Effect of light exposure in the expression of per2, cry1a, per3, and per1b on Z3 cells. (a) Z3 cells were entrained for three LD cycles of 12:12 h. After the third day of entrainment, cells were exposed to constant darkness (DDDD) and mRNA expression of the genes per2, cry1a, per3, and per1b were analyzed every 4 h for 48 h. The eJTK test detected circadian rhythmicity on per3 and per1b, with a p-value of 0.01. No rhythmicity was detected in per2 and cry1a in DDDD. (b) mRNA expression of the genes per2, cry1a, per3 and per1b after a 12-hour exposure of low-intensity light (~ 4.12 × 10¹⁸ photons/s/m²) with subsequent darkness (LDDD). Samples were taken every 4 h for 48 h. Transcript levels were measured against time zero. Yellow bar: light exposure. Grey bar: constant darkness. Means ± SD are shown. Three replicates per group, one experiment.

HK-Spn exposure augments the expression of per2, cry1a, per3, and per1b on zebrafish cells. (a) After entrainment, Z3 cells were exposed to PBS or heat-killed Streptococcus pneumoniae (HK-Spn) and maintained in constant darkness (DDDD). RNA samples were collected every 4 h throughout 48 h, and the relative transcript expression of per2, cry1a, per3, and per1b was measured. (b) Z3 cells were exposed to PBS or heat-killed Streptococcus pneumoniae (HK-Spn) under 12-hour low-intensity light (~ 4.12 × 10¹⁸ photons/s/m²) followed by constant darkness (LDDD). RNA samples were collected every 4 h throughout 48 h, and the relative transcript expression of per2, cry1a, per3, and per1b was measured. All time points were compared against time zero samples. Yellow bar: light period. Grey bar: dark period. Means ± SD are shown. Asterisks (*) indicate statistically significant differences *p < 0.05,** p < 0.01,***p < 0.001, ****p < 0.0001, Two-way ANOVA with Šídák’s multiple comparisons test. Three replicates per group, one experiment.

Light intensity proportionally augments HK-Spn effect on per2 and cry1a but not in per3 and per1b. Z3 cells were entrained and exposed to HK-Spn under two light conditions, low-intensity light (Low-Int light,~4.12 × 10¹⁸ photons/s/m²) and high-intensity light (High-Int light, ~ 3.96 × 10¹⁹ photons/s/m²) for 4 h. Transcript expression of per2, cry1a, per3, and per1b was compared against time zero samples. Means ± SEM are shown. Asterisks (*) indicate statistically significant differences ** p < 0.01,****p < 0.0001, Two-way ANOVA with Tukey’s multiple comparisons test. Six experiments were conducted for dark and low-intensity light conditions, and three were conducted for high-intensity light conditions. Three replicates per group.

Hydrogen peroxide does not replicate light and HK-Spn effects. Relative expression of per2, cry1a, per3, and per1b in Z3 cells incubated with 300 µM hydrogen peroxide (H₂O₂) and HK-Spn for 4 h in the dark. Means ± SEM are shown. Asterisks (*) indicate statistically significant differences ****p < 0.0001, Two-way ANOVA with Tukey’s multiple comparisons test. Three experiments with three replicates per group.

Role of ROS in HK-Spn and light induction of clock genes. (a) Relative expression of per2, cry1a, per3, and per1b of Z3 cells exposed to HK-Spn and incubated in dark and low-intensity light conditions for 4 h. A 2-hour pre-treatment of 6 mM of N-acetyl cysteine (NAC) was used to suppress ROS production. Means ± SEM are shown. Asterisks (*) indicate statistically significant differences *p < 0.05, ***p < 0.001, ****p < 0.0001, Two-way ANOVA with Tukey’s multiple comparisons. Five to eight experiments, three replicates per group. (b) Dichlorodihydrofluorescein (DCF) fluorescence was measured after 2 h of PBS or HK-Spn exposure in dark and low-intensity light conditions. A 2-hour pre-treatment of 6 mM of N-acetyl cysteine (NAC) was used to suppress ROS production. Fold induction of relative fluorescent units was calculated against the dark PBS-treated cells. Means ± SEM are shown. Asterisks (*) indicate statistically significant differences ***p < 0.001,****p < 0.0001, Two-way ANOVA with Tukey’s multiple comparisons test. Four experiments, three replicates per group.