Fig 2: rLRR20 regulates NGAL expression through the E-cadherin/ß-catenin pathway. rLRR20-mediated regulation of NGAL expression through the E-cadherin/ß-catenin pathway was determined using various inhibitors. Effect of short interfering RNA against E-cadherin (si-E-cad) on NGAL expression in HK2s (A) and hRPTECs (B). Effect of Neu-a-E-cad antibody on NGAL expression in HK2s (C) and hRPTECs (D). Effect of ICG-001, a ß-catenin inhibitor, on NGAL expression in HK2s (E) and hRPTECs (F). JW-67, a ß-catenin inhibitor, on NGAL expression in HK2s (G) and hRPTECs (H). Dimethyl sulfoxide (DMSO) was used as the control. * p < 0.05.

Fig 3: Effect of blocking antibody treatment on E- and N-cadherin gene and protein expression. (A) Quantitative PCR analysis of (i) E- and (ii) N-cadherin gene expression in untreated (control) or treated T24 cells with E-blocking antibody for 1 and 5 h, respectively. E-cadherin blocking antibody treatment at the highest concentration tested (25 µm/mL) resulted in a significant upregulation of the E-cadherin gene expression after 5 h compared with control (mean diff. ± SE = 1.8 ± 0.43, p = 0.01). Furthermore, the same treatment resulted in a significant downregulation of N-cadherin gene expression at the same timepoint compared with the 1-h timepoint, indicating a cadherin-switching effect (mean diff. ± SE = 0.23, ±0.05, p = 0.009). (B) Quantitative WB analysis of E-cadherin protein levels in untreated (control) or treated T24 cells with 25µg/mL E-cadherin or 50µg/mL N-cadherin blocking antibody after 24 h. Both antibodies significantly reduced the levels of E-cadherin after 24 h (Control versus E-CAD BA: mean diff± SE = 54.4 ± 19.4, p = 0.03; Control versus N-CAD BA: mean diff± SE = 61.5 ± 19.4, p = 0.03). (C) Quantitative PCR analysis of (i) E- and (ii) N-cadherin gene expression in untreated (control) or treated T24 cells with N- blocking antibody for 1 and 5 h, respectively. N-cadherin blocking antibody treatment at the second highest concentration tested (50 µg/mL) resulted in a nonsignificant upregulation of N- and E-cadherin gene expression at 5 h compared with control (N-cadherin: mean diff ±SE = 0.05 ± 0.17, p = 0.96; E-cadherin: mean diff ±SE = 0.52 ± 0.33, p = 0.32). (D) Quantitative WB analysis of N-cadherin protein levels in untreated (control) or treated T24 cells with 25µg/mL E-cadherin or 50µg/mL N-cadherin blocking antibody after 24 h. Both antibodies significantly reduced the levels of N-cadherin after 24 h (Control versus E-CAD BA: mean diff± SE = 31.4 ± 12.5, p = 0.02; Control versus N-CAD BA: mean diff± SE = 53.8 ± 12.5, p = 0.02). Untreated cells were used for data normalization. One representative WB is shown per condition including a total protein loading control. Whole WBs are shown in Fig. S3. Statistical analysis was performed using 1-way ANOVA followed by Tukey's multiple comparisons (qPCR) or Sidak's multiple comparisons (WB) test; *p < 0.05, **p < 0.01; n = 3 (triplicates).