Fig 2: Effect of AAV-based gene knockdown of PERK and ATF6 in sclera on myopia development.a Distribution of EGFP expression in the scleral whole-mount 28 days after sub-tenon’s capsule injection of adeno-associated virus serotype DJ (AAV-DJ)-EGFP or AAV-DJ-SaCas9 (as non-fluorescence control); scale bar, 1 mm. b Representative Optical Coherence Tomography images 28 days after of each AAV injection; scale bar, 100 µm. c Expression level of IRE1, PERK, and ATF6 protein 28 days after of AAV-SaCas9 with AAV-gRNA against Eif2ak3 (Gene symbol of PERK, gEig2ak3) and/or Atf6 (gAtf6) injections. d Scleral Eif2ak3 and Atf6 knockdown by AAV-DJ-CRISPR/Cas9 injection into sub-tenon’ capsule. After 28 days injection, gene expression in the sclera determined by qPCR (n = 9, respectively). SaCas9 + gScramble group was assigned a value of 1.0. The p values were determined by one-way ANOVA with Tukey HSD. e Effects of scleral Eif2ak3 and/or Atf6 knockdown on axial elongation (sham: n = 5, gScramble: n = 11, gEif2ak3: n = 9, gAtf6: n = 8, gEif2ak3 + gAtf6: n = 8). The p values were determined by two-tailed Generalized Estimating Equations for five groups comparison (sham vs gScramble vs gEif2ak3 vs gAtf6 vs gEif2ak3 + gAtf6) and Student’s two-tailed t-test for two groups comparison (NL vs −30D). NL: No Lens control, −30D: minus 30 D lens-wearing eye. f Effects of scleral Eif2ak3 and/or Atf6 knockdown on refraction (sham: n = 5, gScramble: n = 9, gEif2ak3: n = 9, gAtf6: n = 8, gEif2ak3 + gAtf6: n = 9). The p values were determined by two-tailed Generalized Estimating Equations for five groups comparison and Student’s two-tailed t-test for two groups comparison. g Effects of scleral Eif2ak3 and/or Atf6 knockdown on VCD + RT (sham: n = 5, gScramble: n = 7, gEif2ak3: n = 7, gAtf6: n = 7, gEif2ak3 + gAtf6: n = 7). The p values were determined by two-tailed Generalized Estimating Equations for five groups comparison and Student’s two-tailed t-test for two groups comparison. Source data are provided as a Source Data file.

Fig 3: Lens-induced myopia induces axial elongation and endoplasmic reticulum stress in the sclera.a Changes in axial length during 3-week lens-induced myopia (LIM) in C57BL6J mice (n = 4). NL: No Lens control, −30D: minus 30 D lens-wearing eye. ***p = 0.00062; Student’s two-tailed t-test. b Changes in refraction during 3-week LIM in C57BL6J mice (n = 4); ***p = 0.00029; Student’s two-tailed t-test. c Transmission electron microscopy images of no lens (NL)-wearing (left panels) and minus 30 D lens-wearing (right panels) sclerae. Representative images of three biologically independent samples; N indicates nucleus and allow heads indicate rough endoplasmic reticulum. Scale bars: 1.0 µm. d Immunoblots showing ER sensor protein activation evaluated by phosphorylation levels of IRE1, PERK, eIF2α (downstream of PERK), and the ATF6 precursor (ATF6-P) and cleaved form of ATF6 (ATF6-N). Representative blots from four independent experiments are shown. Age-match: age-matched non-treated control, NL: No Lens-wearing eye, −30D: minus 30 D lens-wearing eye. e Densitometric quantification of blots in Fig. 1d using ImageJ. NL group was assigned a value of 1.0. Each group were n = 4 (each sample pooled 3 sclerae). The p values were determined by one-way ANOVA with Tukey HSD (two-tailed). NS: Not Significant. f UPR target gene expression level determined by quantitative PCR in age-matched control (right gray), No-Lens control (white) and LIM (gray) sclerae for 3-week LIM (n = 7 or 8). NL group was assigned a value of 1.0. The p values were determined by one-way ANOVA with Tukey HSD (two-tailed). Source data are provided as a Source Data file.

Fig 4: Effect of PERK and ATF6 agonizts instillation on myopia development.a Effect of PERK agonist CCT020312 (CCT) and ATF6 agonist AA147 (AA) instillation on IRE1, eIF2 and ATF6 activity in sclera. One hour after eye drop, the sclerae were harvested and subjected to Western blotting. Each compound specifically activated each pathway. b GRP78, ATF4, and EDEM gene expression induced by CCT. Each zero group was assigned a value of 1.0. *p = 0.029, compared to 0 (as a control) group; one-way ANOVA with Tukey HSD (n = 4, respectively). c GRP78, ATF4, and EDEM gene expression induced by AA. Each zero group was assigned a value of 1.0. *p = 0.013, compared to 0 (as a control) group; one-way ANOVA with Tukey HSD (n = 4, respectively). d Effects of 1-week instillation of CCT (100 μM) instillation on refraction; ***p = 0.00055; Student’s two-tailed t-test. e Effects of 1-week instillation of AA (100 μM) instillation on refraction; *p = 0.016; Student’s two-tailed t-test. f Effects of 1-week instillation of CCT plus AA (100 μM, respectively) instillation on refraction; ***p = 0.00001; Student’s two-tailed t-test. g Effects of 1-week instillation of CCT instillation on axial length; *p = 0.041; Student’s two-tailed t-test. h Effects of 1-week instillation of AA instillation on axial length; *p = 0.015; Student’s two-tailed t-test. i Effects of 1-week instillation of CCT plus AA instillation on axial length; ***p = 0.00039; Student’s two-tailed t-test. j Effects of 1-week instillation of CCT instillation on VCD + RT. *p = 0.036; Student’s two-tailed t-test. k Effects of 1-week instillation of AA instillation on VCD + RT. *p = 0.024; Student’s two-tailed t-test. l Effects of 1-week instillation of CCT plus AA instillation on axial length; ***p = 0.0004; Student’s two-tailed t-test. Source data are provided as a Source Data file.

Fig 5: Inhibition of a single UPR pathway with specific inhibitors disrupts the refractive condition of the mouse eyes.a Effect of instillation of IRE1 inhibitor STF080310 (STF; 100 μM), PERK inhibitor GSK2656157 (GSK; 100 μM), and ATF6 inhibitor nelfinavir (NFV; 100 μM) on activation of the UPR pathways by LIM in sclerae. NL: No Lens control, −30D: minus 30 D lens-wearing eye. b Densitometric quantification of blots in Fig. 3a using ImageJ. DMSO-NL group was assigned a value of 1.0. Each group were n = 4 (each sample pooled 3 sclerae). The p values were determined by two-tailed Generalized Estimating Equations for four groups comparison (DMSO vs STF vs GSK vs NFV in each eyes) and Student’s two-tailed t-test for no lens (NL) and −30D lens (−30D) eyes comparison in each treatments. NS: Not Significant. c Effects of STF, GSK, and NFV instillation on axial elongation in NL and −30 D eyes (n = 5 per group); ***p < 0.001 compared to the DMSO group; two-tailed Generalized Estimating Equations. d Effects of STF, GSK, and NFV instillation on refraction in NL and −30 D eyes (n = 5 per group); **p < 0.01 (p = 0.001), ***p < 0.001compared to the DMSO group; two-tailed Generalized Estimating Equations. Source data are provided as a Source Data file.