Research Paper Volume 13, Issue 3 pp 4452—4467

Modeling paraquat-induced lung fibrosis in C. elegans reveals KRIT1 as a key regulator of collagen gene transcription

SKN-1 knockdown inhibits paraquat induction of collagen genes in WT but not kri-1 mutant worms. (A) Paraquat induction of collagen transcription was impaired by skn-1 knockdown. WT C. elegans were fed control RNAi or skn-1 RNAi bacteria on agar plates containing 75 μM paraquat (PQ) from L1 to day-1 of adulthood and total RNA was prepared for RT-qPCR analysis. Shown are the relative expression of col-43, col-80 and col-139 normalized to non-treated controls. Error bars indicate the standard deviation of 3 experiments. P values were obtained by two tailed, paired student’s t-test (*PB) skn-1 knockdown was not additive to kri-1 mutation in regulating collagen gene transcription. Experiments were performed as in (A) except that kri-1 mutant worms were used. P values were obtained by two tailed, paired student’s t-test (**PC) The increase in COL-19::GFP protein levels by paraquat was mitigated by skn-1 knockdown. C. elegans WT and kri-1 mutant expressing COL-19::GFP were fed control RNAi or skn-1 RNAi bacteria on agar plates containing 75 μM paraquat (PQ) from L1 to day-1 of adulthood and the total proteins were prepared for Western blot analysis. Actin serves as a loading control. (D) Quantification of 3 biological replicates of Western blot data shown in (C). Signals on each blot were quantified with ImageJ and normalized to non-treated WT controls. Error bars indicate the standard deviation of 3 biological repeats. P values were obtained by two tailed, paired student’s t-test (*P

Figure 2. SKN-1 knockdown inhibits paraquat induction of collagen genes in WT but not kri-1 mutant worms. (A) Paraquat induction of collagen transcription was impaired by skn-1 knockdown. WT C. elegans were fed control RNAi or skn-1 RNAi bacteria on agar plates containing 75 μM paraquat (PQ) from L1 to day-1 of adulthood and total RNA was prepared for RT-qPCR analysis. Shown are the relative expression of col-43, col-80 and col-139 normalized to non-treated controls. Error bars indicate the standard deviation of 3 experiments. P values were obtained by two tailed, paired student’s t-test (*P<0.05, **P<0.01, ***P<0.001). (B) skn-1 knockdown was not additive to kri-1 mutation in regulating collagen gene transcription. Experiments were performed as in (A) except that kri-1 mutant worms were used. P values were obtained by two tailed, paired student’s t-test (**P<0.01, ***P<0.001, ns, not significant). (C) The increase in COL-19::GFP protein levels by paraquat was mitigated by skn-1 knockdown. C. elegans WT and kri-1 mutant expressing COL-19::GFP were fed control RNAi or skn-1 RNAi bacteria on agar plates containing 75 μM paraquat (PQ) from L1 to day-1 of adulthood and the total proteins were prepared for Western blot analysis. Actin serves as a loading control. (D) Quantification of 3 biological replicates of Western blot data shown in (C). Signals on each blot were quantified with ImageJ and normalized to non-treated WT controls. Error bars indicate the standard deviation of 3 biological repeats. P values were obtained by two tailed, paired student’s t-test (*P <0.05, **P<0.01, ns, not significant).