Naasc

In all experiments, cells were incubated with 1 µM clickable retinoids for 1 hour at 37°C in RPMI medium, without serum, unless otherwise indicated. Cells were fixed using 1% PFA for 30 minutes on ice. 5 µl (100 mM) CuSO4 and 5 µl (1 M) NaAsc (both Sigma Aldrich) we mixed and vortexed for a few minutes until the colour turned from brown to yellow. 2,5 µl THPTA (100 mM) (Sigma Aldrich) was added to this mixture, together with 487.5 µl PBS. After a few minutes, 0,5 µl commercially available azide labelled with alexa fluor 647/488 (ClickChemistryTools) was added and incubated for 3 additional minutes. This is called the ‘click mixture’ and is used for incubation with target cells, in order to visualize clickable retinoids. The azide and alkyne form a triazole, via a copper(I)-catalyzed [2 + 3] cyclo-addition. The interaction between the azide group and alkyne is covalent, resulting in a stable product. Once the interaction is formed with the retinoid probe, the fluorophore cannot leave anymore. Per click condition, around 100 000 cells were treated with 100 ul click mixture for 1 hour at 37°C. Subsequently, cells were washed with 2 mM EDTA, 3% BSA and finally PBS using the centrifuge at 1500 rpm at 4°C.

All reagents and solvents were used without additional purification unless specified otherwise. Poly(ethylene glycol) with molecular mass 5000 Da (PEG5k, M_w/M_n = 1.02) and 11,000 Da (PEG11k, M_w/M_n = 1.04) were purchased from Polysciences, Inc. (Warrington, PA, USA). 2,2-Bis(hydroxymethyl)propionic acid, bis-MPA, (99+%) sodium azide, and NaN₃ (98%, powder) were acquired from Acros. Sodium hydride, NaH, (95%, dry powder), 4-di(methylamino)pyridine, DMAP, (≥99%), copper(I) bromide, CuBr, (98%), propargyl bromide (80 wt.% in toluene), N,N′-dicyclohexylcarbodiimide, DCC, (99%), methyl 3,5-dihydroxybenzoate, 3,5-MDHB, (97%), 3,5-dihydroxybenzyl alcohol, 3,5-DHBA (98%), benzyl bromide (98%), sodium ascorbate, Na-Asc (≥99%), potassium carbonate, K₂CO₃ (≥98%, powder), 18-crown-6 (99%), 2,5-dihydroxybenzoic acid, 2,5-DHBA, (98%), phenylboronic acid, PBA, (95%), 4′-bromoacetophenone, 4′-BAcP, (99%), triethylamine, TEA (99.5%), and tetrahydrofuran, THF (HPLC grade) were all purchased from Sigma-Aldrich (Miwaukee, WI, USA). Dichloro-bis(benzonitrile) palladium, Pd(PhCN)₂Cl₂, (99%) was obtained from Strem Chemicals (Newburyport, MA, USA); diethyl ether, ethyl acetate, EtOAc, hexanes, and methanol (all reagent grade) were received from Pharmco, Inc. (Brookfield, CT, USA), and dichloromethane, DCM, (spectrophotometric grade) was purchased from Spectrum/Gleason Chemicals (Syracuse, NY, USA).