Journal: Frontiers in Oncology

Article Title: MIF promotes cell invasion by the LRP1-uPAR interaction in pancreatic cancer cells

doi: 10.3389/fonc.2022.1028070

Figure Lengend Snippet: Detection of MIF expression in PDAC cell lines Western blot analysis of MIF protein levels in five human PDAC cell lines (A) , MIF expression after CoCl 2 treatment for 24 h compared with vehicle (B) , transduction efficiency of lentivirus-cDNA-MIF (exMIF) or vector (vec) for MIF overexpression and lentivirus-shRNA (shMIF) or nontarget control (shNTC) for MIF knockdown (C) , and MIF expression after CoCl 2 treatment in stably transduced cells (D) . The loading control was assessed by GAPDH blotting. The numbers below the blots are quantitative ratios of the MIF/GAPDH band densities. CoCl 2 concentration used: SW1990, 150 μM; BxPC-3, 200 μM; AsPC-1, 150 μM; PANC-1, 300 μM.

Article Snippet: The lentivirus expressing shRNA targeting MIF (NM_002415) (targeting 5’-GACAGGGTCTACATCAACTAT-3’) and lentivirus vector for full-length MIF overexpression (NM_002415) were synthesized by GeneChem (Shanghai, China).

Techniques: Expressing, Western Blot, Transduction, Plasmid Preparation, Over Expression, shRNA, Control, Knockdown, Stable Transfection, Concentration Assay

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: Figure 1 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: The antibodies used in this study were as follows: anti-Flag (F1804, Sigma-Aldrich Co., St Louis, MO, USA); anti-Proliferating Cell Nuclear Antigen (PCNA) (sc-56; Santa Cruz Biotechnology Inc., Dallas, TX, USA); anti-mouse IgG (sc-2005; Santa Cruz Biotechnology Inc.); anti-NUDT21 (ab183660; Abcam, Cambridge, UK); anticyclin E (ab133266; Abcam); anti-Bax (ab53154; Abcam); anti-Bcl-2 (ab32124; Abcam); anti-phosphatase and tensin homolog deleted on chromosome 10 (PTEN) (ab32199; Abcam); anti-ERK1+ ERK2 (phosphor T202+ Y204) (ab223500; Abcam); and anti-GAPDH (sc-32233; Santa Cruz Biotechnology Inc.) was used as a control. gene knockdown Specif ic shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGTGTCACGT-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: Figure 2 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: The antibodies used in this study were as follows: anti-Flag (F1804, Sigma-Aldrich Co., St Louis, MO, USA); anti-Proliferating Cell Nuclear Antigen (PCNA) (sc-56; Santa Cruz Biotechnology Inc., Dallas, TX, USA); anti-mouse IgG (sc-2005; Santa Cruz Biotechnology Inc.); anti-NUDT21 (ab183660; Abcam, Cambridge, UK); anticyclin E (ab133266; Abcam); anti-Bax (ab53154; Abcam); anti-Bcl-2 (ab32124; Abcam); anti-phosphatase and tensin homolog deleted on chromosome 10 (PTEN) (ab32199; Abcam); anti-ERK1+ ERK2 (phosphor T202+ Y204) (ab223500; Abcam); and anti-GAPDH (sc-32233; Santa Cruz Biotechnology Inc.) was used as a control. gene knockdown Specif ic shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGTGTCACGT-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: Figure 3 K562 cell growth upon knockdown of NUDT21. Notes: (A) Proliferation capabilities were detected by CCK-8 in K562, Jurkat, and HL-60 cells (2×103) 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: The antibodies used in this study were as follows: anti-Flag (F1804, Sigma-Aldrich Co., St Louis, MO, USA); anti-Proliferating Cell Nuclear Antigen (PCNA) (sc-56; Santa Cruz Biotechnology Inc., Dallas, TX, USA); anti-mouse IgG (sc-2005; Santa Cruz Biotechnology Inc.); anti-NUDT21 (ab183660; Abcam, Cambridge, UK); anticyclin E (ab133266; Abcam); anti-Bax (ab53154; Abcam); anti-Bcl-2 (ab32124; Abcam); anti-phosphatase and tensin homolog deleted on chromosome 10 (PTEN) (ab32199; Abcam); anti-ERK1+ ERK2 (phosphor T202+ Y204) (ab223500; Abcam); and anti-GAPDH (sc-32233; Santa Cruz Biotechnology Inc.) was used as a control. gene knockdown Specif ic shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGTGTCACGT-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: Figure 4 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 (1.5×104) 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: The antibodies used in this study were as follows: anti-Flag (F1804, Sigma-Aldrich Co., St Louis, MO, USA); anti-Proliferating Cell Nuclear Antigen (PCNA) (sc-56; Santa Cruz Biotechnology Inc., Dallas, TX, USA); anti-mouse IgG (sc-2005; Santa Cruz Biotechnology Inc.); anti-NUDT21 (ab183660; Abcam, Cambridge, UK); anticyclin E (ab133266; Abcam); anti-Bax (ab53154; Abcam); anti-Bcl-2 (ab32124; Abcam); anti-phosphatase and tensin homolog deleted on chromosome 10 (PTEN) (ab32199; Abcam); anti-ERK1+ ERK2 (phosphor T202+ Y204) (ab223500; Abcam); and anti-GAPDH (sc-32233; Santa Cruz Biotechnology Inc.) was used as a control. gene knockdown Specif ic shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGTGTCACGT-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: Figure 5 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: The antibodies used in this study were as follows: anti-Flag (F1804, Sigma-Aldrich Co., St Louis, MO, USA); anti-Proliferating Cell Nuclear Antigen (PCNA) (sc-56; Santa Cruz Biotechnology Inc., Dallas, TX, USA); anti-mouse IgG (sc-2005; Santa Cruz Biotechnology Inc.); anti-NUDT21 (ab183660; Abcam, Cambridge, UK); anticyclin E (ab133266; Abcam); anti-Bax (ab53154; Abcam); anti-Bcl-2 (ab32124; Abcam); anti-phosphatase and tensin homolog deleted on chromosome 10 (PTEN) (ab32199; Abcam); anti-ERK1+ ERK2 (phosphor T202+ Y204) (ab223500; Abcam); and anti-GAPDH (sc-32233; Santa Cruz Biotechnology Inc.) was used as a control. gene knockdown Specif ic shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGTGTCACGT-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: Figure 6 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: The antibodies used in this study were as follows: anti-Flag (F1804, Sigma-Aldrich Co., St Louis, MO, USA); anti-Proliferating Cell Nuclear Antigen (PCNA) (sc-56; Santa Cruz Biotechnology Inc., Dallas, TX, USA); anti-mouse IgG (sc-2005; Santa Cruz Biotechnology Inc.); anti-NUDT21 (ab183660; Abcam, Cambridge, UK); anticyclin E (ab133266; Abcam); anti-Bax (ab53154; Abcam); anti-Bcl-2 (ab32124; Abcam); anti-phosphatase and tensin homolog deleted on chromosome 10 (PTEN) (ab32199; Abcam); anti-ERK1+ ERK2 (phosphor T202+ Y204) (ab223500; Abcam); and anti-GAPDH (sc-32233; Santa Cruz Biotechnology Inc.) was used as a control. gene knockdown Specif ic shRNA plasmid against NUDT21 targeting 5′-ACCTCCTCAGTATCCATAT-3′ and a non-silencing shRNA control plasmid targeting 5′-TTCTCCGAACGTGTCACGT-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