EMBO/soda-nel huggingface dataset:
Iron supplementation is sufficient to rescue cancer-induced muscle wasting and function. (2022)
Figure 2

Fig. 2. Altered iron metabolism in the skeletal muscle is a feature of cancer-induced cachexia

(A) Gastrocnemius weight normalized to tibial length in C26-tumor bearing mice on day 12 post C26-injection (n=6-7).

(B) TFR1 mRNA levels normalized to 18s (n=6-7)

(C) TFR1 protein expression and densitometric quantification in mouse gastrocnemius (n=5).

(D) Cytosolic aconitase activity in mouse quadriceps (n=4) measured following subcellular fractionation (n=4).

(E) Representative Western blot of IRP2 in mouse gastrocnemius and densitometric quantification (n=5).

(F) Binding of IRE-BPs to the ferritin IRE. The biotin labeled IRE probe was incubated with 6 μg of cytosolic gastrocnemius extracts from mice, in native or reducing conditions (with EDTA 0.5 mM and DTT 1 mM) (n=3).

(G) Representative protein carbonylation blot and densitometric quantification in mouse quadriceps (n=6-7).

(H) Representative Western blot of ferritin Heavy and Light chains in mouse gastrocnemius and densitometric quantification (n=4).

(I-J) ICP-MS quantification of total (I) and protein-bound (J) iron in mouse quadriceps (n=4-5).

Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Iron supplementation is sufficient to rescue cancer-induced muscle wasting and function. (2022)
Figure 3

Fig. 3. Cachectic muscles are characterized by mitochondrial iron deficiency and impaired oxidative metabolism

(A) ICP-MS quantification of mitochondrial iron in mouse quadriceps (n=6-8).

(B) Gastrocnemius heme content quantified by fluorescent heme assay (n=4-6).

(C-D) mRNA levels of mitochondrial iron importer MFRN2 (C) and the rate limiting enzyme of heme synthesis ALAS2 (D) normalized to 18s in mouse gastrocnemius (n=4-5).

(E) Aconitase activity of mouse quadriceps lysates normalized to protein content (n=6-8).

(F) Succinate Dehydrogenase activity staining in transversal sections of mouse gastrocnemius and corresponding intensity quantification (n=3-4). Scale bar= 50µm.

(G) Mitochondrial ATP content in mouse quadriceps (n=5-9).

(H) Representative Western blot and densitometric quantification of phospho-AMPK and total AMPK in mouse gastrocnemius (n=6).

Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Iron supplementation is sufficient to rescue cancer-induced muscle wasting and function. (2022)
Figure 4

Fig. 4. Iron enhances mitochondrial function and prevents cancer-induced myotube atrophy

(A) qPCR analysis of mitochondrial DNA (mtDNA) on nuclear DNA (nDNA) in C2C12 myotubes treated for 48h with C26CM and ferric citrate. (n=6).

(B) Western blot of mitochondrial OXPHOS respiratory complexes in C2C12 myotubes treated for 48H with C26CM and ferric citrate. (n=3)

(C-F) Profile of oxygen consumption rate OCR (C), basal OCR (D) , maximal OCR (E) and OCR used for mitochondrial ATP production (F) in C2C12 myotubes after 48h treatment with C26 CM and ferric citrate supplementation. Data normalized to protein content (n=9-12).

(G-H) Representative microscopic pictures and diameter of C2C12 myotubes stained for myosin heavy chain (G) or human myoblast-derived myotubes (H)after 48h treatment with C26 CM and ferric citrate (n=3 per condition). Scale bars =50 and 100 µm, respectively.

(I) Diameter of C2C12 myotubes treated with rotenone and ferric citrate (n=3).

Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test (D-E), or one-way Anova with Bonferroni's correction (F-G). Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and ##p < 0.01, ###p < 0.001 compared to C26 CM-treated group.
Iron supplementation is sufficient to rescue cancer-induced muscle wasting and function. (2022)
Figure 5

Fig. 5. Iron supplementation prevents cancer-induced cachexia

(A) Representative images of C26-tumor bearing mice receiving saline solution (left) or iron carboxymaltose 15mg/kg I.V. injection (right) taken at day 12 after C26 injection.

(B) Kaplan-Meier depicting the survival of C26-tumor bearing mice after I.V. injection of saline or iron every 5 days post C26-injection (3-month-old Balb/C, n=8-11).

(C) Grip strength of mice measured at day 12 post C26 injection, normalized to average strength of the control group (n=5-9).

(D) Final body weight of C26-tumor bearing mice after iron supplementation at day 12 post C26 injection (n=5-12).

(E) Final weight of total tumor mass extracted from mice after sacrifice (n= 17).

(F) gastrocnemius weight normalized to tibial length of C26-tumor bearing mice after iron supplementation at day 12 post C26 injection (n=5-12).

(G-H) Immunofluorescent staining of myosin heavy chain fast (green) and slow (red) of transversal sections of gastrocnemius (midbelly) with corresponding frequency distribution (G) and average cross-sectional areas (H) (n=3-5). Scale bar=100µm.

(I-K) mRNA levels of Murf 1 (I), Atrogin 1 (J), and REDD1 (K) normalized to GAPDH in gastrocnemius (n=5-14).

Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction, or chi-square test for the survival curves (B). Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.
Iron supplementation is sufficient to rescue cancer-induced muscle wasting and function. (2022)
Figure 6

Fig. 6. Iron supplementation rescues mitochondrial function

(A) Mitochondrial iron quantified by ICP-MS in quadriceps of C26-tumor bearing mice after iron carboxymaltose supplementation (n=7-8).

(B) Aconitase activity of quadriceps lysates normalized to protein content (n=3-7).

(C) Succinate Dehydrogenase activity staining of gastrocnemius transversal sections and resulting intensity quantification (n=3-7).

(D) Mitochondrial ATP content determined by luminescence assay in the quadriceps (n=3-9).

(E) Representative Western blot and densitometric quantification of phospho-AMPK and total AMPK (stripped and re-blotted) in the gastrocnemius of C26-tumor bearing mice after iron carboxymaltose supplementation (n=6).

(F-H) mRNA levels of ACOT 1 (F), MCD (G), and CPT1B (H) normalized to GAPDH in gastrocnemius (n=5-10).

Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.
Aubergine and piRNAs promote germline stem cell self-renewal by repressing the proto-oncogene Cbl (2017)
Figure 1

Figure 1: Intrinsic role of Aub in GSC self-renewal and differentiation

(A) Schematic diagram of a germarium showing the somatic cells (blue) and the germline cells (green). The spectrosomes and fusomes are shown in orange. The different regions of the germarium are indicated. Region 1: dividing cysts; region 2: selection of the oocyte; region 3: egg chamber with posteriorly localized oocyte. GSCs: germline stem cells; CB: cystoblast.

(B-E) Immunostaining of germaria from 7 day-old females with anti-Vasa (green, B-D) or anti-GFP (green, E), and anti-Hts (red). DAPI (blue) was used to visualize DNA. (B) aubHN2/+ was used as a control. (C, D) examples of aubHN2/QC42 germ cell loss and tumor, respectively. (E) Phenotypic rescue of aubHN2/QC42 with UASp-GFP-Aub expressed using nos-Gal4. White arrowheads indicate GSCs; the white arrow indicates GSC loss.

(F) Quantification of mutant germaria with 0 to 1 GSC, or with GSC tumors shown in (B-E). The number of scored germaria (n) is indicated on the right graph. *** p-value <0.001, ns: non significant, using the χ2 test.

(G-H") Germaria containing control (G, G") or aubHN2 mutant (H, H") clonal GSCs stained with anti-GFP (green) and anti-Hts (red), 14 days after clone induction. DAPI (blue) was used to stain DNA. Clonal cells are marked by the lack of GFP. Clonal GSCs and cysts are outlined with dashed line. White arrowheads show clonal GSCs in the control. aub mutant clonal GSCs have been lost (H, H"). Scale bar: 10 μm in B-E and G-H".

(I) Quantification of germaria containing at least one clonal GSC at 7, 14 and 21 days after clonal induction.

(J) Division rate of wild-type and aubHN2 clonal GSCs. The number of scored germaria (n) is indicated. *** p-value <0.001 using the two-tailed Student"s t test.
Aubergine and piRNAs promote germline stem cell self-renewal by repressing the proto-oncogene Cbl (2017)
Figure 2

Figure 2: The role of Aub in GSC self-renewal is partially independent of Chk2 and requires its loading with piRNAs

(A, B) Immunostaining of germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of mnkP6 and mnkP6 aubHN2/QC42 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss.

(C) Quantification of the number of GSCs per non-tumorous germarium, and of germaria with GSC tumors, in the indicated genotypes. The number of scored germaria (n) is indicated. The two-tailed Student"s t test and the χ2 test were used in the left and right panels, respectively. *** p-value <0.001, ** p-value <0.01, * p-value <0.05.

(D-F) Immunostaining of germaria from 7 day-old females with anti-Vasa or anti-GFP (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. aubHN2/QC42; nos-Gal4/+ was used as a negative control. Examples of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-Aub germarium (E), and of lack of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-AubAA germarium (F). White arrowheads indicate GSCs; white arrows indicate GSC loss in (D) and germ cell loss in (F). Scale bar: 10 μm in A, B and D-F.

(G) Quantification of mutant germaria with 0 to 1 GSC shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001, ns: non-significant, using the χ2 test.
Aubergine and piRNAs promote germline stem cell self-renewal by repressing the proto-oncogene Cbl (2017)
Figure 3

Figure 3: Physical and genetic interaction between Aub and the CCR4-NOT complex

(A-B"") Immunostaining of wild-type (A-A"") or nos-Gal/UASp-CCR4-HA (B-B"") germaria with anti-Aub (red), and anti-CCR4 or anti-HA (green). GSCs are shown in (A-A""). White arrowheads in (B-B"") indicate cytoplasmic accumulation of CCR4-HA colocalized with Aub in GSCs.

(C) Co-immunoprecipitation (IP) of NOT1, NOT3 and CCR4 with GFP-Aub in ovaries. Wild-type (WT, mock IP) or nos-Gal4/UASp-GFP-Aub (GFP-Aub) ovarian extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1, anti-NOT3 and anti-CCR4. Inputs are extracts prior to IP.

(D) Co-IP of NOT1 and NOT3 with GFP-Aub or GFP-AubAA in ovaries. Wild-type (WT, mock IP), nos-Gal4/UASp-GFP-Aub (GFP-Aub) or nos-Gal4/UASp-GFP-AubAA (GFP-AubAA) ovarian extracts were immunoprecipitated with anti-GFP in the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1 and anti-NOT3. Inputs are extracts prior to IP.

(E-G) Genetic interaction between aub and twin in GSC self-renewal. Immunostaining of germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of aubHN2/+, twinDG24102 and aubHN2/+; twinDG24102 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss. Scale bar: 10 μm in A-B"" and E-G.

(H) Quantification of mutant germaria with no GSC in 3, 7 and 14 day-old females of the genotypes shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001 using the χ2 test.
Aubergine and piRNAs promote germline stem cell self-renewal by repressing the proto-oncogene Cbl (2017)
Figure 4

Figure 4: Aub represses Cbl expression in the GSCs

(A) Genomic organization of the Cbl locus and Cbl mRNAs. Open boxes represent UTRs and introns, black boxes are exons. The insertion points in the CblEY11427 and CblMB05683 mutants are represented by white triangles. The region encoding the E3 ubiquitin ligase domain (Ring finger) is indicated. The regions used to raised the 8C4 and 10F1 monoclonal antibodies are underlined.

(B-C"") Immunostaining of mosaic germaria with anti-GFP (green), to identify clonal cells by the lack of GFP, and either 8C4 (B-B"") or 10F1 (C-C"") monoclonal anti-Cbl (red). DAPI was used to visualize DNA. White arrowheads indicate aubHN2/+ control GSCs; yellow arrowheads indicate clonal mutant aubHN2 GSCs. Scale bar: 10 μm.

(D-E) Quantification of Cbl protein levels in aubHN2/+ and aubHN2 mutant GSCs using fluorescence intensity of immunostaining with 8C4 or 10F1. Fluorescence intensity was measured in arbitrary units using the ImageJ software. Horizontal bars correspond to the mean and standard deviation. *** p-value <0.001 using the two-tailed Student"s t test.

(F) RNA immunoprecitation (IP) with anti-GFP antibody in wild-type (mock IP) and nos-Gal4/UASp-GFP-Aub ovarian extracts. Cbl mRNA was quantified using RT-qPCR. Normalization was with RpL32 mRNA. Mean of three biological replicates. The error bar represents standard error to the mean. * p-value <0.05 using the two-tailed Student"s t test.

(G) ePAT assay of CblL mRNA. Ovaries from 1-day-old (germarium to stage 8) wild-type, aub and twin mutant females, and from 4- to 7-day-old bam mutant females were used.
Aubergine and piRNAs promote germline stem cell self-renewal by repressing the proto-oncogene Cbl (2017)
Figure 5

Figure 5: GFP-Aub iCLIP in GSCs and targeting by piRNAs, in Cbl mRNAs

(A) GFP-Aub crosslinks in cultured GSCs (Ma et al, 2017), in Cbl mRNAs. Thin boxes are 5"- and 3"UTRs, lines are introns, and thick boxes are exons. The coverage of Cbl mRNAs with iCLIP reads is shown in blue. Significant crosslink clusters are shown in red.

(B, C) Sequences of crosslinked regions in 5"UTR (B) and 3"UTR (C) of CblL mRNA. The nucleotides (nt) in significant crosslink clusters are in red. The sequence, occurrence and origin of anti-sense piRNAs from ovarian and embryonic libraries are indicated. Mismatched nt are in blue.

(D, E) Coverage of CblL (D) and CblS (E) mRNAs with ovarian and embryonic piRNAs. Significant crosslink clusters are indicated in red at the top of the graphs.
Aubergine and piRNAs promote germline stem cell self-renewal by repressing the proto-oncogene Cbl (2017)
Figure 6

Figure 6: Cbl is required for GSC differentiation

(A-E) Cbl expression in germaria. (A) Immunostaining of wild-type germaria with anti-Cbl 8C4. (B-B"") Immunostaining of bamP-GFP germaria that express GFP under the bam promoter, with anti-GFP (green) and anti-Cbl 8C4 (red). (C-C") Immunostaining of germaria expressing GFP-Par1 to label spectrosomes with anti-GFP (green) and anti-Cbl 8C4 (red). DAPI (blue) was used to visualize DNA. White arrowheads indicate GSCs; yellow arrowheads indicate cystoblasts. (D) Quantification of germaria showing similar or increased Cbl protein levels in cystoblasts as compared to GSCs. (E) Quantification of increased Cbl protein levels in cystoblasts using fluorescence intensity of immunostaining with 8C4 antibody. Fluorescence intensity was measured in arbitrary units using ImageJ, in germaria showing increased Cbl levels in cystoblasts. The number of scored cells (n) is indicated. Intensity in GSCs was set to 1. The error bar represents standard deviation. ***p-value <0.001 using the two-tailed Student"s t test.

(F) Western blots of protein extracts from wild-type and Cbl mutant ovaries showing CblL (left panel) and CblS (right panel) revealed with 8C4 and 10F1 antibodies, respectively. A low level of CblS remained, while no CblL was present in the CblEY11427/MB05683 combination. The 10F1 antibody recognizes very poorly CblL in western blot.

(G-J) Cbl is required for germline differentiation. Immunostaining of wild-type and CblEY11427/MB05683 germaria with anti-Hts to visualize spectrosomes and fusomes. White arrowheads indicate GSCs; the white arrow indicates increased number of spectrosomes. (I) Quantification of germaria with increased number of spectrosomes, and (J) quantification of spectrosomes per germarium. The number of scored germaria (n) is indicated in (I). ***p-value <0.001 using the χ2 test in (I), and the two-tailed Student"s t test in (J).

(K-M) Cbl induces GSC differentiation. Immunostaining of germaria overexpressing Cbl with Hsp83-CblL (K) or Hsp83-CblS (L) with anti-Vasa (green) and anti-Hts (red). DNA (blue) was revealed with DAPI. White arrows indicate a cyst in the GSC niche (K, outlined) or the loss of GSCs and germ cells (L). (M) Quantification of germaria with 0 to 1 GSC in the indicated genotypes. The number of scored germaria (n) is indicated. *** p-value <0.001 using the χ2 test. Scale bar: 10 μm in A-C", G, H, K, L.
Aubergine and piRNAs promote germline stem cell self-renewal by repressing the proto-oncogene Cbl (2017)
Figure 7

Figure 7: Regulation of Cbl by Aub in the GSCs is essential for their self-renewal

(A-B) Immunostaining of germaria from aubHN2/QC42 (A) and aubHN2/QC42; CblMB/+ (B) females with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. The white arrow indicates the lack of GSCs; white arrowheads indicate GSCs. Scale bar: 10 μm.

(C) Quantification of mutant germaria with 0 to 1 GSC in the indicated genotypes. The number of scored germaria (n) is indicated. *** p-value <0.001, ** p-value <0.01, * p-value <0.05, using the χ2 test.

(D) Model of Aub function in GSCs. Aub is required intrinsically in GSCs for their self-renewal and differentiation. Aub function in self-renewal depends on translational repression of Cbl mRNA in GSCs through the recruitment of the CCR4-NOT complex. In cystoblasts, this translational repression is decreased, likely through the implication of at least another factor (X). As is the case for other translational controls in the GSC lineage, Aub/CCR4-NOT acts in fine-tuning Cbl levels. Aub function in GSC differentiation depends on activation of the Chk2 DNA damage checkpoint, consistent with a role in transposable element (TE) repression to maintain genome integrity; Ago3 has the same role in GSC differentiation.

Expanded view Figure legends
Aggregation and Disaggregation Features of the Human Proteome (2020)
Figure 2

Figure 2 - Characterization of proteins that aggregate in heat shock.

A Definition of aggregating proteins. Solubility is measured as the log2-transformed ratio of protein intensities in the soluble fraction between heat shocked and mock shocked samples. Proteins with significant reduction in solubility [Benjamini-Hochberg adjusted p-value < 0.05 and solubility < log2(2/3)] are then considered as aggregators. Dotted horizontal line shows the cut-off at solubility of log2(2/3).

B-D Comparisons of physicochemical properties between soluble proteins and aggregators. Hydrophobicity (gravy scores) (B) and isoelectric points (C) are shown as combined violin- and boxplots (p-values are for non-parametric Wilcoxon test). Difference in median amino acid composition between soluble proteins and aggregators is compared to hydrophobicity (gravy score) for each amino acid (D). In D, Pearson coefficient (r) with p-value is shown for the correlation analysis.

E-G Comparison of structural features between soluble proteins and aggregators. Fraction of protein sequence predicted to contain intrinsically disordered regions (E), molecular weight (F) and fraction of protein sequence predicted to contain secondary structure elements (alpha helix, beta sheet or coil) (G) are compared. P-values in E-G are for non-parametric Wilcoxon test.

H Protein complex members in soluble proteins and aggregators. Pie charts show the fraction of proteins annotated to be a member of a protein complex. The number of proteins in each segment is indicated. P-value is for Fisher's exact test.

I Protein complexes involving aggregators. Heatmaps show the absolute difference in solubility change after heat shock between each complex member. Protein complexes with at least 75% of its members quantified and containing at least 60% of its members as aggregators are shown. 'DNA repair complex' = 'DNA repair complex NEIL2-PNK-Pol(beta)-LigIII(alpha)-XRCC1'.

Data information: See 'Materials and Methods' for more detailed description of protein annotations used in B-I. Boxplots indicate median, first and third quartiles with whiskers extended to 1.5 times the interquartile range out from each quartile. Violin plots show the data distribution. Data shown for pre-existing protein fraction (light) quantified with at least two unique peptides from at least two biological replicates.
Aggregation and Disaggregation Features of the Human Proteome (2020)
Figure 4

Figure 4 - Newly synthesized proteins after heat shock.

A-B Accumulation of newly synthesized proteins after mock shock (A) and heat shock (B). Line graphs showing the accumulation of signal intensity (log2-transformed ratios to pre-shock control) in the soluble fraction. Orange lines show the mean ratio.

C Amount of newly synthesized proteins after heat shock compared to mock shock. Line graphs show the heat shock to mock shock ratio during recovery. Orange line show the mean ratio.

D Combined violin- and boxplots show the log2-tranformed ratio to pre-shock of newly synthesized proteins in the soluble fraction after heat shock. Proteins that aggregated in the pre-existing (light) fraction are compared to all other proteins. Boxplots indicate median, first and third quartiles with whiskers extended to 1.5 times the interquartile range out from each quartile. Violin plots show the data distribution. Data from at least two biological replicates.

E Ten proteins with the highest intensity at five hours after heat shock (i.e. strongest upregulation) are listed in order of decreasing intensity. Heat shock proteins are highlighted with red.

F Comparison of heat shock-induced protein synthesis and mRNA synthesis after heat shock (Vihervaara et al, 2017). Ten most upregulated proteins in the proteomics analysis are labelled and highlighted in blue; heat shock proteins are labelled red. HSPA1A and HSPA1B could not be distinguished from one another in the proteomics analysis and are plotted as they would have the same intensity (indicated with a vertical dashed line).

Data information: All proteomics data shown in A-D and F is quantified with at least two unique peptides from at least two biological replicates. Proteomics data shown for newly synthesized proteins (heavy). HS = heat shock. MS = mock shock.