Fig 1: cFOS and NR2F2 transcription factors mediate glucose-dependent repression of GDF15 in human dermis. A) Determination by Simple WES of the protein expression level of proGDF15 in wild-type HDF expressing shcontrol or shGDF15 (N = 3). B) Expression of GDF15 in the skin from human protein expression atlas from EMBL-EBI (https://www.ebi.ac.uk) which includes RNA-seq analyses from tissue samples of 122 human individuals, representing 32 different tissues. The results are expressed as TPM (Transcripts Per Kilobase Million). C) Quantification of GDF15 mRNA transcripts by taqman quantitative PCR in HDF, A549 and HEPG2 (N = 3). D) Quantification of GDF15 mRNA transcripts by taqman quantitative PCR in HDF, 786-O and SN005 cells (N = 3). E) ELISA-Based Quantification of GDF15 secretion in HDFs. HDFs were cultured under conditions of normal (5.5 mM) and high (12 mM) glucose concentrations during 48h. GDF15 levels in the culture supernatants were quantified using an enzyme-linked immunosorbent assay (ELISA) following the manufacturer's instructions. F) Quantification of GDF15 mRNA transcripts by taqman quantitative PCR in HDF grown in 5.55 mM, 12 mM or 25 mM glucose (N = 3). G) GDF15 promoter activity in HDF grown 24 h in DMEM with 5.55 mM,12 mM or 25 mM of glucose or 5.55 mM of galactose (N = 4). H) Dose-dependent relationship between GDF15 promoter activation and glucose concentration in the medium. I) GDF15 gene promoter sequence with identification of the binding site for the FOS transcription factor (Swiss Regulon Expasy). J) Quantification of FOS mRNA transcript by taqman quantitative PCR in HDF cultivated in 5.55 mM or 12 mM glucose (N = 3). K) Determination by Simple WES of FOS protein expression level in HDF cultivated in 5.55 mM or 12 mM glucose (N = 3). L-M) Determination by Simple WES of GDF15 protein expression level in HDF transfected with esiFOS (N = 3). N) Quantification of GDF15 mRNA transcript by taqman quantitative PCR in HDF transfected with esiNR2F2 (N = 3). O) Quantification of GDF15 mRNA transcript by taqman quantitative PCR in HDF expressing sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Normalization of the data to GusB (β-glucuronidase), N = 3. P–S) Quantification of ATF3, ATF4, CHOP and P53 mRNA transcripts by taqman quantitative PCR in HDF expressing siCTRL and esiNR2F2 in 5.5 mM glucose or 12 mM glucose growth medium. Normalization of the data to GusB (β-glucuronidase), N = 3. T) Schematic representation of the NR2F2-MTERF3-GDF15 axis and its control on OXPHOS function in response to glucose stress. All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Ordinary one-way ANOVA with Dunett's test correction was used for panel C, E and N. Unpaired t-test was used for panels A, B, H, I, J, K, L and M.
Fig 2: GDF15 biosynthesis is indispensable for human skin reconstruction. A) The effect of 100 nM GDF15 supplementation was determined using comparative proteomics on HDFs exposed to 12 mM glucose for 48H. The pathway analysis is shown as a bubble volcano plot (significant pathways with -logAdjPvalue>1.3 are shown. The pathways with blue dots are inhibited while pathways with orange dots are activated. The number of proteins detected for each pathway is represented by the diameter of each dot. Activation or inhibition was determined using the Z-score calculated by IPA Qiagen. B) Proteins of the Wound Healing Signaling, AMPK Signaling, Oxidative Phosphorylation or Protein Kinase A Signaling altered by the 12 mM glucose treatment are shown. C) Human skin reconstruction was performed using fiboblasts expressing a shGDF15, wild-type fibroblasts exposed to 12 mM glucose or wild-type fibroblasts exposed to 12 mM glucose and supplemented with 2 nM GDF15. development. Macroscopic view is shown with a scale bar of 0.6 cm, D) Immunofluorescence study of HRS using DAPI marker (blue), Collagen I (yellow) and MKi67 (pink). Scale bar. 50 μM, N = 3. E,F) Migration assay of HDF cultivated in 5.55 mM, 12 mM or 25 mM glucose and HDF cultivated in 12 mM glucose supplemented with 100 nM gdf15, HDF transfected with esiGDF15 or esiTFAM (N = 15). All data were expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Ordinary one-way ANOVA with Dunett's test correction was used for panel F.
Fig 3: GDF15 inhibition by hyperglycemia or shRNA alters mitochondrial biogenesis. A) Metabolomic profile of HDF grown 48H in DMEM with 5.55 mM or 12 mM + 100 nM GDF15 of glucose and HDF expressing a shGDF15 cultivated in 5.55 mM glucose (N = 3). B) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration and uncoupled respiration (CCCP) were determined in HDF grown in 5.55 mM glucose and HDF expressing shGDF15 grown in 5.55 mM glucose or 5.55 mM glucose supplemented with 100 nM gdf15. C) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration and uncoupled respiration (CCCP) were determined in HDF grown in 5.55 mM glucose and supplemented with low doses of gdf15: 20pM, 80pM, 1 nM, 10 nM and 100 nM. D) Mitochondrial respiratory chain proteins (gene loci) specifically activated at the level of chromatin accessibility by GDF15 100 nM. E-H) Quantification of mRNA transcripts by taqman quantitative PCR for NR2F2, GDF15, MTERF3 and TFAM in HDF cultivated with 5.55 mM glucose or 5.55 mM glucose supplemented with 100 nM gdf15. Normalization of the data was performed to GusB (β-glucuronidase), N = 3. I-L) Quantification of mRNA transcripts by taqman quantitative PCR for GDF15, MAPK1, MAPK3, PGC1α (Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha) in HDF cultivated with 5.55 mM glucose supplemented with low doses of gdf15. Normalization of the data was performed to GusB (β-glucuronidase), N = 3 M) Quantification of TFAM mRNA transcripts by taqman quantitative PCR in HDF cultivated with 5.55 mM, 12 mM or 25 mM glucose or in HDF expressing shGDF15 grown in 5.55 mM glucose. Normalization of data was performed to GusB (β-glucuronidase), N = 3. N) Quantification of PGC1α mRNA transcript by taqman quantitative PCR in HDF cultivated with 5.55 mM, 12 mM or 25 mM glucose or in HDF expressing shGDF15 grown in 5.55 mM glucose. Normalization of the data to GusB (β-glucuronidase), N = 3. O) Quantification of PGC1α mRNA transcript by taqman quantitative PCR in HDF transfected with esiNR2F2. Normalization of data to GusB (β-glucuronidase), N = 3. P,Q) Quantification of the total Coenzyme Q10 (oxidized and reduced forms) in HDF cultivated with 5.55 mM, 12 mM or 12 mM glucose medium supplemented with 100 nM gdf15. Analysis was also performed in HDF expressing shGDF15 in 5.55 mM glucose. R) Quantification of TFAM in HDF cultivated with 5.55 mM glucose medium or medium supplemented with 20pM, 100 pM and 100 nM rGDF15. S) Summary of the regulatory network linking TFAM, PGC1α, COQ9 and COQ10. All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Ordinary one-way ANOVA with Dunett's test correction was used for panel A, B, F, G, I-M, P–R. Unpaired t-test was used for panels E, H and O.
Supplier Page from OriGene Technologies for GDF15 Human shRNA Lentiviral Particle (Locus ID 9518)