Bisbenzimide h 33342 trihydrochloride

Cell culture reagents were purchased from Celbio s.r.l. (Milano, Italy). CulturePlate 96/wells plates were purchased from PerkinElmer Life Science (Waltham, MA) and Falcon (BD Biosciences, Bedford, MA). Calcein‐AM, bisBenzimide H 33342 trihydrochloride were obtained from Sigma‐Aldrich (Milan, Italy).
Cell cultures. MDCK‐MDR1, MDCK‐MRP1 and MDCK‐BCRP cells are a gift of Prof. P. Borst, NKI‐AVL Institute, Amsterdam, The Netherlands. Caco‐2 cells were a gift of Dr. Aldo Cavallini and Dr. Caterina Messa from the Laboratory of Biochemistry, National Institute for Digestive Diseases, “S. de Bellis”, Bari (Italy). HT29 and HT29/DX cells from ATCC (Manassas, VA).
MDCK and Caco‐2 cells were grown in DMEM high glucose supplemented with 10 % fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, in a humidified incubator at 37 °C with a 5 % CO2 atmosphere.
HT29 cells were grown in RPMI‐1640 supplemented with 10 % fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin; HT29/DX cells were grown in the above mentioned medium containing 50 nM doxorubicin to maintain the chemoresistant phenotype.^[32]

Cell culture reagents were purchased from Celbio s.r.l. (Milano, Italy). CulturePlate 96/wells plates were purchased from PerkinElmer Life Science; Calcein-AM, bisBenzimide H 33342 trihydrochloride were obtained from Sigma-Aldrich (Milan, Italy).

Recombinant human TGF-β1 was purchased from R&D systems (Shanghai, China). TGF-β1 receptor blocker SB525334 was purchased from Selleck Chemicals (Shanghai, China). The antibodies used included mouse anti-alpha smooth muscle actin antibody (Sigma Aldrich, St. Louis, MO, USA) and goat anti-mouse IgG (whole molecule)-FITC antibody, Thermo (Shanghai, China). BisBenzimide H33342 trihydrochloride (20mg/ml, Sigma Aldrich, St. Louis, MO, USA) was used for nuclear staining. Paraformaldehyde (PFA) was purchased from FuChen Chemical Reagent Factory (Tianjin, China). Bovine Serum Albumin (BSA) was from HWRK Chem Co., LTD (Beijing, China). Other materials included dimethyl sulfoxide (DMSO); DMEM, high glucose, and pyruvate (Invitrogen, State of California, USA); fetal bovine serum (FBS) (ExCell Bio, Shanghai, China); trypsin from bovine pancreas, EDTA, and Triton X-100 (Sigma Aldrich, St. Louis, MO, USA).

HUVECs (1.5 × 10³ cells/well) were seeded in ibidi μ slides (ibidi GmbH, Munich, Germany), treated with MiuA as indicated for 1 h, and subsequently fixed with 4% (v/v) paraformaldehyde. F-actin was stained with rhodamine phalloidin (1:400, R 415, Molecular Probes, Life technologies) and nuclei were labeled with Hoechst stain (bisBenzimide H33342 trihydrochloride, B2261 Sigma-Aldrich). Images were taken using a Zeiss LSM 510 META confocal microscope (Zeiss, Jena, Germany). For the co-localization experiments cells were treated for 30 min with 50 nM MiuA, and subsequently stained as described above with the addition of an anti-cofilin antibody (D3F9, rabbit, Cell Signaling), and a subsequent step of incubation with an Alexa488 labelled secondary antibody (goat anti rabbit, A – 11008, Invitrogen). These images were obtained on an inverted SP8 confocal microscope (Leica, Germany).

HEK-293 human embryonic kidney cells [51 (link)] were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% (v/v) penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in a humidified incubator at 37 °C with 5% CO₂. Mycoplasma contamination was screened using bisBenzimide H 33342 trihydrochloride (14533, Sigma-Aldrich, St. Louis, MO, USA) DNA staining. The HEK-293 cells were plated in 35 mm dishes (density 4 × 10⁴ cells per mL) 16 h before transfection. Cells were transiently transfected using X-tremeGENE™ HP DNA Transfection Reagent according to the manufacturer’s instructions (Roche, Basel, Switzerland), with 250 ng of plasmid (Empty, GFP-only, GFP-tagged WT REST or GFP-tagged MT REST). Live viewing was performed 24 h after transfection, using a Zeiss LSM8800 with Airyscan confocal microscope (Zeiss, Oberkochen, Germany). Cells were spiked with 1 in 100,000 Hoechst for co-visualization of nuclear material. The detector of the confocal was the photomultiplier tube (PMT) and allowed detection of the green fluorescence signal through the Argon laser at 488 nm. Images were visualized and processed using the ZEN Blue Software (latest version) provided by Zeiss (Zeiss, Oberkochen, Germany).

Stage 4 antheridiophores were used to assess sperm production^{36 (link)}. First, 50 μL water was dropped onto the surface of each antheridiophore and left for 10 min. After pipetting five times, 30 μL water was placed in a 1.5-mL tube with 1.5 μL bisBenzimide H33342 trihydrochloride (1 mg/mL) (Sigma-Aldrich). After staining for 10 min, 10 μL solution was drawn to a C-Chip (NanoEnTek) to check fluorescence by confocal microscopy (OLYMPUS U-HGLGPS BX53).