MCF10A cells stably overexpressing TSPO.

<p>Stable vector control (pLXSN) and TSPO-FLAG overexpressing cells (TSPO) were generated as described under <i>Materials and Methods</i>. <b>A</b>. Expression of TSPO was detected by immunoblotting using antibodies against FLAG tag (<i>top</i> panel) or TSPO (<i>bottom</i> panel), respectively, with actin as a loading control. <b>B</b>. Immunostaining of ectopically expressed and endogenous TSPO using antibodies against FLAG tag (<i>top</i> panel) or TSPO (<i>bottom</i> panel). MitoTracker Red was used to stain mitochondria and cells were imaged by confocal microscopy. Scale bar: 10 μm.</p

Overexpression of TSPO enhances proliferation during mammary epithelial morphogenesis.

<p>Control MCF10A-pLXSN cells and MCF10A-TSPO cells were seeded and cultured in Matrigel as described under <i>Materials and Methods</i>. <b>A</b>. On day 15, the cultures were fixed and stained with 4′,6′-diamidino-2-phenylindole (DAPI, <i>blue</i>) and anti-Ki-67 (<i>green</i>). Representative fluorescent images of control vector (pLXSN) and TSPO expressing acini are shown. Scale bar: 50 μm. Expanded image of a single acinus from pLXSN control or TSPO are shown. Scale bar: 10 μm. <b>B</b>. Cultures were scored for the number of acini containing 0, 1 to 5, or more than 5 Ki-67-positive cells based on at least 250 acini from each condition, combined from three independent experiments. Error bar: SD. P-value was determined by Student's <i>t</i>-test. * p<0.05 indicates a significant difference between TSPO and pLXSN control.</p

Overexpression of TSPO increases acini size.

<p>Control MCF10A-pLXSN and MCF10A-TSPO cells were seeded in Matrigel as described under <i>Materials and Methods</i>. <b>A</b>. Images were acquired on day 15 and day 20 of culture. Scale bar: 50 μm. <b>B</b>. Maximal cross-sectional area in pixels of individual acini was determined using ImageJ software, and plotted as a box plot. <i>Black line</i>, median value; <i>box</i>, interquartile range; <i>solid square</i>, mean; <i>open circles</i>, outliers. Data are from a representative set of three independent experiments in which ∼100 acini per condition were measured. P-value determined by Student's <i>t</i>-test. *** p<0.001 indicates a significant difference between TSPO and pLXSN control. <b>C</b>. The cross-sectional area of stable TSPO-overexpressing acini relative to the control acini ( = 100%). Each column represents results from 3 independent experiments in which a total of at least 300 acini per condition were measured. Error bar: SD. P-value was determined by Student's <i>t</i>-test. * p<0.05 indicates significant difference between TSPO and pLXSN control.</p

Combination of TSPO ligands and lonidamine potentiates apoptosis in breast cancer cells.

<p>BT549 (<b>A</b>) or MDA-MB-231 (<b>B</b>) cells were treated with vehicle (DMSO) or different concentrations of PK 11195 combined with different concentrations of lonidamine (LON) as indicated for 24 h. <b>C.</b> MDA-MB-231 cells were treated with DMSO or Ro5-4864 combined with different concentrations of LON, as indicated, for 24 h. Cell death was determined using trypan blue exclusion assays as described in <i>Materials and Methods</i>. Column: Mean of three independent experiments. Error bar: SD. <b>D</b>. MDA-MB-231 cells were treated with DMSO, PK 11195 or Ro5-4864 alone or in combination with LON at the indicated concentrations for 24 h and cellular lysates were generated. Immunoblotting of cellular lysates was performed using antibodies against PARP. Actin was used as loading control. PK: PK 11195; Ro: Ro5-4864.</p

Overexpression of TSPO leads to partial filling of lumen during morphogenesis.

<p>Control MCF10A-pLXSN and MCF10A-TSPO cells were seeded and cultured in Matrigel. On day 20, cultures were fixed and stained with DAPI and anti-cleaved caspase-3 as described in <i>Materials and Methods</i>. <b>A</b>. Schematic diagram (<i>top</i> panel) of serial confocal cross sections of an acinus structure showing the relative position of the sections with respect to z-axis. Serial confocal cross-sections images of MCF10A-pLXSN and MCF10A-TSPO acini stained with DAPI (<i>bottom</i> panel). Scale bar: 20 μm. <b>B</b>. Representative images of cleaved caspase-3 staining (<i>green</i>) were acquired using confocal microscopy. Scale bar: 25 μm. <b>C</b>. The number of viable cells (active caspase-3 negative) per lumen was quantified, and plotted as a box plot. <i>Black line</i>, median value; <i>box</i>, interquartile range; <i>solid square</i>, mean; <i>open circles</i>, outliers. Data are from a representative set of three independent experiments in which ∼90 acini per condition were measured. <b>D</b>. The percent of active caspase-3 positive cells in the lumen was quantified based on at least 250 acini combined from three independent experiments. Error bar: SD. P-value was determined by Student's <i>t</i>-test. ** p<0.01, *** p<0.001 indicate significant differences between TSPO and pLXSN control.</p

TSPO promotes breast cancer cell migration.

<p><b>A.</b> Control or TSPO expression vectors were transiently expressed in MCF7 cells. HA-tagged TSPO was detected by immunoblotting using an antibody against the HA tag, with actin as a loading control (<i>top</i> panel). The control and HA-TSPO-expressing MCF7 cells were allowed to migrate toward 10% FBS for 24 h in a transwell assay as described under <i>Materials and Methods.</i> Duplicate wells were used for each of three independent experiments. The results are expressed relative to the migration of control cells ( = 100%). Column (<i>bottom</i> panel): Mean of three experiments. Error bar: SD. P value was determined by Student's <i>t</i>-test. * p<0.05.indicates a significant difference between control and TSPO-overexpressing MCF7 cells. Control siRNA and siRNA sequences against TSPO (siTSPO #1 or siTSPO #2) were used to transfect MDA-MB-231 cells (<b>B</b>) or BT549 (<b>C</b>). The extent of silencing was determined by immunoblotting with antibodies against TSPO<b>,</b> with actin used as a loading control (<i>top</i> panel). Control and TSPO-depleted cells were then subjected to transwell assays as described under <i>Materials and Methods</i> Duplicate wells were used for each of three independent experiments. The results are expressed relative to the migration of control cells ( = 100%). Column (<i>bottom</i> panel): Mean of three experiments. Error bar: SD. P-value was determined by Student's <i>t</i>-test. * p<0.05 and ** p<0.01 indicate significant differences between control and TSPO-depleted cells.</p

A Copper-Catalyzed Reductive Defluorination of β‑Trifluoromethylated Enones via Oxidative Homocoupling of Grignard Reagents

An efficient copper-catalyzed reductive defluorination of β-trifluoromethylated enones via an oxidative homocoupling of Grignard reagents is reported. The reaction proceeded smoothly with a wide range of substrates, including the ones bearing aromatic heterocycles, such as furyl and thienyl ring systems in high yields (80–92%, except one example) under mild conditions. This provides a practical method for synthesis of <i>gem</i>-difluoroolefin ketones. It is also worth noting that this homocoupling process of Grignard reagents using β-trifluoromethylated enones as “oxidizing reagents” is effective for both the Csp²–Csp² and Csp³–Csp³ bond formations, including for substrates whose β-hydride elimination of the corresponding transition metal alkyl complex is particularly facile, affording coupling products with high yields (83–95%), simultaneously

Supplementary Material from UV_{185+254 nm} photolysis of typical thiol collectors: decomposition efficiency, mineralization and formation of sulfur byproducts

The decomposition of toxic flotation reagents upon UV185+254 nm irradiation was attractive due to operational simplicity and no dosage of oxidants. In this work, the degradation of typical thiol collectors (potassium ethyl xanthate (PEX), sodium diethyl dithiocarbamate (SDD), O-isopropyl-N-ethyl thionocarbamate (IET) and dianilino dithiophoshoric acid (DDA)) was investigated by the UV185+254 nm photolysis. The degradation efficiencies and mineralization extents of collectors were assessed. The formation of CS2 and H2S byproducts was studied, and the mechanisms of collector degradation were proposed under UV185+254 nm irradiation. The PEX, SDD and IET were decomposed with nearly 100% removal upon 75 min of UV185+254 nm irradiation. The decomposition rate constants decreased in the order SDD > PEX > IET ≫ DDA, and the DDA was the refractory collector. After 120 min of UV185+254 nm irradiation, 15‒45% of carbon and 25‒75% of sulfur of collectors were completely mineralized, and the mineralization extent decreased in the order PEX > SDD > IET > DDA. The percentage of gaseous sulfur (CS2 and H2S) was ranged from 0.48 to 4.85% for four collectors, showing the fraction of emitted sulfur byproducts was small. The aqueous CS2 concentration increased in first 10‒20 min, and was decreased to a low level of 0.05‒0.1 mgl−1 at 120 min. Two mechanisms, i.e. direct UV254 nm photolysis and indirect oxidation with free radicals, were responsible for collector decomposition in the UV185+254 nm photolysis

DataSheet1_Analysis of Electromagnetic Interference and Shielding in the μLED Optrode Based on Finite Element Method.docx

Monolithic integrated μLED optrode has promising applications in optogenetics due to their ability to achieve more optical channels in a smaller footprint. The current used to drive the μLED will cause electromagnetic interference (EMI) noise to the recording electrodes at a very close distance. Utilizing a grounded metal shielding layer between the active device and the electrode can potentially reduce the interference. In this paper, multi-dimensional μLED optrode models are set up according to the real device. By numerically analyzing the electromagnetic interference between the μLED and recording electrodes, several optimized shielding schemes are evaluated by simulations and experiments. Some important process and layout parameters that may influence the shielding effect are studied through the finite element method (FEM). Different circuit models based on the corresponding test environment are built to analyze the simulation and experiment results. A new PCB with a shielding layer has been designed and initially verified. The proposed novel computational model can analyze EMI quantitatively, which could facilitate the design of low-noise μLED optrode with reasonable shielding and packaging.</p

Redox-Neutral Cyclization of 2‑Isocyanobiaryls through Photoredox/PPh₃ Dual Catalysis

The photoredox/PPh3-mediated cyclization of 2-isocyanobiaryls has been developed. A substantial range of functional-group-rich phenanthridine derivatives were synthesized at room temperature in a highly selective and atom-economic manner. Mechanistic studies suggested that the cyclization process is probably mediated both by Ph3P radical cation with key 1,2-hydride transfer and hydrogen atom generated through O–H bond homolytic cleavage of Ph3P–OH radical intermediate