HCT116 cells were cultured in McCoy's 5A modified medium (Gibco, Invitrogen, Paisley, UK) containing 10% foetal calf serum (Gibco, Invitrogen), unless otherwise specified. The 786-O and HEK-293 cells were cultured in 10% foetal calf serum-containing Dulbecco's modified Eagle's medium. Cells were incubated at 37°C in 5% CO2. Rapamycin treatment was carried out 24 h after seeding. Cells were treated for 24 h typically, or 72 h for cell proliferation analysis, with 3 nM (HCT116) or 100 nM (HEK-293) rapamycin, diluted from a stock solution in dimethyl sulfoxide (R8781; Sigma, Gillingham, UK), or as specified for dose–response curves. Founder cell lines were obtained from ATCC (LGC Standards, Teddington, UK) and used within 6 months of resuscitation (<25 passages). si435 used to knockdown PAT4 expression and the scrambled control siRNA (Figure 1 ) were previously described in Heublein et al.14 (link)
R8781
Manufactured by Merck Group
Sourced in United Kingdom
R8781 is a laboratory equipment product. It is designed for specific laboratory tasks. The core function of R8781 is to perform essential operations in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Foetal calf serum, by Thermo Fisher Scientific (1 mentions)
Mccoy s 5a modified medium, by Thermo Fisher Scientific (1 mentions)
C6628, by Merck Group (1 mentions)
E1383, by Merck Group (1 mentions)
T7402, by Merck Group (1 mentions)
B 1080, by Merck Group (1 mentions)
Red blood cell lysis buffer, by BioLegend (1 mentions)
Ack lysis buffer, by BioLegend (1 mentions)
4 protocols using r8781
1
Rapamycin treatment in cell lines
2
Chemotherapeutic and Autophagy Modulators Protocol
3
Generating Murine Bone Marrow-Derived Macrophages
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Hind legs were collected from C57/B6J mice (aged 6–15 weeks) and cleaned using a scalpel, followed by flushing of the bone marrow with a 1 mL syringe, phosphate-buffered saline (PBS) and 25 G needle. Red blood cells were lysed using ACK lysis buffer following the manufacturer’s instructions (10× red blood cell lysis buffer, 420301; BioLegend). Bone marrow cells were cultured on non-tissue culture-treated 10-cm Petri dishes in complete Iscove’s modified Dulbecco’s media (IMDM), 10% FBS, 50 µM β-mercaptoethanol and supplemented with 25 ng/mL of macrophage colony-stimulating factor (315-02; Peprotech). BMDMs were supplemented with macrophage colony-stimulating factor every 3 days until day 7 of differentiation. BMDMs were activated with 1 ng/mL of LPS purchased from Sigma (E. coli O111:B4 LPS25) for either 5 or 18 h according to experiment type. Further experiments were performed with the glutaminase inhibitor CB-839 (Merck, AMBH2D6FB23B) (1 µM per well) or rapamycin (R8781; Sigma) (50 nM per well) with their respective vehicle controls.
4
Visualizing ER-TGN Contact Sites
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To explore ER-TGN contact sites, the rapamycin system was used to produce a tight association between the FKBP and FRB domains (Bohdanowicz and Fairn, 2011 (link)). Based on this, transfection of HEK-SAC1GFP cells with mCherry-pHR-TcRb-FKBP, CFP-TGN38-FRB, and YFP-P4M was performed followed by rapamycin addition (5 µM at room temperature; R8781; Sigma-Aldrich). This synthetic system allows ER recruitment to the TGN and a consequent decrease in Golgi PI(4)P (Dickson et al., 2014 (link)). Under constant perfusion, cells were tracked for ∼15 min with images taken every ∼5 s. Positive ER-TGN junction creation was only considered when the ER signal localized at the TGN increased with respect to its signal outside the TGN. This increase was measured with ImageJ/Fiji software as described above in one-stack images. Mean PI(4)P intensity was also quantified in the TGN compartment in each cell by creating a TGN-englobing mask and then applying it on the PI(4)P image. PI(4)P inside the TGN was always normalized to the cytoplasm signal in order to discard any difference due to the transfection efficiency.
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