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: Laboratory investigation; a journal of technical methods and pathology

Article Title: Basal cells of the human airways acquire mesenchymal traits in idiopathic pulmonary fibrosis and in culture.

doi: 10.1038/labinvest.2015.114

Figure Lengend Snippet: Figure 1 Basal cell reactivity, hyperplasia and upregulation of EMT markers adjacent to fibroblastic foci. Epithelium adjacent to fibroblastic foci shows increased reactivity and mesenchymal phenotype. Sparse staining of CK14 is seen in control samples, whereas CK14 is strongly expressed in epithelium overlying fibroblastic foci. P63, a restricted transcription factor for basal cells is expressed in epithelial cells located both basally and in the top layer indicating formation of metaplasia in IPF samples. E-cadherin is abundant in epithelium both in control and IPF samples. In addition, E-cadherin is present in cells within the foci. N-cadherin is expressed in lower basal epithelial cells in IPF samples whereas control samples are negative. Vimentin is expressed in both epithelium and mesenchyme in IPF samples. Bar 100 μm.

Article Snippet: Lentiviral transduction was performed as previously described.28 In short, plasmids containing scrambled hairpin (pLKO.1 shSCR; Addgene plasmid 17920)29 or shRNA against p63 (shp63alpha pLKO.1 puro; Addgene plasmid 19120)30 were used with packaging plasmids (psPAX2 and pMD2.G) (Addgene plasmids 12260 and 12259, respectively) to generate viral titer in HEK-293 T cells using Arrest-in (Open Biosystems).

Techniques: Staining, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Basal cells of the human airways acquire mesenchymal traits in idiopathic pulmonary fibrosis and in culture.

doi: 10.1038/labinvest.2015.114

Figure Lengend Snippet: Figure 5 Knockdown (KD) of p63 abrogates UG-induced EMT of VA10 cells. p63 KD cells are unable to undergo UG-induced EMT. The expression of the mesenchymal markers N-cadherin and Thy-1 is abrogated in treated KD cells, whereas scrambled control cells show a division into two cellular sub-populations much like the mother cell line VA10. The expression of Vimentin can still be detected in treated KD cells, although to a lesser extent than in treated scrambled cells. Bars 100 μm.

Article Snippet: Lentiviral transduction was performed as previously described.28 In short, plasmids containing scrambled hairpin (pLKO.1 shSCR; Addgene plasmid 17920)29 or shRNA against p63 (shp63alpha pLKO.1 puro; Addgene plasmid 19120)30 were used with packaging plasmids (psPAX2 and pMD2.G) (Addgene plasmids 12260 and 12259, respectively) to generate viral titer in HEK-293 T cells using Arrest-in (Open Biosystems).

Techniques: Knockdown, Expressing, Control

Journal: Scientific reports

Article Title: Nox4 is a Target for Tuberin Deficiency Syndrome.

doi: 10.1038/s41598-018-21838-4

Figure Lengend Snippet: Figure 1. ROS levels are increased in human angiomyolipomas and in Tsc2+/− mouse model. (A) H2O2 levels and (B) Superoxide production in renal angiomyolipoma samples (AML) from TSC patients (n = 8) and normal kidney biopsy (n = 17) were shown, measured by Amplex red assay and lucigenin chemiluminescence, respectively. (C) H2O2 levels and (D) Superoxide production in control (n = 6) and Tsc2+/− mouse (n = 10) kidneys was measured by Amplex red and lucigenin chemiluminescence assays, respectively. (E) ROS levels were measured by flow cytometry with DCF dye in proximal tubular epithelial cells with siScramble (orange and light blue) or siTSC2 (green and dark blue) (left panel). The top right panel shows the western blotting for tuberin protein levels in these cells. The bottom right panel showed the quantification of the data for DCF dye measurement. (F) ROS levels were measured with DHE dyes, scale bar: 200 μm and the intensity of staining per cells were quantified (right panel). Mean ± SE of 3 independent experiment, *p < 0.05 vs siScr; (G) Superoxide production was measured in siScramble or siTSC2 transfected cells by lucigenin chemiluminescence. The data are presented as the mean ± S.E of 3 repeats. *p < 0.05; **p < 0.01; ***p < 0.001 in comparison with normal human kidney (A,B), or control mice (C,D), or cells receiving scramble siRNA (E–G).

Article Snippet: Lentiviral pLKO.1-puro vectors encoding shRNA specific for TSC2 (#15478) or control scramble (#1864) were purchased 1 2SCIENTIfIC RepoRts | (2018) 8:3781 | DOI:10.1038/s41598-018-21838-4 from Addgene (Cambridge, MA).

Techniques: Amplex Red Assay, Control, Flow Cytometry, Western Blot, Staining, Transfection, Comparison

Journal: Scientific reports

Article Title: Nox4 is a Target for Tuberin Deficiency Syndrome.

doi: 10.1038/s41598-018-21838-4

Figure Lengend Snippet: Figure 2. Tuberin regulates Nox4 protein expression. (A) RT-PCR analysis of the Nox isoforms in human proximal tubular epithelial cells with primers listed in Supplementary Table 1. (B) Nox4 protein levels were determined by western blotting in human proximal tubular epithelial cells transfected with Scramble or TSC2 siRNA; (C) mRNA levels were measured by qRT-PCR in cells transfected with Scramble or TSC2 siRNA; The data are presented as the mean ± S.E, in comparison with cells receiving scramble siRNA (n = 4). (D) Proximal tubular epithelial cells stably expressing TSC2 shRNA were infected with adenoviruses expressing GFP or TSC2; tuberin and Nox4 protein levels were measured in these cells by western blotting. (E) Nox4 RNA stability was measured by qRT-PCR in Actinomycin D (5 μg/mL)-treated shTSC2 cells. (F) Protein stability of Nox4 was measured by western blotting using cycloheximide (CHX) (100 μg/mL)-treated shTSC2 cells for indicated times. Lower panel showed the percentage changes of protein during the CHX treatment; (G) Nox4 and tuberin protein levels were measured in RK3E and LEF2 cells by western blotting; (H) Nox4 and tuberin protein levels were measured in LEF2 cells infected with adenoviruses containing GFP or TSC2; (I) Nox4 and tuberin protein levels were measured in control and Tsc2+/− mouse kidney cortices; (J) Nox4 protein levels were measured in renal angiomyolipoma samples from TSC patients and normal human kidney biopsies. In all western blotting assays, β-actin or GAPDH serves as loading controls.

Article Snippet: Lentiviral pLKO.1-puro vectors encoding shRNA specific for TSC2 (#15478) or control scramble (#1864) were purchased 1 2SCIENTIfIC RepoRts | (2018) 8:3781 | DOI:10.1038/s41598-018-21838-4 from Addgene (Cambridge, MA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Quantitative RT-PCR, Comparison, Stable Transfection, shRNA, Infection, Control

Journal: Cell reports

Article Title: Gain-of-function p53 R172H mutation drives accumulation of neutrophils in pancreatic tumors, promoting resistance to immunotherapy

doi: 10.1016/j.celrep.2021.109578

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pLKO.1 hygro , Gift from Bob Weinberg , Addgene Cat# 24150.

Techniques: Blocking Assay, Marker, Recombinant, Mutagenesis, TA Cloning, SYBR Green Assay, Staining, Software, Expressing

Journal: Cell reports

Article Title: Gain-of-function p53 R172H mutation drives accumulation of neutrophils in pancreatic tumors, promoting resistance to immunotherapy

doi: 10.1016/j.celrep.2021.109578

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pLKO.1 hygro , Gift from Bob Weinberg , Addgene Cat# 24150.

Techniques: Blocking Assay, Marker, Recombinant, Mutagenesis, TA Cloning, SYBR Green Assay, Staining, Software, Expressing