Supplementary information for the manuscript. Removal of damaged proteins during ES cell fate specification ... Figure S3. The 19S regulator does not bind 20S ...
Supplementary information for the manuscript
Removal of damaged proteins during ES cell fate specification requires the proteasome activator PA28 Malin Hernebring, Åsa Fredriksson, Maria Liljevald, Marija Cvijovic, Karin Norrman, John Wiseman, Henrik Semb, Thomas Nyström
b
20 nM Epoxomicin
c 100
100
% viable cells
Relative #cells
a
50
0 20 nM Epoxomicin
50
0 20 nM Epoxomicin
Figure S1. Effect of 20 nm epoxomicin on differentiating ES cells. (a) Cleaved Caspase‐3 (in red; detected by IHC using rabbit anti‐cleaved caspase‐3, 1:1000 from R&D Systems; and donkey anti‐rabbit A594, 1:500 from Molecular probes, Invitrogen) and DAPI (blue) in epoxomicin treated and differentiated (3 days) ES cells (left). An apoptotic cells is shown to the right (caspase‐3 localized to the nucleus). (b‐c) Effect of 20nM epoxomicin treatment on the growth rate (b) and viability (c) of differentiating ES cells presented as the relative number of cells per plate after 3 days of subsequent differentiation and proteasome inhibition. The number of cells on the untreated plates was set to 100% in (b), while the percentage of viable cells on the plates is presented in (c). Cell number and viability were both estimated using Countess® Automated Cell Counter (Invitrogen) according to the manufacturer’s instructions. Error bars represent SEM (n=3).
Days of differentiation 0
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kDa 50 37
25 20
15 β5i detection
β5 detection
Figure S2. β5 and β5i are processed (around 23 kDa rather than 28‐30kDa) suggesting that β5i is incorporated into the 20S. SDS‐PAGE followed by Western blot of β5i (middle blot and in red in the merged blot to the left) and β5 (right blot and in green in the merged blot to the left) in undifferentiated and differentiated ES cells. Neither the β5 nor the β5i blots displayed a 28.5kDa1 or 30kDa2 protein band which would correspond to their unprocessed form.
References Figure S2 1.
2.
Früh, K. et al. Alternative exon usage and processing of the major histocompatibility complex‐encoded proteasome subunits. Journal of Biological Chemistry 267, 22131‐ 22140 (1992). Frentzel, S., Pesold‐Hurt, B., Seelig, A. & Kloetzel, P.‐M. 20 S proteasomes are assembled via distinct precursor complexes processing of LMP2 and LMP7 proproteins takes place in 13–16 S preproteasome complexes. J Mol Biol 236, 975‐981 (1994).
Days of differentiation 0
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5
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α7 (20S) detection Rpn7 (19S) detection
Figure S3. The 19S regulator does not bind 20S during PA28‐20S favouring conditions. Native gel blot of samples extracted and run to optimize PA28‐20S interaction. The red signal corresponds to detection of Rpn7 (19S subunit) and green to α7 (20S subunit). The blot in the middle displays the α7 signal alone and the blot to the right Rpn7 alone.
1.0
0.5
miRNA: lacZ
Luc
PA28α
Figure S4. miRNA targeting PA28α does not affect the levels of protein carbonyls in undifferentiated ES cells. Levels of carbonylated proteins in undifferentiated ES cells treated with miRNA targeting PA28α and the non‐targeting controls lacZ and Luc. 24 h after electroporation, blasticidin was added to the ES medium containing LIF and the cells were subjected to 4 days of selection. The mean value was set to 1.0 and error bars represent SEM (n=3).
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b
Figure S5. Supplementary information of statistical analysis of figure 1a. “df0” denotes undifferentiated cells, “df3” cells that have been differentiating for 3 days, and “df5” cells after 5 days of differentiation. (a) Boxplot of the carbonyl levels during differentiation (y‐axis relative levels of protein carbonyls, x‐axis different days of differentiation). (b) Plot visualizing a post hoc Tukey test (y‐axis: 3 analysed groups: df3‐df0, df5‐df0 and df5‐df3; x‐axis: difference in mean levels of Days of differentiation (DF)). An analysis of variance (ANOVA) yielded significant variation among different groups of days of differentiation (F(2,7)=23.367, p=.00079). A post hoc Tukey test showed significant difference at p
