Journal: The FASEB Journal

Article Title: Sphingosine phosphate lyase regulates myogenic differentiation via S1P receptor-mediated effects on myogenic microRNA expression

doi: 10.1096/fj.13-233155

Figure Lengend Snippet: Changes in SphK1 and SPL during differentiation of C2C12 cells. C2C12 cells were propagated under proliferation conditions until time 0, when the medium was replaced with differentiation medium containing 2% horse serum. Cells were harvested at d 0–5. Whole-cell extracts were evaluated by immunoblotting with antibodies specific for MHC, myogenin, SPL, SphK1 (SK1), and GAPDH loading control. A) Representative immunoblot. B) Quantification of autoradiogram. All bands were normalized to the loading control for the corresponding time point. Experiment is representative of 3 similar experiments.

Article Snippet: SPL knockdown (KD) in C2C12 cells The lentiviral vector pLKO.1 (Addgene plasmid 10878; Addgene, Cambridge, MA, USA) was used to clone small hairpin RNAs targeting the murine SPL gene Sgpl1 , according to pLKO.1 protocol ( http://www.addgene.com ), as we described for human SPL-KD cells ( 29 ).

Techniques: Western Blot

Journal: The FASEB Journal

Article Title: Sphingosine phosphate lyase regulates myogenic differentiation via S1P receptor-mediated effects on myogenic microRNA expression

doi: 10.1096/fj.13-233155

Figure Lengend Snippet: Generation of SPL-KD and vector control C2C12 lines. A) Phase-contrast images of C2C12 cells after lentiviral transduction to knock down murine SPL. Scale bar = 100 μm. B) SPL knockdown was confirmed by immunoblotting extracts of cells maintained in proliferation or differentiation conditions. Actin was used as a loading control. C) SPL enzyme activity. n = 3/group. SPL-specific activity is expressed as picomoles product formation per milligram protein per minute. *P < 0.05. D) Intracellular S1P concentration was determined by mass spectrometry and normalized to phospholipid content (PC). Data are presented as percentage control cell levels, n = 3/group. *P < 0.05. E) Extracellular S1P concentration was determined as in D; n = 4/group. Data are presented as percentage control levels. *P < 0.05. F) Expression of S1PR1–5 was compared in SPL-KD and control cells by qRT-PCR, with results normalized to GAPDH; n = 3/group. These experiments were performed ≥3 times with similar results. *P < 0.05.

Article Snippet: SPL knockdown (KD) in C2C12 cells The lentiviral vector pLKO.1 (Addgene plasmid 10878; Addgene, Cambridge, MA, USA) was used to clone small hairpin RNAs targeting the murine SPL gene Sgpl1 , according to pLKO.1 protocol ( http://www.addgene.com ), as we described for human SPL-KD cells ( 29 ).

Techniques: Plasmid Preparation, Transduction, Western Blot, Activity Assay, Concentration Assay, Mass Spectrometry, Expressing, Quantitative RT-PCR

Journal: Virus Genes

Article Title: Overexpression of m6A-factors METTL3, ALKBH5, and YTHDC1 alters HPV16 mRNA splicing

doi: 10.1007/s11262-022-01889-6

Figure Lengend Snippet: A Linearized HPV16 genome (numbers refer to the HPV16 reference strain GeneBank: K02718.1). Early and late genes are indicated. P97: HPV16 early promoter. P670: HPV16 late promoter. Black oval: splice donor. White oval: splice acceptor. pAE: HPV16 early polyadenylation site. pAL: HPV16 late polyadenylation site. LCR: HPV16 long control region. B Schematic representation of HPV16 subgenomic plasmid pBELsLuc. Transcription from pBELsLuc is driven by the human cytomegalovirus immediate early promoter (CMV). Secreted luciferase (sLuc) gene was integrated in the L1 gene following the poliovirus 2A internal ribosomal entry site (IRES) sequence. C Schematic structures of a subset of HPV16 alternatively spliced mRNAs produced from pBELsLuc. Arrows indicate HPV16 RT-PCR primers. D Effect of METTL3, METTL14, WTAP, or all three combined on HPV16 E2 mRNA splicing was monitored by RT-PCR with indicated primer pair on RNA extracted from HeLa cells transfected with pBELsLuc and pMETTL3, pMETTL14, pWTAP or pMETTL3, pMETTL14 and pWTAP combined. D Effect of METTL3 on HPV16 E2 mRNA splicing was monitored by RT-PCR with indicated primer pair on RNA extracted from HeLa cells transfected with pBELsLuc and empty pUC plasmid (−) or pMETTL3 in triplicates. E Effect of METTL3 on HPV16 E2 mRNA splicing was monitored by RT-PCR with indicated primer pair on RNA extracted from HeLa cells transfected with pBELsLuc and pUC (−) or pMETTL3 in triplicates. The RT-PCR was optimized for detection also of the larger E1 mRNAs with retained E1-encoding intron (E1). Effect of YTHDC1, METTL3, or ALKBH5 on HPV16 E2- ( F ), E4- ( G ), or L1- ( H ). mRNA splicing was monitored by RT-PCR with indicated primer pair on RNA extracted from HeLa cells transfected with pBELsLuc and empty pUC plasmid (−), pYTHDC1, pMETTL3, or pALKBH5 in duplicates. I RNA immunoprecipitation of RNA extracted from C33A2 cells using IgG or anti-m6A antibody followed by RNA extraction and RT-PCR with indicated primers. Input: RT-PCR on RNA extracted from C33A2 cells in the absence of immunoprecipitation. J Western blot with anti-ALKBH5 or actin antibody on proteins extracted from C33A2 cells infected with lentivirus pLKO.1-vector or pLKO.1 with shRNA to ALKBH5 (shAKBH5-1). K Effect of ALKBH5 knock-down on HPV16 E2 mRNA splicing (880^2709) was monitored by RT-PCR with RT-PCR primers 773S and E2qas on RNA extracted from C33A2 cells infected with pLKO.1-vector or pLKO.1 with shRNA to ALKBH5 (shALKBH5-1). The RT-PCR was optimized for detection also of the larger E1 mRNAs with retained E1-encoding intron (E1). L Effect of ALKBH5 knock-down on HPV16 L1/L1i mRNA splicing was monitored by RT-PCR with RT-PCR primers 773S and L1as on RNA extracted from C33A2 cells infected with pLKO.1-vector or pLKO.1 with shRNA to ALKBH5 (shALKBH5-1)

Article Snippet: Lentivirus for the short hairpin RNA (shRNA)-mediated knockdown of ALKBH5 was generated by co-transfection of HEK293T cells with a pLKO.1-vector or pLKO.1-vector carrying specific shRNA together with the packaging vector pMISSION-GAG-POL and a vesicular stomatitis virus G protein expressing vector pMISSION-VSV-G. pLKO.1 was purchased from Sigma-Aldrich (SHC001).

Techniques: Plasmid Preparation, Luciferase, Sequencing, Produced, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunoprecipitation, RNA Extraction, Western Blot, Infection, shRNA

Journal: Current issues in molecular biology

Article Title: Identification and Characterization of Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma Using the SORE6 Reporter.

doi: 10.3390/cimb43020041

Figure Lengend Snippet: Figure 3. Experimental manipulation of Sox2 and Oct4 in SupM2 SORE6 clones. (A) FACS analysis showing the effect on SORE6 reporter activity of overexpressing Sox2 in single-cell clone 5-4 (SupM2 SORE6−). (B) FACS analysis showing the effect on SORE6 reporter activity of overexpressing shSox2 in single-cell clone 6-5 (SupM2 SORE6+). (C) FACS analysis showing the effect of Oct4 downregulation on SORE6 reporter activity in clone 5-4 (SupM2 SORE6−). (D) FACS analysis showing the effect of shOct4 transfection on SORE6 reporter activity in single-cell clone 6-5 (SupM2 SORE6+). All results shown are representative of three independent experiments and were also repeated in a second batch of single-cell clones. Side panels show the fold change of GFP in log scale (mean ± SEM). Bottom panels of each figure are the Western blots showing the effect of transient transfection with Sox2, Oct4, shSox2, and shOct4 plasmids. Empty vector (EV) was included as a negative control. ** p < 0.01, *** p < 0.001.

Article Snippet: The Oct4 expression vector (pLVEF1a-hOCT4-IRES-Neo) was purchased from BiOSETTIA (San Diego, CA, USA). shRNA for Sox2 (#26353) and the Sox2 expression vector (#16577) were purchased from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Activity Assay, Transfection, Western Blot, Plasmid Preparation, Negative Control

Journal: Current issues in molecular biology

Article Title: Identification and Characterization of Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma Using the SORE6 Reporter.

doi: 10.3390/cimb43020041

Figure Lengend Snippet: Figure 6. SORE6−and SORE6+ clones are biochemically distinct. (A) The subcellular localization of Sox2, Oct4, and c-Myc in SORE6−and SORE6+ cells derived from SupM2, assessed by the nuclear cytoplasmic fractionation assay. (B) The DNA pull-down assay was performed to assess Sox2, Oct4, and c-Myc transcriptional activity in SORE6−and SORE6+ cells using a biotin-labeled SORE6 probe.

Article Snippet: The Oct4 expression vector (pLVEF1a-hOCT4-IRES-Neo) was purchased from BiOSETTIA (San Diego, CA, USA). shRNA for Sox2 (#26353) and the Sox2 expression vector (#16577) were purchased from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Derivative Assay, Fractionation, Pull Down Assay, Activity Assay, Labeling