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The recombinant antibody H90-10 detects the endogenous human heat-shock protein 90 beta (Hsp90β) by immunoprecipitation (IP) and Western blotting.

miR-22 is one of the most abundant miRNAs in the liver and alterations of its hepatic expression have been associated with the development of hepatic steatosis and insulin resistance, as well as cancer. However, the pathophysiological roles of miR-22-3p in the deregulated hepatic metabolism with obesity and cancer remains poorly characterized. Herein, we observed that alterations of hepatic miR-22-3p expression with non-alcoholic fatty liver disease (NAFLD) in the context of obesity are not consistent in various human cohorts and animal models in contrast to the well-characterized miR-22-3p downregulation observed in hepatic cancers. To unravel the role of miR-22 in obesity-associated NAFLD, we generated constitutive Mir22 knockout (miR-22KO) mice, which were subsequently rendered obese by feeding with fat-enriched diet. Functional NAFLD- and obesity-associated metabolic parameters were then analyzed. Insights about the role of miR-22 in NAFLD associated with obesity were further obtained through an unbiased proteomic analysis of miR-22KO livers from obese mice. Metabolic processes governed by miR-22 were finally investigated in hepatic transformed cancer cells. Deletion of Mir22 was asymptomatic when mice were bred under standard conditions, except for an onset of glucose intolerance. However, when challenged with a high fat-containing diet, Mir22 deficiency dramatically exacerbated fat mass gain, hepatomegaly, and liver steatosis in mice. Analyses of explanted white adipose tissue revealed increased lipid synthesis, whereas mass spectrometry analysis of the liver proteome indicated that Mir22 deletion promotes hepatic upregulation of key enzymes in glycolysis and lipid uptake. Surprisingly, expression of miR-22-3p in Huh7 hepatic cancer cells triggers, in contrast to our in vivo observations, a clear induction of a Warburg effect with an increased glycolysis and an inhibited mitochondrial respiration. Together, our study indicates that miR-22-3p is a master regulator of the lipid and glucose metabolism with differential effects in specific organs and in transformed hepatic cancer cells, as compared to non-tumoral tissue.

Additional file 2: Figure S2. Carbon flux and total quantitation of target metabolites. (a) Schematic metabolic map showing the flow and distribution of 13C atoms in metabolites of the TCA cycle when cells consume 13C-Gln. Note that most of these metabolites traced with 13C-Gln were found to be upregulated in TRAP1 KO cells. (b, c) Total quantitation of target metabolites in WT and KO HEK293T and A549 cells. Note that this is “total” quantitation and should not be confused with 13C tracing. Total quantitation must be combined with the information provided in Additional file 4: Table S2 to infer metabolites with increased 13C incorporation. Data points on bar graphs indicate metabolite concentration per 106 cells from each biological replicate (n = 2).

Additional file 12: Figure S5. An extension of Figure 5 showing TRAP1-GST pulldown MS strategy and analysis, and a control experiment for mitochondrial lysis conditions. (a) TRAP1-GST pulldown strategy. (b) Venn diagram of the proteins identified by the MS analysis. Note that TRAP1 peptides are the only unique ones in the TRAP1-GST pulldown samples compared to the GST controls. (c) TRAP1 complexes from mitochondria, lysed with the indicated buffers, analysed by native PAGE and SDS-PAGE. The standard lysis buffer contained 1 mM DTT and 0.1% Triton X-100 (first lane); variations as indicated. IGEPAL, IGEPAL CA-630 (Sigma-Aldrich #I30211).

Additional file 6: Figure S4. Analysis of the whole cell proteome and TRAP1-associated proteins. (a) Control immunoblot performed to check TRAP1 WT and mutant expression in the KO cells used for the IP-MS experiments. (b, c) Comparative relative abundance of proteins immunoprecipitated with the indicated TRAP1 ATPase muatnts or WT TRAP1. The scatterplot was generated as mentioned in the legend to Fig. 4a. (d, e) Scatter plots comparing the levels (LFQ intensities) of the 3679 high confidence proteins between WT and KO HEK293T or HCT116 cells. Note that proteins highlighted in red above or below the 2-fold cutoff did not change consistently between the two cell lines.

Additional file 5: Figure S3. TRAP1 truncation and point mutants. (a) Schematic representation of the constructs for expression of mitochondrially targeted TRAP1 and EGFP. (b) Fluorescence micrographs showing proper targeting of mitoEGFP to mitochondria. Mitochondria are revealed with MitotrackerRED. (c) Expression analysis of TRAP1 truncation mutants by immunoblotting with an antibody to their HA-tag. (d) ATPase activity assay for the TRAP1 double mutant E115A/R402A. (e) Quantitation of basal respiration rates in WT versus KO HEK293T cells expressing the indicated proteins. Note that all ATPase mutants can rescue the KO phenotype to WT levels.

Dans la 2ème moitié du 20ème siècle, bien des efforts ont été déployés pour développer l’agriculture des pays francophones d’Afrique subsaharienne et de Madagascar (AFM). Ces efforts ont donné lieu à de nombreuses études critiques fondées notamment sur les résultats obtenus par les agricultures pratiquées, souvent jugés décevants. Dans ces régions, l’histoire des institutions et des structures de recherche, de la fin de la guerre de 1939-45 à nos jours, a également été très étudiée. From the publications of agronomy researchers working in French-speaking countries of sub-Saharan Africa and Madagascar, this article describes the evolution of thought in this discipline between 1946 and 1984. Initially, the desire to quickly make available to farmers and planters results to intensify the practices will lead to many experimental trials of transposed crop techniques of agriculture in temperate countries. The know-how of local farmers will be given little consideration. Gradually, faced with the difficulties encountered in applying these unsuitable techniques, the researchers will engage in work aimed more at understanding the functioning of plant communities and cropping systems, benefiting from advances in agronomic theory and methods of increasing more elaborate to analyze these operations.

The recombinant antibody AI179 against the Myc tag detects a Myc-tagged protein exogenously expressed in human cells by Western blotting.

The recombinant antibodies AF291and AE391 against the standard HA tag detect an HA-tagged protein exogenously expressed in human cells by Western blotting. However, similarly to one of the original monoclonal antibodies, they also recognize a number of unrelated endogenous proteins.

From the publications of agronomy researchers working in French-speaking countries of sub-Saharan Africa and Madagascar, this article describes the evolution of thought in this discipline between 1946 and 1984. Initially, the desire to quickly make available to farmers and planters results to intensify the practices will lead to many experimental trials of transposed crop techniques of agriculture in temperate countries. The know-how of local farmers will be given little consideration. Gradually, faced with the difficulties encountered in applying these unsuitable techniques, the researchers will engage in work aimed more at understanding the functioning of plant communities and cropping systems, benefiting from advances in agronomic theory and methods of increasing more elaborate to analyze these operations.; Dans la 2ème moitié du 20ème siècle, bien des efforts ont été déployés pour développer l’agriculture des pays francophones d’Afrique subsaharienne et de Madagascar (AFM). Ces efforts ont donné lieu à de nombreuses études critiques fondées notamment sur les résultats obtenus par les agricultures pratiquées, souvent jugés décevants. Dans ces régions, l’histoire des institutions et des structures de recherche, de la fin de la guerre de 1939-45 à nos jours, a également été très étudiée.

Complex patterns of protein-protein interactions (PPInts) are involved in almost all cellular processes. This has stimulated the development of a wide range of methods to characterize PPInts in detail. Methods with fluorescence resonance energy transfer can be technically challenging and suffer from several limitations, which could be overcome by switching to luminescence resonance energy transfer (LRET) with lanthanide ions such as Tb3+. With LRET, energy transfer between PPInt partners works over a larger distance and with less topological constraints; moreover, the long-lived luminescence of lanthanides allows one to bypass the short-lived background fluorescence. We have developed a novel LRET method to investigate PPInts between partners expressed as fusion proteins with genetically encoded donor and acceptor moieties. Upon UV excitation of a tryptophan within a lanthanide binding peptide, the Tb3+ luminescence is harnessed to excite either a green or a red fluorescent protein. We demonstrate the usefulness of the LRET assay by applying it to analyze the interactions of the molecular chaperones HSP70 and HSP90 with their common co-chaperone HOP/Sti1. We recapitulate the previously described interaction specificities between the HSP70/HSP90 C-termini and tetratricopeptide repeat domains of HOP/Sti1 and demonstrate the impact of single point mutants on domain-domain interactions.