Supplementary Table 1. Location of lens opacification in Aldh1a1(-/-), Aldh3a1(-/-) single and Aldh1a1(-/-)/Aldh3a1(-/-) double knockout mice (DKO) at different ages. A. One to three months of age Genotype Cornea Anterior Lens Mid Lens Posterior Lens Wild type (n = 18) 0 ± 0 0.11 ± 0.08 0 ± 0 0.17 ± 0.09 Aldh1a1(-/-) (n = 15) 0 ± 0 0.13 ± 0.09 0.07 ± 0.07 0.07 ± 0.07 Aldh3a1(-/-) (n = 16) 0 ± 0 1.19 ± 0.28* 1.19 ± 0.25* 0.63 ± 0.13* DKO (n = 14) 0 ± 0 1.43 ± 0.23* 1.50 ± 0.25* 0.93 ± 0.20* B. Six to nine months of age Genotype Cornea Anterior Lens Mid Lens Posterior Lens Wild type (n = 23) 0 ± 0 0.09 ± 0.06 0.17 ± 0.08 0.17 ± 0.08 Aldh1a1(-/-) (n = 16) 0 ± 0 0.63 ± 0.23 0 ± 0 0.06 ± 0.06 Aldh3a1(-/-) (n = 12) 0 ± 0 1.58 ± 0.29* 1.42 ± 0.23* 0.75 ± 0.28* DKO (n = 17) 0 ± 0 1.82 ± 0.25* 1.71 ± 0.14* 1.24 ± 0.18*
These data are based on a grading scale ranging from 0 to 3 (0 = no opacity, 1 = mild, 2 = moderate and 3 = severe). Three measurements were ascertained for each region per mouse in a blinded fashion. All repeated measurements were identical, so all comparisons were based on one measurement for each region per mouse. An overall significant difference in lens opacification was observed for all regions except the cornea when comparing all mouse genotypes (p < 0.01). Results were similar for either one-way ANOVA or Kruskal-Wallis Test (based on ranks). *p < 0.05 based on the post hoc Bonferroni t- test when comparing the designated group to the wild type mice for the specified region and age group
Supplementary Table 2. Cataract formation and corneal edema in Aldh1a1(-/-), Aldh3a1(-/-) single and double knockout mice following UVB exposure. A. 12 hours Anterior Lens Mid Lens Posterior Lens UVB Exposure (Difference ± SD) (Difference ± SD) (Difference ± SD) Genotype UV (0.05 J/cm 2 ) Wild type 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Aldh1a1(-/-) 0.50 ± 0.0 * 0.17 ± 0.3 0.50 ± 0.3 * Aldh3a1(-/-) 0.50 ± 0.3 * 0.50 ± 0.3 * 0.17 ± 0.0 * DKO 0.17 ± 0.3 0.50 ± 0.0 * 1.67 ± 0.3 * UV (0.20 J/cm 2 ) Wild type 0.0 ± 0.0 0.13 ± 0.6 0.17 ± 0.6 Aldh1a1(-/-) 0.67 ± 0.3 * 0.50 ± 0.0 0.67 ± 0.0 Aldh3a1(-/-) 1.0 ± 0.3 * 0.67 ± 0.0 0.67 ± 0.0 DKO 0.83 ± 0.3 * 0.50 ± 0.0 1.87 ± 0.3 B. 48 hours UVB Exposure Anterior Lens (Difference ± SD) Mid Lens (Difference ± SD) Posterior Lens (Difference ± SD) Genotype UV (0.05 J/cm 2 ) Wild type 0.33 ± 0.6 0.50 ± 0.5 0.33 ± 0.6 Aldh1a1(-/-) 1.17 ± 0.3 1.0 ± 0.0 0.83 ± 0.0 Aldh3a1(-/-) 1.33 ± 0.0 * 0.50 ± 0.3 0.50 ± 0.3 DKO 1.63 ± 0.3 * 0.83 ± 0.3 1.0 ± 0.0 Corneal Edema UV (0.2 J/cm 2 ) Wild type N.D a N.D N.D 1.0 ± 0 Aldh1a1(-/-) N.D N.D N.D 1.5 ± 0 Aldh3a1(-/-) N.D N.D N.D 2.0 ± 0 DKO N.D N.D N.D 2.5 ± 0
These data are based on a grading scale ranging from 0 to 3 (0 = no opacity, 1 = mild, 2 = moderate and 3 = severe). Three measurements were ascertained for each region per mouse in a blinded fashion. All animals (one to three months old) were exposed to 0.05 J/cm 2 and 0.2 J/cm 2 and examined for lens opacification 12 (Table 5A) and 48 hrs (Table 5B) after UVB exposure. All values represent the difference and standard deviation (SD) in opacity between UVB exposure compared to no UV for each group of mice; (n = 3 per group). Asterisks (*) indicate that the difference in opacity attributed to UV exposure is statistically significant (p<0.05) when compared to the difference in the corresponding wild type mouse, as determined by Student s unpaired t-test. a It was not possible to examine the lens due to the edema in the cornea.
Supplementary Figure 1. Generation of Aldh1a1/Aldh3a1 deficient mice. (A) Southern blot analysis of wild type [WT, 10.3 kb] and double knockout mice [DKO, 5.6 kb] for the Aldh1a1(-/- ) allele. Fifteen μg of genomic DNA was loaded in each lane. (B) Results of PCR analysis for double knockout [DKO, 198 bp] and wild type mice [WT, 280 bp] for the Aldh3a1(-/-) allele. One μg of genomic DNA was loaded in each lane.
Supplementary Figure 2. Slit lamp biomicroscopy of a double knockout mouse of more than 12 months of age. (A) Clear cornea. The anterior and posterior cataracts are not visible because the camera is focused on the cornea. (B) Anterior lens capsule showing cortical region with punctate opacities (arrow) and a relatively clear embryonal nucleus (arrow head). (C) Posterior cortex with punctate lens opacities (arrows). Opacities had refractile quality; the cornea and the anterior capsule are out of focus. A faint annular cataract with relatively clear embryonal nucleus (arrow head) is evident. (D) Posterior lens capsule with punctate, refractile opacities (arrows).
Supplementary Figure 3. Detection of 4-HNE metabolizing enzymes and antioxidant defenses in cornea and lens. (A) Western blot analysis of transketolase (TKT), catalase, alcohol dehydrogenase (ADH), aldose reductase (AR), and Cu,Zn-SOD in corneal extracts (total lysates) from two month old wild type [WT], Aldh1a1(-/-) [1a1(-/-)] and Aldh3a1(-/-) [3a1(-/-)] single and double knockout [DKO] mice. Ten μg cellular protein was loaded per lane. (B) Western blot analysis of transketolase (TKT), aldose reductase (AR), glutathione peroxidase (GPX), and Cu,Zn-SOD in lens extracts (total lysates) from two months old wild type [WT], Aldh1a1(-/-) [1a1(-/-)] and Aldh3a1(-/-) [3a1(-/-)] single and DKO mice. Thirty μg cellular protein was loaded per lane and equal loading was confirmed by probing the membranes with β- actin antibody.
Supplementary Figure 4. Slit lamp biomicroscopy and corresponding densitometry tracings of UVB-exposed mice. (A) Wild type, (B) Aldh1a1(-/-), (C) Aldh3a1(-/-) and (D) Aldh1a1(-/-)/Aldh3a1(-/-) mice were exposed to 0.05 J/cm 2 (10A) and 0.2 J/cm 2 (10B). Pictures were taken 48 hrs after UVB exposure. Arrows show opacities in the anterior subcapsular portion of the lens. Arrow heads show corneal edema. Cataract and corneal edema were scored during the examination (Supplementary Tables 2A & 2B).