8 well chamber slide

In 8-well chamber slides (BD Bioscience, Shanghai, China, catalog number: 354108), 30 µL growth factor reduced BD Matrigel (BD Biosciences, catalog number: 354230) was added per well. Slides were placed in a cell culture incubator for at least 15 min to solidify the Matrigel. Next, 2000 C4-2B cells/well or 4000 LNCaP cells/well were seeded in RPMI-1640 medium containing 10% FBS with DMSO or 10 μM enzalutamide (Beyotime Biotechnology) and 2% Matrigel. Media were replenished every 3 days. Chamber slides were incubated for 2 (C4-2B) or 3 (LNCaP) weeks, and then photographed. Image J program (NIH, USA) was used to measure the diameter of each sphere. Spheres with a diameter larger than 80 μm were counted.

To assess the effects of siGSK3β with and without siRTP801 on RGC survival and neurite outgrowth in the presence of activated glia, mimicking the in vivo injury-induced scenario, retinal cell cultures were prepared from animals 5 days after ONC [21 (link)]. Briefly, adult male Sprague-Dawley rats were killed by CO₂ overdose and retinae was harvested and dissociated into single cell suspensions using a papain dissociation kit, following the manufacturer’s instructions (Worthington Biochemicals, Lakewood, NJ, USA) [21 (link)]. Retinal cells were plated at a density of 125,000 cells per well in 8-well chamber slides (BD Biosciences, Watford, UK), pre-coated with poly-d-lysine and laminin in sNBA (Invitrogen), and were incubated at 37 °C and 5% CO₂. Cultures were allowed to settle overnight before treatment the next day. The identity of wells was masked from the investigator and experiments were performed in duplicate and repeated on 3 independent occasions.

Cells on 8-well chamber slides (BD Biosciences) were fixed with 4% PFA, washed with PBS, and permeabilized with 0.2% Triton X-100 for 10 min. Cells were incubated with primary antibodies in antibody diluent (Dako, Glostrup, Denmark) overnight at 4°C and then with Alexa Fluor-488 goat anti-rabbit and mouse IgG (Invitrogen) secondary antibodies. Cells were mounted with ProLong Gold Antifade Reagent with DAPI (Life Technologies, Carlsbad, CA, USA). Images were acquired using a Carl Zeiss confocal microscope (Weimar, Germany).

Patient-derived organoids were cultured in 8-well chamber slides (BD Biosciences, East Rutherford, NJ, USA), fixed in 4% PFA for 30 min, and washed with PBS twice. We used whole-mount blocking buffer (WBB, containing 5% FBS, 0.2% BSA, 0.3% Triton X-100 in PBS) for blocking and permeabilizing the samples for 30 min. Samples were incubated with primary antibodies at 4 °C overnight in WBB, they were washed with PBS, labeled secondary antibodies were applied overnight at 4 °C and the organoids were mounted with ProLong Diamond antifade mountant containing DAPI (Thermo Fisher Scientific). We used a Leica TCS SP8 confocal microscope for imaging and the ImageJ software for analysis and quantification. The antibodies used are listed in Table S1.

Three-dimensional (3-D) culture of cells on matrigel basement membrane was carried out as described [37 ]. Briefly, 4 x10³ cells were resuspended in modified growth medium containing 2% growth factor-reduced matrigel (BD Biosciences) without or with drugs (2 nM fulvestrant, 20 nM Dasatinib, 100 μg/ml MK0646, or combinations) or vehicle DMSO, and seeded onto matrigel in 8-well chamber slides (BD Bioscience). Medium with drugs was replaced every 2 days. Photographs of representative fields were taken as indicated. Acini were photographed and counted in 10 randomly chosen fields. Quantitative analysis of the number and total area of acini were performed with AlphaVIEW SA software. Data expressed as means of triplicates, representative of two independent experiments.