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. 2015 Jul 30;34(18):2334–2349. doi: 10.15252/embj.201591711

Figure 3. Chronic proteotoxic stress is associated with a shift towards more oxidizing conditions in the cytosol.

Figure 3

  1. Ratio-metric analysis of cytosolic roGFP in 4-day-old C. elegans upon expression of Q40-RFP in the cytosol of muscle tissue. The co-expression of polyQ in the same tissue is associated with a shift towards oxidizing conditions. Twenty nematodes each were analysed. The data are presented as means ± SD with a P-value of 1.94 × 10−11. Scale bars are 15 μm.
  2. Expression of Q77-mCherry, but not Q19, is associated with a shift towards oxidizing conditions in the cytosol in HeLa cells. 16, 31, 33, 27, 47 and 13 HeLa cells were analysed for fully oxidized, Q19-mCherry, Q77-mCherry, HttQ22-mCherry, HttQ134-mCherry and fully reduced conditions, respectively. The upper row shows the overlay images of cyto-roGFP1 upon excitation at 405 and 488 nm. The middle row shows the red channel for the polyQ-mCherry fusion proteins to show the aggregation propensity of Q77-mCherry and HttQ134-mCherry. The bottom row displays the overlay images from the upper two rows. On the top left, the 405/488 nm overlay image of the DTT (5 mM) control is shown and on the top right the respective overlay image of the DPS control (1 mM). Scale bars are 10 μm. The data are presented as means ± SD and are shown on the left. The ratio-metric analyses between HttQ22, HttQ134, Q19 and Q77 passed the ANOVA (F = 14; P > 0.0001) and the post hoc Tukey's HSD test confirmed a significant difference between HttQ22 versus HttQ134, HttQ22 versus Q19, HttQ134 versus Q19 and Q19 versus Q77. The difference between HttQ22 versus Q77 and HttQ134 versus Q77 is non-significant.
  3. Ratio-metric analyses of cytosolic HyPer in 4-day-old nematodes expressing Q40-RFP (right column) or RFP alone (left column, control) in muscle tissue. The increase in the ratio indicates a higher cytosolic concentration of H2O2 upon Q40-RFP expression. The data are presented as means ± SD with a P-value of 1.23 × 10−7.
  4. Ratio-metric analysis of cytosolic roGFP in muscle tissue in 4-day-old nematodes upon RNAi-mediated knockdown of ire-1 or skn-1. Depletion of ire-1 and skn-1 is associated with a shift towards oxidizing conditions in the cytosol. Twenty nematodes each were analysed. The data are presented as means ± SD. The ratio-metric analyses between the cyto-roGFP control, cyto-roGFP ire-1 RNAi, cyto-roGFP tunicamycin (Tm) and cyto-roGFP skn-1 RNAi passed the ANOVA (F = 31; P > 0.0001) and the post hoc Tukey's HSD test confirmed a significant difference between control versus ire-1 RNAi, control versus Tm, control versus skn-1 RNAi, ire-1 RNAi versus Tm and Tm versus skn-1 RNAi. The difference between ire-1 RNAi versus skn-1 RNAi is non-significant.
Source data are available online for this figure.