Instat software version 3

InStat software version 3.05 (GraphPad, San Diego, CA, USA) was used to analyze the data. The significant differences between the two samples in the PO activity and OfPPO expression assays were analyzed using a t test. One-way analysis of variance and Tukey’s multiple comparisons were used to analyze caste- and tissue-specific gene expression and RNA interference assays. A p value < 0.05 indicated a significant difference. For the survival assay, GraphPad Prism version 8.0.2 (GraphPad Software, San Diego, CA, USA) was used to analyze the data according to the log-rank (Mantel–Cox) test.

Allele and genotype frequencies were evaluated by gene count, using an online statistical analysis tool for the evaluation of SNPs (https://www.snpstats.net/start.htm, last access: 25 November 2022). The data were tested for goodness of fit between observed and expected genotype frequencies, according to Hardy-Weinberg equilibrium, by Pearson’s distribution and χ² tests. Significant differences in allele, homozygous, and heterozygous genotype distributions among groups were calculated using Fisher’s exact test (adjusted for age and gender). Multiple logistic regression models were applied, using dominant (major allele homozygotes versus heterozygotes plus minor allele homozygotes) and recessive (major allele homozygotes plus heterozygotes versus minor allele homozygotes) models. Odds ratios (OR), 95% confidence intervals (95% C.I.), and p values (p-value cutoff < 0.05) were determined using GraphPad InStat software version 3.06 (GraphPad, San Diego, California, USA) and the above-mentioned online statistical analysis tool.

Allele and genotype frequencies were evaluated by gene count, using an online statistical analysis tool applied to the evaluation of SNPs (https://www.snpstats.net/start.htm, last access on 30 September 2022). Data were tested for goodness of fit between observed and expected genotype frequencies according to Hardy-Weinberg equilibrium, by Pearson’s distribution and χ2 tests. Significant differences in allele, homozygous and heterozygous genotype distributions among groups were calculated by using Fisher’s exact test (adjusted by age and sex). Multiple logistic regression models were applied using dominant (major allele homozygotes versus heterozygotes plus minor allele homozygotes) and recessive (major allele homozygotes plus heterozygotes versus minor allele homozygotes) models. Odds ratios (OR), 95% confidence intervals (95% C.I.) and p values (p-value cutoff < 0.05) were determined using GraphPad InStat software version 3.06 (GraphPad, San Diego, CA, USA) and the abovementioned online statistical analysis tool. Haplotype frequency estimation (frequency threshold for rare haplotype: 0.01) and haplotype association with response estimation functions at https://www.snpstats.net/start.htm (last access on 30 September 2022) were applied to search for comparison of haplotype frequencies between patient and control groups at the IL-1 gene cluster.

All tests were carried out on four independent cell cultures, derived from four different cornea donors, and performed in triplicates for each of the treatments. The levels of the cytokines protein contents and mRNA expression were calculated as a ratio relative to medium. Statistical analysis and multiple comparisons were performed by one-way ANOVA using the InStat software version 3.0 (GraphPad Software Inc, San Diego, CA, USA).
The median effective concentration (EC 50) of RV-D1 was calculated with sigmoid Emax model. The pharmacokinetic modeling was performed using WinNonlin® 6.2 software (Pharsight Corporation, USA).

Statistical significance was calculated using two-tailed Fisher’s exact test, an ordinary analysis of variance, and InStat software version 3.0 (GraphPad, Los Angeles, CA, USA). A two-sided p value of <0.05 was considered to indicate statistical significance.

All tests were carried out on three independent cell cultures, and performed in triplicate for each of the treatments. In the experiments of the effect of ALA on nitrite secretion in HCF and effect of ALA on NOS-2 mRNA expression tests, we carried out the tests on four independent cell cultures, and performed triplicates for each of the treatments.
Statistical analysis and multiple comparisons were performed by one-way ANOVA using the InStat software version 3.0 (InStat software Inc., Graphpad, San Diego, CA). The differences in mean values among treatment groups were determined by ANOVA adjusted for multiple comparison by the Tukey test, ANOVA adjusted for multiple comparison by the Student-Newman-Keuls test, and ANOVA with multiple comparison by the Bonferroni t-test.