Journal: British journal of cancer

Article Title: Inactivation of the WASF3 gene in prostate cancer cells leads to suppression of tumorigenicity and metastases.

doi: 10.1038/sj.bjc.6605850

Figure Lengend Snippet: Figure 7 Knockdown of WASF3 expression suppresses MMP9 expression levels and inhibits MMP9 release from prostate cancer cell lines. (A) RT–PCR analysis of PC3 and DU145 cells showing knockdown of WASF3 also show down-regulation of MMP9, but not MMP2 expression levels. Cont ¼ parental cells. (B) Secretion of the pro- and active form of MMP9 from PC3 and DU145 cells were measured in the supernatants of actively growing cells using the Fluorokine MAP assay procedure. Cells from each clone showing knockdown of WASF3 also showed reduced MMP9 levels compared with parental (control) and non-knockdown cells that did not.

Article Snippet: After 24 h, the media were collected in tubes and centrifuged for 10 min at 10 000 g. The pro- and active MMP-9 levels released into the media were measured using a Fluorokine MAP human MMP9 kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: The FASEB Journal

Article Title: Suppression of cigarette smoke induced MMP1 expression by selective serotonin re‐uptake inhibitors

doi: 10.1096/fj.202001966RR

Figure Lengend Snippet: Validation of and NMLSMR targets identified by the high throughput assay. A, To test for reproducibility of the assay, 293T cells were transfected with the MMP‐1/pGL3 reporter plasmid. Cells were seeded in three 96‐well plates using an interleaved format and treated with 5% CSE media (high‐signal) 2% CSE (medium‐signal) no CSE (low‐signal). After a 24‐h incubation, the luciferase activity was measured in each well. Representative data from days 1–3 is shown. The calculated Z' factor was 0.71 ± 0.05 SEM, n = 5. B, As expected, the ERK inhibitor PD98059 inhibited luciferase expression in a dose‐dependent fashion in positive controls exposed to 5% CSE ( *** p ≤ 0.001, significantly different from control; ### p ≤ 0.001, significantly different from promoter + CSE; ## p ≤0.01, significantly different from promoter + CSE). C, Data from the high throughout screen displaying each compound tested as a block dot. The y‐axis illustrates the change in MMP1 expression compared with expression levels in CSE exposed in HEK293 cells. The drugs in the −100% range (red shaded area) are capable of nullifying all CSE induced MMP1 transcriptional activity

Article Snippet: MMP‐1 Fluorokine E kits (R&D Systems F1M00) were used to determine MMP‐1 levels and performed per manufacturer's protocol.

Techniques: Biomarker Discovery, High Throughput Screening Assay, Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Expressing, Control, Blocking Assay

Journal: Nature Chemical Biology

Article Title: Correlating chemical sensitivity and basal gene expression reveals mechanism of action

doi: 10.1038/nchembio.1986

Figure Lengend Snippet: Figure 2 | MoA analysis reveals new mechanisms of small-molecule metabolism. (a) Expression-sensitivity correlations for the compound austocystin D and CYP2J2 expression. Reactive functionalities are indicated with a red arrow. (b) Cytotoxicity of austocystin D and a selective CYP2J2 inhibitor (CYP2J2i) across renal CCLs with different expression levels of CYP2J2, and effects of co-treatment of austocystin D with CYP2J2i. Cell viability values are normalized to vehicle-only (DMSO) treatment, with each point representing the mean of n = 2 independent experiments with two technical replicates each. (c) Cytotoxicity of austocystin D in MCF7-ER-Snail-16SA cells either induced to undergo epithelial-to-mesenchymal transition (blue) or treated with vehicle (black). Each point is the mean of n = 2 technical replicates. Two independent inductions are shown. (d) Expression-sensitivity correlations for the compound RITA and SULT1A1 expression. (e) Protein expression of SULT1A1 and sensitivity to RITA of six renal CCLs. CCLs described in ref. 23 as capable (*) or incapable (‡) of metabolizing RITA into a cytotoxic form are indicated. Each point is mean ± s.d. for n = 3 independent experiments. For uncropped gel image, see Supplementary Figure 8b. (f) Sulfotransferase enzyme activity of varying amounts of recombinant SULT1A1 at a fixed concentration of RITA in the presence of the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate. Each point is mean ± s.d. for n = 3 independent experiments.

Article Snippet: Recombinant human SULT1A1 (R&D Systems) activity in the presence of 0.2 mM 3′-phosphoadenosine-5′phosphosulfate and the indicated concentrations of RITA was measured by a phosphatase-coupled assay using the Universal Sulfotransferase Activity Kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Activity Assay, Recombinant, Concentration Assay