). (K) Survival curves of the mice in panel I. The P value was calculated by log-rank (Mantel-Cox) test (n = 5). (L) Bioluminescence imaging of NSG (NOD-SCID/IL2Rgammanull) mice which received transplants of Cas9-expressing MV4-11 cells transduced with the indicated sgRNA. Representative images are shown on the indicated day after transplantation. (M) Quantification of bioluminescence from panel L. Values represent photons per second (p/s) of bioluminescent signal detection (mean ± SEM). The P value was calculated by unpaired Student t test (n = 5). (N) Survival curves of the mice in panel L. The P value was calculated by log-rank (Mantel-Cox) test (n = 5). sgNeg1, sgNeg2, shREN: negative controls. sgRpa3: positive control.

Article Snippet: Plasmid constructions The lentiviral single-guide RNA (sgRNA) expression vectors (LRG2.1, Addgene_108098; LRG, Addgene_65656) and the lentiviral Cas9 (LentiV_Cas9_puro, Addgene_108100) or luciferase (Lenti-luciferase-P2A-Neo, Addgene_105621) expression vectors were described previously.

Techniques: CRISPR, Western Blot, Infection, Retroviral, Transduction, Control, Imaging, Expressing, Transplantation Assay, Positive Control