Fv3000rs inverted confocal microscope

To generate spheroids for the use in the NFs sensitivity assay, 2000 NDF, 2000 HaCaT, and 2000 NDF + 2000 HaCaT were seeded per well on day 0. On day1 post‐seeding, the spheroids were transfected with 10 µm of TGFBR1 siRNA duplexes (Thermofisher Scientific) using Lipofectamine 2000 (ThermoFisher Scientific), according to manufacturer's protocol, and incubated at 37 °C for 24 h. The transfection media was then replaced with 100 µL of 1% FBS DMEM with or without TGFβ1 (10 ng mL⁻¹) and incubated at 37° for 24 h. Following these treatments, the spheres were incubated in 100 µL of 1% FBS DMEM containing either KRT14‐NF and GAPDH‐NF (HaCaT spheres) or FSP1‐NF and GAPDH‐NF (NDF spheres) for another 16 h. The spheroids were then washed once with PBS and stained with Hoechst 33 342. Cy3 filter was used to visualize the signals of KRT14‐NF and FSP1‐NF, while GAPDH‐NF signal was detected using Cy5 filter on the Olympus FV3000RS inverted confocal microscope. For all samples, Z‐stacks were collected and processed using FIJI software to obtain the sum slices projection images.

In situ hybridization was performed with the ViewRNA ISH Tissue 2-Plex Assay Kit (Thermo Fisher) according to the manufacturer’s instructions. Briefly, the paraformaldehyde-fixed cells were permeablized with protease and hybridized with the commercial Smyca tilling Type 1 probe set (Thermo Fisher, Catalog #VA1-3016541) at 40 °C for 2 h. After washing out the unlabeled probes, the hybridization signal was amplified by 2 steps of branch DNA hybridization at 40℃ for 40 min. These branch DNA amplifiers were then labeled by alkaline phosphatase, and fluorescence was developed by incubating with Fast Red substrates at 40 °C for 60 min. The slide was then counterstained with DAPI and examined by an Olympus FV3000RS inverted confocal microscope equipped with 60x/1.40 oil objective lens (Olympus Objective Lens, PlanApo N). Images were collected by an Olympus FV3000 FV31S-SW (v 2.40) software.

Immortalized normal human keratinocytes (KCs, N/TERT‐1, courtesy of J. G. Rheinwald) and human primary hair follicle DPCs (Adult, Cell Application Inc.) were used to generate the 3D spheroid cultures. KCs and DPCs were grown in Keratinocyte serum‐free media (KSFM) and DMEM supplemented with 10% fetal bovine serum (Hyclone) and 0.14 mg mL⁻¹ bone pituitary extract (Gibco), respectively. Single cell suspensions were seeded into low attachment 96‐well plates (Corning) and incubated at 37 °C for spheroid formation.
To generate spheroids, 2000 KCs, 2000 DPCs and 2000 KCs + 2000 DPCs were seeded into each well to generate KC, DP, and the KCDP spheres. The spheroids were collected at day 3 post‐seeding and incubated in 100 µL of 1% FBS DMEM containing a mixture of KRT14‐NF, FSP1‐NF, and GAPDH‐NF (2–5 µL mL⁻¹, at the concentration of OD 0.1–0.125) for 16 h. The spheroids were then washed once with PBS and stained with Hoechst 33 342 to stain the nuclei. Cyanine 3 (Cy3), 6‐FAM, and Cy5 filters were used to visualize the signals of KRT14‐NF, FSP1‐NF, and GAPDH‐NF, respectively, on the Olympus FV3000RS inverted confocal microscope. For all samples, Z‐stacks were collected and processed using FIJI software to obtain the sum slices projection images.