Fig. 6 | Nature Communications

Fig. 6

From: Lysosomal protease deficiency or substrate overload induces an oxidative-stress mediated STAT3-dependent pathway of lysosomal homeostasis

Fig. 6

Direct STAT3 involvement in regulation of lysosomal hydrolase genes under conditions of protease activity/lysosomal substrate imbalance. a Agarose gel analysis of representative ChIP-PCR products for AEP, CtsD and CtsL promoters in WT and AEP-deficient MEFs. Full data and gel calibration markers can be seen in Supplementary Fig. 6. b Quantitative PCR analysis of DNA from STAT3 ChIP of WT (blue circles) and AEP-deficient (red squares) MEFs expressed as fold enrichment. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t-test. c AEP activity in WT MEF treated overnight with different concentrations of MVO26630 in the presence (dashed line) or absence (solid line) of 100 μM S3I-201. Data are the average ± SD of three independent experiments. d Quantitative PCR analysis of the mRNA levels for different lysosomal proteases in WT (blue circles) and STAT3c 3T3 (red squares) cells. Data are the average ± SD of n = 6 biologically independent samples for all samples, except for AEP where n = 5 and CtsL where n = 3. Statistical significance was calculated using a two-sided unpaired t-test. e Quantitative PCR analysis of the mRNA levels for different lysosomal proteases in WT MEFs treated (red squares) or not (blue circles) with 30 mg/ml BSA overnight. Data are the average ± SD of n = 4 biologically independent samples for all samples, except for AEP and CtsD where n = 3. Statistical significance was calculated using a two-sided unpaired t-test. f AEP and CtsD/E activities measured in WT and STAT3c 3T3 cells. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t-test. g Activities of AEP, CtsB/L and CtsD/E in WT MEF treated or not with 30 mg/ml BSA overnight in the presence or absence of 100 μM S3I-201. Data are the average ± SD of n = 3 biologically independent samples. h Quantitation of P-STAT3 and LC3II levels in WT MEFs treated with Rapamycin (1 μM) or Torin1 (1 μM) for 4 h or with MVO26630 (50 μM) or BSA (30 mg/ml) for 16 h. Data are the average ± SD of n = 25 microscopic fields for P-STAT3 and n = 14 for LC3II. Statistical significance was calculated using a Dunnett’s multiple comparison test. ns = not significant. (Scale bar = 20μm)

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