a, Illustration of the absorption spectrum of CRY2 in vitro. Cryptochrome 2 was optimally activated by 350–475 nm light23. A sharp drop in absorption and activation was seen for wavelengths greater than 480 nm. Spectrum was adapted from ref. 23. b, Impact of illumination duty cycle on LITE-mediated gene expression. Varying duty cycles (illumination as percentage of total time) were used to stimulate 293FT cells expressing LITEs targeting the KLF4 gene, to investigate the effect of duty cycle on LITE activity. KLF4 expression levels were compared to cells expressing GFP only. Stimulation parameters were: 466 nm, 5 mW cm−2 for 24 h. Pulses were performed at 0.067 Hz with the following durations: 1.7% = 0.25 s pulse, 7% = 1 s pulse, 27% = 4 s pulse, 100% = constant illumination. (mean ± s.e.m.; n = 3–4 biological replicates.) c, The transcriptional activity of CRY2PHR/ CIB1 LITE was found to vary according to the intensity of 466 nm blue light. Neuro 2a cells were stimulated for 12 h at a 7% duty cycle (1 s pulses at 0.067 Hz). All Neurog2 mRNA levels were measured relative to cells expressing GFP only (mean ± s.e.m.; n = 3–4 biological replicates). d, Light-induced toxicity measured as the percentage of cells positive for red-fluorescent ethidium homodimer-1 versus calcein-positive cells (mean ± s.e.m.; n = 3 biological replicates; **P < 0.01). e, We compared the activation domains VP16 and p65 in addition to VP64 to test the modularity of the LITE CIB1–effector component. Neurog2 upregulation with and without light by LITEs using different transcriptional activation domains (VP16, VP64 and p65). Neuro 2a cells transfected with LITE were stimulated for 24 h with 466 nm light at an intensity of 5mW cm−2 and a duty cycle of 7% (1 s pulses at 0.067 Hz). All three domains produced a significant light-dependent Neurog2 mRNA upregulation (P < 0.001). We selected VP64 for subsequent experiments due to its lower basal activity in the absence of light-stimulation (mean ± s.e.m.; n = 3–4 biological replicates).