Journal: Stem Cell Research & Therapy

Article Title: A puromycin selectable cell line for the enrichment of mouse embryonic stem cell-derived V3 interneurons

doi: 10.1186/s13287-015-0213-z

Figure Lengend Snippet: Generation and identification of Sim1-Puro cell line. a Red recombineering was utilized to insert the region approximately 2 kb upstream to 10 kb downstream of Exon 1 of the Sim1 gene from RP23-223 M2 BAC into the pStartK backbone, generating the Sim1-pStartK plasmid. b After AscI cut sites were introduced by another red recombination reaction, a PAC-PGK-Neo cassette was inserted into the open reading frame of Sim1 Exon1in the Sim1-pStartK plasmid. Using gateway recombination, the pStartK backbone was replaced with the pWS-TK3 backbone to introduce the negative selection gene, TK. 5’ and 3’ homology arms are labeled. c CRISPR/Cas9 targeting of the Sim1 gene was used to generate a double stranded break at the desired recombination location. Sim1-Puro-pStartTK recombined into the Sim1 locus as shown by the dotted lines. 5’ and 3’ homology arms are labeled. d The Sim1-Puro cell line with PAC in Sim1 Exon 1. Junction PCR (JPCR) primer for approximately 2.6 kb was used to screen for the desired recombination event. e JPCR bands of positive (+) and negative (−) clones with a 1 kb ladder. f Copy number assay shows Sim1 clones have one copy of PAC by comparison with RW4 ESCs (0 copy) and Hb9-Puro ESC (1 copy) controls. AMP, Ampicillin resistance gene; AscI, Restriction enzyme site; attB1 & attB2, Gateway recombination results; HA, Homology arm; JPCR, Junction PCR; ori, Origin of replication; PAC, Puromycin resistance gene; PGK, Phosphoglycerate kinase promoter sequence; Neo, Neomycin resistance gene; Sim1 ATG, Translation start in Sim1 Exon1; TK, Thymidine kinase

Article Snippet: Approximately 1 × 10 7 RW4 ESCs were resuspended in electroporation buffer with 10 μg of Sim1-Puro-pStartTK vector and 200–300 ng of a Cas9 guide RNA vector (deemed gSim1.MS8.mSim1.g6a, with guide RNA (Fig. , Cas9 Guide RNA) targeting 5’-gtccatcattcgtgtcttcc cgg-3’ near the Sim1 start codon (Fig. , Cas9 Target)) in the MLM3636 plasmid (Addgene plasmid #43860) and 200–300 ng of the Cas9 nuclease expression plasmid p3s-Cas9HC (Addgene plasmid #43945).

Techniques: Plasmid Preparation, Introduce, Selection, Labeling, CRISPR, Clone Assay, Sequencing

Journal: Cancer Management and Research

Article Title: Knockdown of NUDT21 inhibits proliferation and promotes apoptosis of human K562 leukemia cells through ERK pathway

doi: 10.2147/CMAR.S173496

Figure Lengend Snippet: Expression levels of NUDT21 in human leukemia. Notes: ( A ) mRNA expression levels of NUDT21 in bone marrow samples from healthy control subjects (HC1–HC15) and CML patients (P1–P15). The mRNA ( B ) expression levels of NUDT21 were evaluated by qRT-PCR in human normal PBMCs, human leukemia cells K562, Jurkat, and hl-60 cells. The protein ( C and D ) expression levels of NUDT21 were evaluated by Western blotting in human normal PBMCs, human leukemia cells K562, Jurkat, and hl-60 cells. GAPDH was used as internal control; the data are the mean ± SD for duplicate experiments. *** P < 0.001. Abbreviations: CML, chronic myelocytic leukemia; HC, healthy control; qRT-PCR, quantitative reverse polymerase chain reaction.

Article Snippet: Specific shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGT-GTCACGT-3′ were synthesized by Santa Cruz Biotechnology Inc.

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title: Knockdown of NUDT21 inhibits proliferation and promotes apoptosis of human K562 leukemia cells through ERK pathway

doi: 10.2147/CMAR.S173496

Figure Lengend Snippet: Knockdown of NUDT21 in leukemia cells. Notes: Western blotting ( A and B ) and qPCR ( C ) were used to confirm the knockdown effects of shRNAs against NUDT21 in K562, Jurkat and HL-60 cells. The data are the mean ± SD for duplicate experiments; *** P <0.001. Abbreviation: qRT-PCR, quantitative reverse polymerase chain reaction.

Article Snippet: Specific shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGT-GTCACGT-3′ were synthesized by Santa Cruz Biotechnology Inc.

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title: Knockdown of NUDT21 inhibits proliferation and promotes apoptosis of human K562 leukemia cells through ERK pathway

doi: 10.2147/CMAR.S173496

Figure Lengend Snippet: K562 cell growth upon knockdown of NUDT21. Notes: ( A ) Proliferation capabilities were detected by CCK-8 in K562, Jurkat, and HL-60 cells (2×10 3 ) for 5 days after transfection of shRNA control or NUDT21 shRNA. ( B ) K562 cells were transfected with shRNA control or NUDT21 shRNA for 48 hours and re-seeded in 96-well plate for BrdU assays. ( C ) Cell cycle phase distributions were detected by flow cytometry in K562 cells after transfection of shRNA control or NUDT21 shRNA. ( D ) Western blotting analysis of the protein expressions of PCNA and cyclin E after transfection of shRNA control or NUDT21 shRNA. The data are expressed as mean ± SD for duplicate experiments. * P <0.05; ** P <0.01; *** P <0.001. Abbreviations: CCK-8, Cell Counting Kit-8; BrdU, bromodeoxyuridine.

Article Snippet: Specific shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGT-GTCACGT-3′ were synthesized by Santa Cruz Biotechnology Inc.

Techniques: Knockdown, CCK-8 Assay, Transfection, shRNA, Control, Flow Cytometry, Western Blot, Cell Counting

Journal: Cancer Management and Research

Article Title: Knockdown of NUDT21 inhibits proliferation and promotes apoptosis of human K562 leukemia cells through ERK pathway

doi: 10.2147/CMAR.S173496

Figure Lengend Snippet: K562 cell apoptosis upon knockdown of NUDT21. Notes: ( A ) The activities of Caspase3/7 were determined by Caspase-Glo 3/7 assays in K562 cells (1.5×10 4 ) after transfection of shRNA control or NUDT21 shRNA. ( B ) The percentage of annexin V-positive revealed apoptosis. Percentages of cells undergoing apoptosis in different groups are shown. ( C and D ) Bax and Bcl-2 protein were determined by Western blot analysis in K562 cells after transfection of shRNA control or NUDT21 shRNA. Data are expressed as mean ± SD for duplicate experiments. ** P <0.01; *** P <0.001. Abbreviation: PI, propidium iodide.

Article Snippet: Specific shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGT-GTCACGT-3′ were synthesized by Santa Cruz Biotechnology Inc.

Techniques: Knockdown, Transfection, shRNA, Control, Western Blot

Journal: Cancer Management and Research

Article Title: Knockdown of NUDT21 inhibits proliferation and promotes apoptosis of human K562 leukemia cells through ERK pathway

doi: 10.2147/CMAR.S173496

Figure Lengend Snippet: Deregulation of signaling pathways upon knockdown of NUDT21. Notes: ( A ) Heatmap of 697 differentially expressed genes after knockdown of NUDT21 in K562 cells. ( B ) Enrichment analysis of differentially expressed genes in GO terms. ( C ) Enrichment analysis of differentially expressed genes in KEGG pathways. ( D ) The histogram showing the relative protein expressions of the key signaling components in K562 cells after transfection of shRNA control or NUDT21 shRNA. Data are expressed as mean ± SD for duplicate experiments. Abbreviations: GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomics.

Article Snippet: Specific shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGT-GTCACGT-3′ were synthesized by Santa Cruz Biotechnology Inc.

Techniques: Protein-Protein interactions, Knockdown, Transfection, shRNA, Control

Journal: Cancer Management and Research

Article Title: Knockdown of NUDT21 inhibits proliferation and promotes apoptosis of human K562 leukemia cells through ERK pathway

doi: 10.2147/CMAR.S173496

Figure Lengend Snippet: Potential mechanisms underlying the effects of NUDT21 on K562 cell growth. Notes: ( A ) The protein levels of p-ERK1/2 and PTEN were shown in K562 cells transfected with shRNA control or NUDT21 shRNA using Western blot analysis. ( B ) The mRNA levels of PTEN were compared between K562 cells of shRNA control group and NUDT21 shRNA group using qRT-PCR analysis. ( C ) BrdU incorporation in K562 cells treated with honokiol or control. ( D ) Percentages of cells undergoing apoptosis (annexin V-positive) in different groups were shown. Data are expressed as mean ± SD for duplicate experiments. * P <0.05; ** P <0.01; *** P <0.001. Abbreviations: BrdU, bromodeoxyuridine; PI, propidium iodide; PTEN, phosphatase and tensin homolog deleted on chromosome 10; qRT-PCR, quantitative reverse polymerase chain reaction.

Article Snippet: Specific shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGT-GTCACGT-3′ were synthesized by Santa Cruz Biotechnology Inc.

Techniques: Transfection, shRNA, Control, Western Blot, Quantitative RT-PCR, BrdU Incorporation Assay, Polymerase Chain Reaction

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Calreticulin Promotes Proliferation and Migration But Inhibits Apoptosis in Schwann Cells

doi: 10.12659/MSM.900956

Figure Lengend Snippet: The difference in calreticulin (CRT) expression in transfected Schwann cells (SCs). SCs were transfected with pcDNA3.1 (pcNC), pcDNA3.1-CRT (pc-CRT), non-silencing small interfering RNA (siNC), or specific small interfering RNA against CRT (siCRT). After transfection, cells were harvested for quantitative real-time (qRT)-PCR ( A ) and Western blot analysis ( B ). Data presented are the mean of at least 3 independent experiments. Error bars indicate SD. ** P <0.01; *** P <0.001.

Article Snippet: Specific siRNA against CRT (siCRT) targeting 5′-GGA GCA GUU UCU GGA CGG A-3′ and a non-silencing siRNA (siNC) targeting 5′-TTC TCC GAA CGT GTC ACG T-3′ were synthesized by GenePharma (Shanghai, China).

Techniques: Expressing, Transfection, Small Interfering RNA, Quantitative RT-PCR, Western Blot

Journal: OncoTargets and therapy

Article Title: SPAG9 is involved in hepatocarcinoma cell migration and invasion via modulation of ELK1 expression

doi: 10.2147/OTT.S98727

Figure Lengend Snippet: SPAG9 and ELK1 expression in HCC tissue specimens and cell lines. Notes: ( A ) IHC staining of SPAG9 and ELK1 in hepatocarcinomas and adjacent noncancerous liver tissues. Weak SPAG9 and ELK1 staining can be observed in the adjacent noncancerous liver tissues (left panel) compared to the strong SPAG9 cytoplasmic staining and ELK1 nuclear staining in cancerous tissues (right panel) (magnification: 200×). ( B ) Protein and ( C ) mRNA levels of SPAG9 and ELK1 were examined in HCCLM3, HuH7, and HepG2 cell lines using Western blot and qRT-PCR analyses, respectively. ( D ) Mann–Whitney U -testing of IHC scores (*** P <0.001) (the bars show standard error), and ( E ) Spearman rank correlation analysis of SPAG9 and ELK1 expression in HCC tissues. Abbreviations: SPAG9, sperm-associated antigen 9; IHC, immunohistochemistry; qRT-PCR, quantitative real-time polymerase chain reaction; ELK1, ETS-like gene 1, tyrosine kinase; HCC, hepatocellular carcinoma.

Article Snippet: SPAG9 -specific siRNA targeting 5′-GCAATGACTCAGATGCATA-3′, ELK1 -specific siRNA targeting 5′-CACATCCCTTCTATCAGCGTGGATG-3′, and control scrambled siRNA were chemically synthesized from RIBO-BIO (1451493202, S151113152330; Guangzhou, People’s Republic of China).

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot, Quantitative RT-PCR, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: SPAG9 is involved in hepatocarcinoma cell migration and invasion via modulation of ELK1 expression

doi: 10.2147/OTT.S98727

Figure Lengend Snippet: The relationship between SPAG9 expression and the clinical features of HCC

Article Snippet: SPAG9 -specific siRNA targeting 5′-GCAATGACTCAGATGCATA-3′, ELK1 -specific siRNA targeting 5′-CACATCCCTTCTATCAGCGTGGATG-3′, and control scrambled siRNA were chemically synthesized from RIBO-BIO (1451493202, S151113152330; Guangzhou, People’s Republic of China).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: SPAG9 is involved in hepatocarcinoma cell migration and invasion via modulation of ELK1 expression

doi: 10.2147/OTT.S98727

Figure Lengend Snippet: Reduction in SPAG9 expression suppresses cell migration and invasion of HCC cell lines. Notes: ( A and B ) SPAG9 siRNA-treated cells had decreased mRNA ( A ) and protein ( B ) levels compared to the control cells. ( C ) Results from the wound-healing assay showed that SPAG9 depletion decreased cell migration of HCCLM3 and HuH7 cells. ( D ) Results from the transwell migration and matrigel invasion assay showed that SPAG9 depletion decreased cell migration and invasion of HCCLM3 and HuH7 cells; * P <0.05, ** P <0.01. Abbreviations: SPAG9, sperm-associated antigen 9; siRNA, small interfering RNA; HCC, hepatocellular carcinoma; h, hour.

Article Snippet: SPAG9 -specific siRNA targeting 5′-GCAATGACTCAGATGCATA-3′, ELK1 -specific siRNA targeting 5′-CACATCCCTTCTATCAGCGTGGATG-3′, and control scrambled siRNA were chemically synthesized from RIBO-BIO (1451493202, S151113152330; Guangzhou, People’s Republic of China).

Techniques: Expressing, Migration, Control, Wound Healing Assay, Invasion Assay, Small Interfering RNA

Journal: OncoTargets and therapy

Article Title: SPAG9 is involved in hepatocarcinoma cell migration and invasion via modulation of ELK1 expression

doi: 10.2147/OTT.S98727

Figure Lengend Snippet: SPAG9 regulates ELK1 expression and p38 activation in HCC cell lines. Notes: ( A ) Western blot and ( B ) qRT-PCR analyses revealed that siRNA knockdown of SPAG9 decreased protein and mRNA levels of SPAG9 and ELK1 , respectively, in HCCLM3 and HuH7 cell lines. ( C ) Western blot analysis showed that SPAG9 depletion decreased SPAG9 and p-p38 levels in HCCLM3 and HuH7 cells. Abbreviations: SPAG9, sperm-associated antigen 9; siRNA, small interfering RNA; qRT-PCR, quantitative real-time polymerase chain reaction; ELK1, ETS-like gene 1, tyrosine kinase; p-p38, phosphorylated protein-38; HCC, hepatocellular carcinoma.

Article Snippet: SPAG9 -specific siRNA targeting 5′-GCAATGACTCAGATGCATA-3′, ELK1 -specific siRNA targeting 5′-CACATCCCTTCTATCAGCGTGGATG-3′, and control scrambled siRNA were chemically synthesized from RIBO-BIO (1451493202, S151113152330; Guangzhou, People’s Republic of China).

Techniques: Expressing, Activation Assay, Western Blot, Quantitative RT-PCR, Knockdown, Small Interfering RNA, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: SPAG9 is involved in hepatocarcinoma cell migration and invasion via modulation of ELK1 expression

doi: 10.2147/OTT.S98727

Figure Lengend Snippet: SPAG9-induced migration and invasion of HCC cells is dependent on ELK1. Notes: ( A ) Transwell migration and matrigel invasion assay revealed that ELK1 transfection rescued the inhibition in cell migration and invasion induced by SPAG9 depletion. ( B ) The expression of ELK1 was significantly upregulated in the siCtrl + ELK group and siSPAG9+ ELK1 group compared to the controls. ( C ) Transwell migration and matrigel invasion assay revealed that ELK1 depletion decreased the migration and invasion in HuH7 cells. ( D ) Western blot showed that siRNA knockdown of ELK1 decreased the protein level of ELK1, while SPAG9 expression was not changed in HuH7 cells. * P <0.05. Abbreviations: SPAG9, sperm-associated antigen 9; siSPAG9, SPAG9 siRNA; siCtrl, control siRNA; siRNA, small interfering RNA; ELK1, ETS-like gene 1, tyrosine kinase; HCC, hepatocellular carcinoma.

Article Snippet: SPAG9 -specific siRNA targeting 5′-GCAATGACTCAGATGCATA-3′, ELK1 -specific siRNA targeting 5′-CACATCCCTTCTATCAGCGTGGATG-3′, and control scrambled siRNA were chemically synthesized from RIBO-BIO (1451493202, S151113152330; Guangzhou, People’s Republic of China).

Techniques: Migration, Invasion Assay, Transfection, Inhibition, Expressing, Western Blot, Knockdown, Control, Small Interfering RNA

Journal: Molecular Cancer

Article Title: Increased accumulation of hypoxia-inducible factor-1α with reduced transcriptional activity mediates the antitumor effect of triptolide

doi: 10.1186/1476-4598-9-268

Figure Lengend Snippet: HIF-1α silencing partially prevented the proliferation inhibition and apoptosis induction by triptolide . A . siRNAs targeting three different sequences of the HIF-1α gene were used to effectively silence this gene in SKOV-3 cells (Western blotting). GFP siRNA was used as scrambled control. B . The SKOV-3 cells transfected with HIF-1α or GFP siRNA were treated with triptolide for 48 h and then subjected to SRB assays for the proliferation inhibition. C . The SKOV-3 cells transfected with HIF-1α or GFP siRNA were treated with triptolide for 36 h and then assayed for apoptotic induction by flow cytometry as described in the Materials and Methods section. The representative histograms were from three independent experiments with similar results. D . The apoptosis rate from C. Data shown in B and D were expressed as mean ± SD, n = 3. The significant difference between triptolide-treated groups and hypoxia-control groups was analyzed by Student t test. * P < 0.05.

Article Snippet: siRNAs (HIF-1α siRNA-1 targeting 5'-CUG AUG ACC AGC AAC UUG ATT-3' [ ], HIF-1α siRNA-2 targeting 5' -GCU CAA UUU AUG AAU AUU ATT-3', HIF-1α siRNA-3 targeting 5'-GAA GGA ACC UGA UGC UUU ATT-3' and GFP (scrambled) siRNA targeting 5'-GAC CCG CGC CGA GGU GAA GTT-3') were obtained from GenePharma (Shanghai, China).

Techniques: Inhibition, Western Blot, Control, Transfection, Flow Cytometry