Hcx fl fluotar 40x 0

HeLa cells that were incubated with SirReal2 (20 and 50 μM), SirReal6 (50 μM), AGK2 (Sigma-Aldrich, 20 μM) or DMSO as a control in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS, antibiotics and DMSO (1% (v/v)) for 4 h, were fixed with ice-cold methanol (10 min), washed with PBS and blocked with PBS supplemented with 0.1% (v/v) Triton-X-100 and 5% (v/v) FCS (30 min). Cells were then stained with an anti-acetyl-α-tubulin antibody (Sigma-Aldrich, T6793) and then probed with a secondary Alexa 546 conjugated anti-mouse-antibody (Invitrogen). Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Coverslips were mounted with FluoroMount (Sigma-Aldrich) and sealed with DPX Mountant (Sigma-Aldrich). Images of the mounted samples were acquired on a Leica DM500 microscope equipped with a Leica DFC 395 FX camera and HBO 100 W lamp40 (link). The microscope was run with the Leica Application Suite 4.4.0 software. Chroma UV filter set (No. C40888) and Leica N2.1 filter set (No. 513832) were used for DAPI and Alexa 546 signal acquisition, respectively, with a HCX FL Fluotar 40x/0.75 (dry) objective. Further details about the equipment and the settings that were used to acquire the images are found in the Supplementary Methods section. Unprocessed images are found in Supplementary Fig. 9.

HeLa cells (ATCC^® CCL-2™, American Type Culture Collection) were grown in Dulbecco’s modified eagle medium supplemented with 10% fetal calf serum and 100 μg/ml kanamycin in a humidified incubator at 37 °C with 5% CO₂. Cells were grown on 12-mm-diameter glass coverslips for microscopic analysis. For the evaluation of the BiFC signal, HeLa cells were transfected with different mVenus constructs of TPPP/p25 (0.3 μg of each plasmid) using Turbofect (Invitrogen) transfection reagent according to the manufacture’s protocol. Monoclonal tubulin antibody (Sigma T9026) and an Alexa 546 conjugated mouse secondary antibody (Thermo Fisher Scientific A11030) were used for the detection of the tubulin signal. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images of the mounted samples were acquired on a Leica DM IL 500 microscope equipped with Leica DFC 395 FX camera and HBO 100w lamp. The equipment software was Leica Application Suite 4.4.0. Chroma UV filter set (No. C40888), Chroma 41028 HQ NB GFP filter set (No. C21116) and Leica filter N2.1 (No. 513832) was used for DAPI, BiFC and Alexa 546 signal acquisition, respectively, using a HCX FL Fluotar 40x/0.75 (dry) objective.