F8775 25ml

Cells were washed twice with PBS and fixed with 4% formaldehyde (Sigma F8775-25ML) in PBS for 30 min at room temperature, washed again three times with PBS and permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min. They were then washed three times with PBS and blocked with 3% BSA (Sigma A9418) in PBS for 1 h. Thereafter, cells were incubated with a primary antibody and a secondary antibody for 1 h with three washes in between. Nuclei were counterstained with 4,6-diamidino-2-phenylindole or Hoechst 33342. Images were acquired using an Olympus IX81 inverted fluorescence microscope equipped with an Olympus 1 × 2-UCB camera or the Zeiss LSM 710 system. The AhR fluorescence intensity ratio between the nucleus and cytoplasm was analysed with ImageJ.

In experiments where cross-links were introduced a few key steps were performed differently. Fixation of tissue was done on the sRIN slide for 5 or 10 min at RT using PBS (09-9400, Medicago) containing 4% formaldehyde (F8775-25ML, Sigma-Aldrich), then washed briefly in PBS (09-9400, Medicago), dried for 1 min at 37 °C and then continued with propan-2-ol (A461-1, Fisher Scientific) treatment before HE staining. A tissue-specific (mouse olfactory bulb) permeabilization was performed before reverse transcription in situ. The permeabilization reactions were done, on the sRIN slide in a sealed hybridization cassette (AHC1X16, ArrayIT Corporation), first by adding a mixture of 1x Exonuclease I buffer (B0293S, NEB) and 0.2 mg/ml BSA (B9000S, NEB) for 30 min at 37 °C. Subsequently, tissue was briefly washed in 0.1 × SSC buffer (S6639, Sigma-Aldrich), treated with 0.1% pepsin (P7000, Sigma-Aldrich) dissolved in 0.1 M HCl (318965, Fluka) for 10 min at 37 °C, washed with 0.1 × SSC buffer (S6639, Sigma-Aldrich) and then reverse transcription in situ was performed immediately.

HeLa cells are cultured in Dulbecco’s modified Eagle’s medium (11-965-092, Fisher Scientific Ltd. Canada), supplemented with 10%, fetal bovine serum (F7524-500ML, Sigma-Aldrich, USA), and 1% (volume/volume) penicillin-streptomycin (15-140-122, Thermo Fisher Scientific Inc., USA)^{29 (link)}. Cells in 2D are cultured and passaged at 80% confluence and medium change is performed every 48 hours. For the formation of 3D cancer spheroids, cells are trypsinized, resuspended to a density of $2\times {10}^6$ cell/ml, and seeded in 3% (weight/volume) agarose (16500500, ultra-pure agarose, Invitrogen, USA) microwell arrays resulting in average 267 cells per spheroid. Half medium change is done daily. At day 3, spheroids are collected and stained with $10\mathrm{\mu }\text{M}$ CellTracker Red CMTPX (C34552, Thermo Fisher Scientific Inc., USA) by 45 min incubation in a serum-free medium and washed twice with cell culture medium. Both 2D and 3D cultures are done in a humidified atmosphere with 5% carbon dioxide at 37 ${}^{\circ }$ C. For long-term storage, 3D spheroids stained with CMTPX are fixated at room temperature with 4% formaldehyde (F8775-25ML, Sigma-Aldrich, USA) for 15 min and then washed twice with Dulbecco’s phosphate-buffered saline (DPBS). For imaging experiments, the spheroids are immobilized and maintained using the developed microfluidic chip (Fig. 6a).