Journal: Biochemical pharmacology

Article Title: Involvement of mTOR kinase in cytokine-dependent microglial activation and cell proliferation.

doi: 10.1016/j.bcp.2009.06.097

Figure Lengend Snippet: Fig. 5. Effects of the mTOR inhibitor RAD001 on COX activity and expression. (A) Microglial cells were incubated for 48 h in plain medium or medium containing TII; when indicated RAD (0.1–1 nM) was added to the cells at the beginning of the experiment. PGE2 release in the incubation media was assessed by RIA. Data are expressed as pg/mL of PGE2, and are means SEM of 4 replicates per each group. The experiment is representative of three different experiments run in the same conditions. *P < 0.05 vs. Controls. Whole-cell lysates were prepared from microglial cells treated as described above. 20 mg of protein were analyzed by western blot for the expression of COX1 (B) and COX2 (D) enzyme. Line 1: Controls; line 2: TII; line 3: TII/0.1 nM RAD; line 4: TII/1 nM RAD; line 5: 1 nM RAD. TII significantly increased intracellular levels of COX1 (C) while having a slight but not significant stimulatory effect on COX2 (E). 1 nM RAD significantly reduced COX1 intracellular levels, both under basal conditions and after stimulation with TII (C). **P < 0.01 and ***P < 0.001 vs. Controls; §P < 0.05 vs. TII.

Article Snippet: COX1 and COX2 rabbit polyclonal antibodies were from Cayman (Ann Arbor, MI, USA); b-actin (clone AC-74) mouse monoclonal antibody was from Sigma– Aldrich; and rabbit polyclonal anti-phospho [ser-2448] mTOR was purchased from Novus Biological (Littleton, CO, USA).

Techniques: Activity Assay, Expressing, Incubation, Western Blot

Journal: Andrology

Article Title: Differential roles of cyclooxygenase enzymes in the regulation of murine juvenile undifferentiated spermatogonia.

doi: 10.1111/andr.13537

Figure Lengend Snippet: FIGURE 3 Characterization of C18-4Cox1-KD1 cells and KD-associated morphological and gene changes. (A) Expression of cyclooxygenase (Cox)1 on populations of transfected cells with varying concentrations of shRNA and lipofectamine 3000 showing that knockdown “B” had the most downregulated expression of Cox1. (B) Cox1 protein expression on immunoblot, comparing wildtype cells (WT), scrambled cells (Scrb), knockdown “B” clone, and an isolated clonal population of C18-4Cox1-KD1. C18-4Cox1-KD1 Cox1 protein expression validated against the total protein. (C) Gene expression of Cox1 in C18-4Cox1-KD1 and Scrambled controls, (D) Immunofluorescence staining of Cox1 in C18-4Cox1-KD1 and Scrambled controls. Green: Cox1, red: alpha-tubulin, blue: DAPI. Scale in µm. (E) Brightfield visualization illustrating morphological differences between C18-4Cox1-KD1 and Scrambled controls. (F) Differences in gene expression between C18-4Cox1-KD1 and Scrambled controls of Jam-1, Mmp2, Stra8, and Kit. Results are presented as fold change of controls. mRNA expression data were normalized to Glyceraldehyde 3-phosphate dehydrogenase (Gapdh); N = 3 independent experiments conducted in triplicates. Significant difference relative to controls with t-test: * (p ≤0.05), ** (p < 0.01), *** (p < 0.001).

Article Snippet: Cox1 shRNA plasmid for a mouse (sc-35097-SH) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotech.

Techniques: Expressing, Transfection, shRNA, Knockdown, Western Blot, Isolation, Gene Expression, Immunofluorescence, Staining

Journal: Andrology

Article Title: Differential roles of cyclooxygenase enzymes in the regulation of murine juvenile undifferentiated spermatogonia.

doi: 10.1111/andr.13537

Figure Lengend Snippet: FIGURE 5 Activation of Notch3 signaling pathway in C18-4Cox1-KD1. (A) Immunofluorescence (IF) images of Notch3 expression in C18-4Cox1-KD1 and Scrambled controls. Green: Notch3, red: alpha-tubulin, blue: DAPI. Scale in µm. (B) Immunoblots of Notch3, Hes1, cyclooxygenase (Cox)1, and Cox2 expression in C18-4Cox1-KD1 and Scrambled controls are cropped at indicated bands, and protein expression, indicated by values under the bands, is normalized to alpha-tubulin. (C) Quantification of Notch3, Hes1, Cox1, and Cox2 protein expression on western blot normalized against the total protein. Results are presented as fold change of controls. (D) IF images of Hes1 expression in C18-4Cox1-KD1 and Scrambled controls. Green: Hes1, red: alpha-tubulin, blue: DAPI. Hes1-positive cells were counted and the data show the percentage of Hes-positive cells as fold of control samples. N = 3 independent experiments. Significant difference relative to controls with t-test: * (p ≤0.05), ** (p < 0.01), *** (p < 0.001).

Article Snippet: Cox1 shRNA plasmid for a mouse (sc-35097-SH) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotech.

Techniques: Activation Assay, Immunofluorescence, Expressing, Western Blot, Control

Journal: Nature Communications

Article Title: Pparg promotes differentiation and regulates mitochondrial gene expression in bladder epithelial cells

doi: 10.1038/s41467-019-12332-0

Figure Lengend Snippet: Validation of gene expression changes from RNA-Seq experiments. a , c Expression of Cpt2 and Krt5 in the urothelium of Pparg fl/fl controls ( a ) and Shh Cre ;Pparg fl/fl mutants ( c ). b , d Cox1 and Krt5 expression in a control ( b ) and in the Shh Cre ;Pparg fl/fl mutant urothelium ( d ). e , g Sod1 and Krt5 expression in a Pparg fl/fl control urothelium ( e ) and in the urothelium of an Shh Cre ;Pparg fl/fl mutant ( g ). f , h Sod2 and Krt5 expression in the urothelium of a Pparg fl/fl control ( f ) and in a Shh Cre ;Pparg fl/fl mutant urothelium ( h ). i , l Uchl1 and Krt5 expression in the urothelium of a wild-type adult tongue ( i ) and in the urothelium of a Shh Cre ;Pparg fl/fl mutant ( l ). j , m Expression of Sprr1a and Krt5 in the urothelium of a control ( j ) and in a urothelium of a Shh Cre ;Pparg fl/fl mutant ( m ). k , n Cldn8 and Krt5 expression in a control urothelium ( k ) and in a Shh Cre ;Pparg fl/fl mutant ( n ). o , q )Krt6 and Krt5 expression in a control urothelium ( o ) and in a Shh Cre ;Pparg fl/fl mutant ( q ). p , r Uchl1 and Krt5 expression in a control urothelium ( p ) and in a Shh Cre ;Pparg fl/fl mutant ( r ) urothelium. Scale bars: 50 μm. Adult wild type, n = 6; adult mutant, n = 5

Article Snippet: The following primary antibodies were used in these studies: p63 mouse IgG (clone 4A4, Santa Cruz Biotechnology, sc8431, 1:100) or rabbit IgG (GenTex, GTX102425, 1:300), CK5 rabbit IgG (Covance, AF-138, PRB-160P, 1:300), or chicken IgY (Covance, SIG-3475, 1:300), Ck20 mouse IgG2a, kappa, clone Ks20.8 (Dako, M7019, 1:250), Ki67 rabbit IgG (Abcam, ab15580, 1:300), Tom20 rabbit IgG (Santa Cruz, SC-11415, 1:2000), E-cadherin goat IgG (R&D System, AF748, 1:300), PPARG rabbit IgG (Cell Signaling Technology, #2435, 1:100), FABP4 goat IgG (R&D Systems, AF1443, 1:1000), Krt14 chicken IgY (BioLegend, 906001, 1:500) or rabbit IgG (BioLegend, 905301, 1:300), ZO1 rabbit IgG (Thermo Fisher Scientific, 40-2300, 1:100), Krt6a rabbit (LSbio, LS-B12036, 1:500), Krt10 mouse IgG1 (Santa Cruz, Sc-53252, 1:500), CLDN8 rabbit IgG (GeneTex, GTX77832, 1:50), UCHL1 mouse IgG1 (Santa Cruz, sc-271639, 1:200), SNAIL+SLUG rabbit IgG (Abcam, ab180714, 1:200), laminin rabbit (Sigma, L9393, 1:100), actin, a smooth muscle mouse (Sigma, C6198, 1:300), NF-κB p65 rabbit IgG (Abcam, ab19870, 1:300), CD45 rat IgG2b (BD Sciences, 550539, 1:50), F4/80 rat IgG2a (Thermo Fisher Scientific, 14-4801-82, 1:100), CD3 rat IgG1 (Abcam, ab11089, 1:50), CD19 rat IgG2a (Thermo Fisher Scientific, 14-0194-82, 1:500), SPRR1A rabbit IgG (Biorbit, orb1053, 1:200), COX1 rabbit (Novus, NBP1-85500, 1:200), SOD1 mouse IgG (Santa Cruz, sc-17767, 1:200), SOD2 rabbit IgG (Abcam, ab13533, 1:500), and CPT2 rabbit IgG (Genetex, GTX33117, 1:50).

Techniques: Biomarker Discovery, Gene Expression, RNA Sequencing, Expressing, Control, Mutagenesis