Fig 2: mRNA levels of CASP family markers (A-E), BCL2 family markers (F-H) and other apoptosis-related markers (I-M) in limbal stromal cells (LSCs) and in Aniridia-LSCs (AN-LSCs), after treatment with 0 µM, 50 µM and 75 µM Cobalt chloride (CoCl2).CASP3, CASP7, CASP8, CASP9 and CASP10 mRNA levels (A-E), BCL2, BAX and BID mRNA levels (F-H) and CDKN1A, CDKN1B, TNFα, XIAP and BIRC5 mRNA levels (I-M) are shown. Values are expressed on a logarithmic scale (Log 2) with geometric mean±geometric standard deviation. Two-way ANOVA followed by Dunnett’s test was used, significant p values are indicated. BCL2 mRNA levels were significantly higher in AN-LSCs compared to LSCs (p = 0.0291). In LSCs, BCL2 mRNA level significantly decreased (p = 0.0102) and CDKN1B level significantly increased using 75 µM CoCl2 treatment. In addition, in AN-LFCs, 75 µM CoCl₂ treatment significantly decreased CASP3, CASP9 and BCL2 mRNA levels (p = 0.0494; p = 0.0414; p = 0.0218).

Fig 3: KGN cells are susceptible to CBD-induced cell death. A Application of CBD in different concentrations (1 µM, 5 µM, 10 µM, 15 µM and 30 µM) or the solvent control EtOH for 6 h (gray line with circles; n = 8) and 24 h (lead line with triangles; n = 12) and measurement of cell survival (in % to solvent control EtOH) by means of ATP assays was used to determine the CBD IC50 for KGN cells. Results are shown as interpolated dose-effect curves based on identified mean ± SEM cell survival for each concentration applied. Inset depicts variance of interpolated CBD IC50 values for 6 h (gray) and 24 h incubation (lead) of all performed experiments. B Representative micrographs captured after 6 h treatment with 11 µM CBD (a) or the solvent control EtOH (b; EtOH ctrl.). Presence of CBD induced first signs of cell death, such as intracellular vacuolization and nuclear condensation, while the solvent control EtOH had no effects on cellular morphology or appearance. Scale bar 50 µM. C Treatment of KGN cells with 11 µM CBD for 6 h resulted in increased levels of the characteristic apoptotic cell death markers cleaved (cl) initiator caspase (Casp) 8 (c) and effector clCasp3 (d), as identified by means of Western Blotting. β-Actin served as internal loading control. Representative bands from KGN cells treated either with the solvent control EtOH or 11 µM CBD, respectively. Cropped blot images are shown; original gel images provided in Supplementary Fig. 9