Raw data of qRT-PCR and western blot analyses of proteasome subunits and GFP-CL1 degradation in Podospora anserina

<p>Dataset 1 : Raw data of qRT-PCR analysis of the PaPre3 gene used in Figure 1A<br>CP values of the reference gene PaPorin and of the target gene PaPre3 are displayed for juvenile middle-aged and senescent samples</p> <p>Dataset 2 : Raw data of qRT-PCR analysis of the PaPre2 gene used in Figure 1A<br>CP values of the reference gene PaPorin and of the target gene PaPre2 are displayed for juvenile middle-aged and senescent samples</p> <p>Dataset 3 : Raw data of qRT-PCR analysis of the PaUmp1 gene used in Figure 1A<br>CP values of the reference gene PaPorin and of the target gene PaUmp1 are displayed for juvenile, middle-aged and senescent samples</p> <p>Dataset 4 : Raw data of qRT-PCR analysis of the PaPre3 gene used in Figure 2A<br>CP values of the reference gene PaPorin and of the target gene PaPre3 are displayed. The wild type CP is the mean CP value of juvenile samples displayed in Dataset 1</p> <p>Dataset 5 : Raw data of qRT-PCR analysis of the PaPre2 gene used in Figure 2B<br>CP values of the reference gene PaPorin and of the target gene PaPre2 are displayed. The wild type CP is the mean CP value of the juvenile samples displayed in Dataset 2</p> <p>Dataset 6: Raw data of western blot displayed in Figure 1B probed with α-PaPRE2. Fluorescence was detected at 700 nm and 800 nm. Both signals are merged in the displayed image. Green signal represents fluorescence at 800 nm generated by anti-rabbit 800 antibody bound to α-PaPRE2. Red signal represents fluorescence at 700 nm. Lane 1 (from left to right): Thermo Fischer PageRulerTM (Cat# 26616) Prestained protein ladder. Lanes 2 – 7: Samples described in Figure 1B.</p> <p>Dataset 7: Raw data of western blot displayed in Figure 1B probed with α-PaPRE3. Fluorescence was detected at 700 nm and 800 nm. Both signals are merged in the displayed image. Green signal represents fluorescence at 800 nm generated by anti-rabbit 800 antibody bound to α-PaPRE3. Red signal represents fluorescence at 700 nm. Lane 7 (from left to right): Thermo Fischer PageRulerTM Prestained protein ladder. Lanes 1 – 6: Samples described in Figure 1B.</p> <p>Dataset 8: Raw data of western blot displayed in Figure 1B probed with α-PaPUP1. Fluorescence was detected at 700 nm and 800 nm. Both signals are merged in the displayed image. Green signal represents fluorescence at 800 nm generated by anti-rabbit 800 antibody bound to α-PaPUP1. Red signal represents fluorescence at 700 nm. Lane 10 (from left to right): Thermo Fischer PageRulerTM Prestained protein ladder. Lanes 1 – 6: Samples described in Figure 1B. Lanes 7 - 9 are not relevant to this study.</p> <p>Dataset 9: Raw data of western blot displayed in Figure 1B probed with α-PaHSP60. Fluorescence was detected at 700 nm. Red signal represents fluorescence at 700 nm generated by anti-mouse 700 antibody bound to α-PaHSP60. Lane 1 (from left to right): Thermo Fischer PageRulerTM Prestained protein ladder. Lanes 2 - 7: Samples described in Figure 1B.</p> <p>Dataset 10: Raw data of western blot displayed in Figure 2C probed with α-PaPRE3. Fluorescence was detected at 700 nm and 800 nm. Both signals are merged in the displayed image. Green signal represents fluorescence at 800 nm generated by anti-rabbit 800 antibody bound to α-PaPRE3. Red signal represents fluorescence at 700 nm. Lane 1 (from left to right): Thermo Fischer PageRulerTM Prestained protein ladder. Lanes 3 – 5 and lane 9: Samples described in Figure 1C. The other lanes are not relevant to this study.</p> <p>Dataset 11: Raw data of western blot displayed in Figure 2D probed with α-PaPRE2. Fluorescence was detected at 700 nm and 800 nm. Both signals are merged in the displayed image. Green signal represents fluorescence at 800 nm generated by anti-rabbit 800 antibody bound to α-PaPRE2. Red signal represents fluorescence at 700 nm. Lane 1 (from left to right): Thermo Fischer PageRulerTM Prestained protein ladder. Lanes 3 and 4: Samples described in Figure 1D. Lane 2 is not relevant to this study.</p> <p>Dataset 12: Raw data of western blot displayed in Figure 2D probed with α-PaPRE3. Fluorescence was detected at 700 nm and 800 nm. Both signals are merged in the displayed image. Green signal represents fluorescence at 800 nm generated by anti-rabbit 800 antibody bound to α-PaPRE3. Red signal represents fluorescence at 700 nm. Lane 1 (from left to right): Thermo Fischer PageRulerTM Prestained protein ladder. Lanes 3 and 4: Samples described in Figure 1D. Lane 2 is not relevant to this study.</p> <p>Dataset 13: Raw data of western blot displayed in Figure 3B probed with α-GFP. Fluorescence was detected at 700 nm. Red signal represents fluorescence at 700 nm generated by anti-mouse 700 antibody bound to α-GFP. Lane 1 (from left to right): Thermo Fischer PageRulerTM Prestained protein ladder. Lanes 9 and 10: Samples described in Figure 3B.</p> <p>Dataset 14: Raw data of western blot displayed in Figure 3C probed with α-GFP. Fluorescence was detected at 700 nm. Red signal represents fluorescence at 700 nm generated by anti-mouse 700 antibody bound to α-GFP. Lane 1 (from left to right): Thermo Fischer PageRulerTM Prestained protein ladder. Lanes 5 and 6: Samples described in Figure 3C.</p> <p> </p

Data_Sheet_1_Quercetin-Induced Lifespan Extension in Podospora anserina Requires Methylation of the Flavonoid by the O-Methyltransferase PaMTH1.pdf

<p>Quercetin is a flavonoid that is ubiquitously found in vegetables and fruits. Like other flavonoids, it is active in balancing cellular reactive oxygen species (ROS) levels and has a cyto-protective function. Previously, a link between ROS balancing, aging, and the activity of O-methyltransferases was reported in different organisms including the aging model Podospora anserina. Here we describe a role of the S-adenosylmethionine-dependent O-methyltransferase PaMTH1 in quercetin-induced lifespan extension. We found that effects of quercetin treatment depend on the methylation state of the flavonoid. Specifically, we observed that quercetin treatment increases the lifespan of the wild type but not of the PaMth1 deletion mutant. The lifespan increasing effect is not associated with effects of quercetin on mitochondrial respiration or ROS levels but linked to the induction of the PaMth1 gene. Overall, our data demonstrate a novel role of O-methyltransferase in quercetin-induced longevity and identify the underlying pathway as part of a network of longevity assurance pathways with the perspective to intervene into mechanisms of biological aging.</p

Additional file 2 of The autophagy interaction network of the aging model Podospora anserina

Nucleotide sequence of the vector pGAD-HA. The sequence is provided as text-based FASTA file. (FASTA 8 kb

Transcript analysis of <i>Pa_1_16400</i> (<i>PaCtr1</i>: A, B), <i>Pa_4_4770</i> (<i>PaCtr2</i>: C, D), and <i>Pa_3_10440</i> (<i>PaCtr3</i>: E, F), <i>Pa_5_11970</i> (G, H), <i>Pa_3_1710</i> (I, J).

<p>Results from the SuperSAGE analysis (<b>A, C, E, G, I</b>) are shown as ‘tags per million’ (tpm) for wild type (WT) and grisea (gr). The results of the qRT-PCR (<b>B, D, F, H, J</b>) are relative expression levels of the three copper transporter genes of the wild type (WT, n = 5 in <b>B</b>, <b>D</b>, <b>F</b>, <b>J</b>; n = 6 in <b>H</b>) and the grisea mutant (gr, n = 6) grown in standard medium, and wild type (WT+Cu, n = 3) and grisea (gr+Cu, n = 3) grown on copper-supplemented medium, normalized to the expression level of the gene coding for mitochondrial PORIN The error bars represent the standard deviation.</p

Copper-dependent standard and alternative respiration in <i>P. anserina</i>.

<p>While respiration of the wild type predominantly proceeds via the standard copper-dependent pathway, respiration in the grisea mutants does mainly follow the alternative route utilizing the di-iron containing alternative oxidase.</p

Transcript analysis of <i>Pa_2_7880</i> (<i>PaMth1</i>: A, B) and <i>Pa_2_7310</i> (C, D).

<p>The results of the SuperSAGE analysis (<b>A, C</b>) are shown as tags per million (tpm) for wild type (WT) and grisea (gr). The results of the qRT-PCR (<b>B, D</b>) are relative expression levels of the gene of the wild type (WT, n = 6) and of grisea (gr, n = 6) grown in standard medium, and wild type (WT+Cu, n = 3) and grisea (gr+Cu, n = 3) grown on copper supplemented medium, normalized to the expression level of the gene coding for mitochondrial PORIN. The error bars represent the standard deviation.</p

Results of the reference simulation.

<p><i>(A)</i> Selectivity functions for all processes as function of quality . <i>(B)</i> Initial random distribution of in quality state-space at time min. <i>(C)</i> Equilibrium distribution of in quality state-space at time min. <i>(D)</i> Transition rates of all processes normalized to their individual maximal values as function of time. The blue, red and green curves are on top of each other. <i>(E)</i> Average quality of mitochondria as function of time over all states (blue) and over active states (green). Error bars correspond to the standard deviation of the distribution and are plotted single-sided for reasons of clarity. <i>(F)</i> Fraction of mitochondria in the non-active state (red) and in active states (green) as function of time.</p

Results of the reference simulation in the presence of molecular damage.

<p><i>(A)</i>–<i>(C)</i> Random molecular damage: <i>(A)</i> Transition rates of all processes normalized to their individual maximal values as function of time. The blue, red and green curves are on top of each other. <i>(B)</i> Average quality of mitochondria as function of time over all states (blue) and over active states (green). Error bars correspond to the standard deviation of the distribution and are plotted single-sided for reasons of clarity. <i>(C)</i> Fraction of mitochondria in the non-active state (red) and in active states (green) as function of time. <i>(D)</i>–<i>(F)</i> Infectious molecular damage: the same quantities as in <i>(A)</i>–<i>(C)</i> are plotted.</p

Mitochondrial infectious damage adaptation (MIDA) model.

<p>Schematic representation showing the loss of mitochondrial quality over the fictive life span of an organism according to the MIDA model (green) versus a non-MIDA model (blue). In the MIDA model fusion–fission rates are reduced when a certain degree of molecular damage has accumulated (green arrow). In the non-MIDA model this adaptation was omitted. Stage I is characterized by high fusion–fission rates, low levels of accumulated random molecular damage, yet a high removal rate of those few dysfunctional mitochondria. Stage II represents the time/age when already a significant amount of molecular damage has accumulated. This damage is propagated and enhanced by ongoing fusion and fission cycles, representing a distinct type of damage, termed ‘infectious molecular damage’. At some time point the latter outweighs the benefit of mitochondrial dynamics and mitophagy in removing dysfunctional mitochondria. Decelerating mitochondrial dynamics (green arrow), in the MIDA model, slows down the accumulation of dysfunctional mitochondria compared to the situation in the non-MIDA model. Still, this adaptation in the MIDA model renders the system less capable of dealing with additional random molecular damage. Assuming a certain survival threshold (dotted line) this results in a net life span extension. Reaching this threshold marks stage III and cell death. The rates for mitophagy, quality decay, and mitochondrial biogenesis under homeostatic conditions are kept constant over all stages. The full simulation and used parameters are provided in the Supporting Information.</p

Stress-dependent opposing roles for mitophagy in aging of the ascomycete <i>Podospora anserina</i>

<p>Mitochondrial dysfunction is causatively linked to organismal aging and the development of degenerative diseases. Here we describe stress-dependent opposing roles of mitophagy, the selective autophagic degradation of mitochondria, in aging and life-span control. We report that the ablation of the mitochondrial superoxide dismutase which is involved in reactive oxygen species (ROS) balancing, does not affect life span of the fungal aging model <i>Podospora anserina</i>, although superoxide levels are strongly increased and complex I-dependent respiration is impaired. This unexpected phenotype depends on functional autophagy, particularly mitophagy, which is upregulated during aging of this mutant. It identifies mitophagy as a prosurvival response involved in the control of mitohormesis, the well-known beneficial effect of mild mitochondrial oxidative stress. In contrast, excessive superoxide stress turns mitophagy to a prodeath pathway and leads to accelerated aging. Overall our data uncover mitophagy as a dynamic pathway that specifically responds to different levels of mitochondrial oxidative stress and thereby affects organismal aging.</p