Tmr star

Cells were grown at 37 °C and 5.0% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 2 mM l-glutamine, 1 mM sodium pyruvate and 10% (vol/vol) fetal bovine serum. Cells were routinely passaged after 2–3 d or upon reaching 80% confluency. Before seeding cells, type number 1 eight-well LabTek chambered coverslips (Thermo Fisher Scientific) were cleaned with 0.1 M hydrofluoric acid to improve cell attachment. Twenty-four hours before imaging, cells seeded in LabTek chambers were transiently transfected using FuGene HD (Promega) or TransIT-X2 (Mirus) transfection reagents according to manufacturer’s guidelines. Cells transfected with HaloTag or SNAP_f-tag fusion constructs were labeled before imaging. Growth medium was exchanged with staining solution containing either 20 nM HTL-SiR or 200 nM TMR-Star (New England Biolabs) in DMEM and incubated between 20 min (HaloTag labeling) and up to 1 h (SNAP_f-tag labeling).
For mitochondria staining, cells were incubated with 200 nM MitoTracker Orange CMTMRos (Thermo Fisher Scientific) in DMEM for 1 h according to manufacturer’s guidelines. All measurements were performed in Leibovitz L15 medium (Sigma-Aldrich). All used CIPs were purified by preparative HPLC. Lyophilized products were dissolved in DMSO, and stocks were diluted in L15 medium. Final DMSO concentrations were kept below 2% for all experiments.

Cell lines expressing CENP-A-SNAP were pulse labeled as previously described³⁰ . For the primary siRNA screen, HeLa-CENP-A-SNAP cells were grown in 6 well plates and pulse labeled with tetra-methyl-rhodamine-conjugated SNAP substrate (TMR-Star; New England Biolabs) at 4 µM final concentration, labeling all pre-existing CENP-A molecules at the centromere. This was followed by a quenching step with bromothenylpteridine (BTP; New England Biolabs) at 2 µM final concentration to prevent any further fluorescent labeling of nascent CENP-A following TMR-Star washout. The cells were then trypsinized (Trypsin-EDTA, Gibco) and 1.5 × 10⁵ cells were directly seeded onto 1 chamber Lab-Tek slides spotted with the siRNA library as described above. The chambers were then incubated at 37 °C, 5% CO2 for 48 h. At the end of 48 h, cells were labeled with Oregon-Green SNAP substrate (New England Biolabs) at 4 µM final concentration for labeling of the newly synthesized CENP-A molecules. Finally the cells were co-extracted (pre-extraction and fixation) in 4% paraformaldehyde, 0.2% Triton X and Hoeschst (1 µg/ml) at room temperature for 30 min.

Cell lines expressing CENP-A-SNAP were quench-pulse labelled as previously described [37 (link)]. Briefly, cells were quenched with a non-fluorescent bromothenylpteridine (BTP; New England Biolabs) at 2 µM final concentration and kept in culture for 5 h and 30 min. Cells were then pulse labelled with tetra-methyl-rhodamine-conjugated SNAP substrate (TMR-Star; New England Biolabs) at 4 µM final concentration, labelling all newly synthesized CENP-A molecules at the centromere, and fixed for immunofluorescence.

SNAP-tagged NKA in MDCK-II cells was tagged with cell permeable dye TMR-STAR (New England Biolabs, Inc.). SNAP-tagged MDCK-II cells were grown in MFKP for a week to let the cells mature into a monolayer. Normal growth media was perfused every day on both apical and basal side using a syringe. On the day of experiment, cells were incubated for 30 min with 3 µM TMR-STAR dye solution in normal growth media. The cells were then washed three times very gently using normal media to get rid of residual TMR-STAR in the device. MFKPs were then incubated for 1 h in the incubator before imaging.
A mcirocapillary (MC) (Sigma P2049-1PAK) in port-0 and a 1 ml syringe (BD 309659) with 400–500 µL media in port-3 were connected. By gently pushing the plunger media was let into the MC and appropriate pressure gradient on the basal side was achieved by changing the height to fluid in the MC. The same setup was used to perform live-cell imaging of cells under fluid pressure and to condition cells for qPCR experiments.
Confocal imaging (Zeiss) was used to take fluorescence images of one field of view (slice) on the baso-lateral domain of SNAP-tagged MDCK-II cells and F-tractin MDCK-II cells at 1 min interval. The intensity analysis was done using FIJI (ImageJ version 1.41).