Journal: The journal of headache and pain

Article Title: Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

doi: 10.1186/s10194-023-01658-2

Figure Lengend Snippet: Fig. 2 AdipoR1 mediates the adiponectin-induced increase in IT. A, B Protein abundance of adipoR1 (A) and adipoR2 (B) in the brain and TGs of intact mice. GAPDH was used as an equal loading control. Representative blots of at least 3 independent experiments are shown. C, Colocalization of adipoR1 (red) with NeuN, GS, NF200, CGRP, IB4 or Cav3.2 (green) in TG sections. Arrows show the colocalization. Scale bar, 50 µm. D, Protein abundance of adipoR1 in TG cells treated with control siRNA (NC-siRNA) or adipoR1 siRNA (adipoR1-siRNA). Representative blots of at least 3 independent experiments are shown. **p < 0.01 (vs. NC-siRNA), unpaired t test. E, Representative traces (left) and bar chart (right) revealing that adipoR1-siRNA treatment prevented the adiponectin-induced IT increase (n = 11 cells). Adiponectin at 100 nM significantly enhanced IT in cells transduced with NC-siRNA (n = 12 cells). **p < 0.01 (vs. control + NC-siRNA), unpaired t test

Article Snippet: The human adipoR1 cDNA obtained from Origene was cloned into the pCMV6-AC-GFP vector.

Techniques: Quantitative Proteomics, Control, Transduction

Journal: The journal of headache and pain

Article Title: Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

doi: 10.1186/s10194-023-01658-2

Figure Lengend Snippet: Fig. 3 The AdipoR1-induced IT increase is mediated by the protein kinase CK2α. A Time course of IT changes (left) and bar chart (right) showing the effect of adiponectin at 100 nM on IT in cells dialyzed with GDP-β-S (1 mM) (n = 11 cells). Insets indicate representative current traces. Numbers represent points used for exemplary traces. B Coimmunoprecipitation of adipoR1 with RACK1 in the TG. Representative blots of at least 3 independent experiments are shown. C Protein abundance of RACK1 in TG cells treated with RACK1-siRNA or NC-siRNA. Representative blots of at least 3 independent experiments are shown. **p < 0.01 (vs. NC-siRNA), unpaired t test. D Summary of results revealing that treatment with either NC-siRNA (n = 10 cells) or RACK1-siRNA (n = 11 cells) did not affect the adiponectin-induced IT response. *p < 0.05 (vs. control + NC-siRNA), #p < 0.05 (vs. control + RACK1-siRNA), unpaired t test. E Interaction of adipoR1 with PKCK2α in the TG. Representative blots of at least 3 independent experiments are shown. F-G Time course of IT changes showing the effect of adiponectin at 100 nM on IT in cells preincubated with CX-4945 (F, 10 μM) or TBB (G, 20 μM). Insets indicate representative current traces. Numbers represent points used for exemplary traces. H Summary of the results revealing the effect of adiponectin at 100 nM on IT in the presence of CX-4945 (n = 8 cells) or TBB (n = 11 cells). ***p < 0.001 (vs. control), paired t test. I Protein abundance of CK2α in NC-siRNA- or CK2α-siRNA-treated groups. Representative blots of at least 3 independent experiments are shown. **p < 0.01 (vs. NC-siRNA), unpaired t test. J Representative traces (left) and bar chart (right) revealing that treatment with CK2α-siRNA prevented the adiponectin-induced IT increase (n = 11 cells). Adiponectin at 100 nM significantly enhanced IT in cells transduced with NC-siRNA (n = 12 cells). *p < 0.05 (vs. control + NC-siRNA), unpaired t test

Article Snippet: The human adipoR1 cDNA obtained from Origene was cloned into the pCMV6-AC-GFP vector.

Techniques: Quantitative Proteomics, Control, Transduction

Journal: The journal of headache and pain

Article Title: Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

doi: 10.1186/s10194-023-01658-2

Figure Lengend Snippet: Fig. 5 PKCβ1 is involved in the adipoR1-induced IT response. A Determination of mRNAs of classic PKC isoforms (PKCα, PKCβ1, PKCβ2 and PKCγ) in the TG of mice. No signal was detected in the reactions without RT (− RT). B Bar graph revealing the effect of 100 nM adiponectin on IT in the presence of LY333531 (200 nM, n = 10 cells), HBDDE (2 µM, n = 8 cells), PKCβ1 inhibitory peptide (PKCβ1-IP, 10 µM, n = 9 cells), or PKCβ2-IP (10 µM, n = 9 cells). ***p < 0.001 (vs. control), paired t test. C Protein abundance of PKCβ1 in TG cells treated with NC-siRNA or PKCβ1-siRNA. Representative blots of at least 3 independent experiments are shown. **p < 0.01 (vs. NC-siRNA), unpaired t test. D Bar graph revealing that treatment with PKCβ1-siRNA prevented the adiponectin-induced IT increase (n = 9 cells). Adiponectin at 100 nM still significantly enhanced IT in cells transduced with NC-siRNA (n = 12 cells). *p < 0.05 (vs. control + NC-siRNA), unpaired t test. E Immunofluorescence analysis of PKCβ1 translocation mediated by 100 nM adiponectin. Arrows in white indicate the line-scanned area. Data are representative of 3 independent experiments. F Immunoblot analysis of PKCβ1 expression in cytoplasmic and membrane fractions isolated from TG cells treated with 100 nM adiponectin. GAPDH served as a control for protein loading. α-Na+/K+ ATPase was used as an indicator for membrane contamination of cytosolic extracts. Representative blots of at least 3 independent experiments are shown. **p < 0.01 (vs. control), unpaired t test

Article Snippet: The human adipoR1 cDNA obtained from Origene was cloned into the pCMV6-AC-GFP vector.

Techniques: Control, Quantitative Proteomics, Transduction, Immunofluorescence, Translocation Assay, Western Blot, Expressing, Membrane, Isolation

Journal: The journal of headache and pain

Article Title: Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

doi: 10.1186/s10194-023-01658-2

Figure Lengend Snippet: Fig. 8 Peripheral adipoR1 participated in mechanical pain hypersensitivity. A Escape threshold after intra-TG injection of vehicle or adiponectin at 0.1 nmol, 1 nmol or 5 nmol. *p < 0.05 (vs. vehicle) at the corresponding time point, two-way ANOVA. B, C Administration of adipoR1-siRNA (B) or PKCβ1-siRNA (C) prevented adiponectin-induced mechanical hypersensitivity. *p < 0.05 (vs. vehicle); #p < 0.05 (vs. APN) in NC-siRNA-treated groups, two-way ANOVA. D Pretreatment with TTA-P2 (1 nmol) or Z941 (0.5 nmol) attenuated adiponectin (1 nmol)-induced mechanical hypersensitivity. *p < 0.05 (vs. vehicle), #p < 0.05 (vs. APN) at the 1-h time point, two-way ANOVA. E Escape threshold to mechanical stimuli in the sham- or CCI-ION-operated groups. ***p < 0.01 (vs. sham) at the corresponding time point, two-way ANOVA. F Protein abundance of adipoR1 in TGs 14 days following CCI-ION or sham surgery. *p < 0.05 (vs. sham), unpaired t test. Representative blots of at least 3 independent experiments are shown. G Intra-TG administration of adipoR1-siRNA 14 days after CCI-ION significantly attenuated mechanical hypersensitivity in CCI-ION mice. *p < 0.05 and **p < 0.01 (vs. NC-siRNA), two-way ANOVA. H Effect of Cav3.2-siRNA vs. NC-siRNA (Day 0) on adipoR1-siRNA (intra-TG injection on Day 3)-induced alleviation of mechanical allodynia in CCI-ION mice. Intra-TG injection of adipoR1-siRNA did not have additive effects to Cav3.2-siRNA on mechanical allodynia in CCI-ION mice. ** p < 0.01 (vs. CCI-ION) at -14 days, # p < 0.05 and ## p < 0.01 (vs. NC-siRNA) at the 3-day point in CCI-ION mice, + p < 0.05 (vs. Cav3.2-siRNA) at the 0-day point in CCI-ION mice, two-way ANOVA

Article Snippet: The human adipoR1 cDNA obtained from Origene was cloned into the pCMV6-AC-GFP vector.

Techniques: Injection, Quantitative Proteomics

Journal: The journal of headache and pain

Article Title: Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

doi: 10.1186/s10194-023-01658-2

Figure Lengend Snippet: Fig. 9 Illustration of proposed mechanisms of adipoR1 signaling on Cav3.2 channels. Adiponectin acts through the G protein-independent adipoR1, leading to the activation of protein kinase CK2α subunits. CK2α stimulates the downstream conventional PKCβ1, which selectively modulates Cav3.2 channel activity, resulting in an IT increase. The signaling cascade mediated by adipoR1 contributes to TG neuronal hyperexcitability and nociceptive behaviors of adiponectin. Importantly, adipoR1 was upregulated in the injured TG, and blockade of Cav3.2 attenuated adipoR1-mediated pain hypersensitivity in CCI-ION-induced neuropathic pain behaviors. Neither PKA, CaMKII nor novel PKC isoforms are involved in the adiponectin-mediated IT response. Nevertheless, whether PKCβ1 directly phosphorylates Cav3.2 channels or acts via some intermediate signaling molecules needs to be further explored

Article Snippet: The human adipoR1 cDNA obtained from Origene was cloned into the pCMV6-AC-GFP vector.

Techniques: Activation Assay, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: The Role of Monoubiquitination in Endocytic Degradation of Human Ether-a-go-go-related Gene (hERG) Channels under Low K+ Conditions

doi: 10.1074/jbc.m110.198408

Figure Lengend Snippet: FIGURE 9. Scheme of role of Ub in endocytic degradation of hERG channels. Under low K conditions, mature hERG channels adopt a non-conducting con- formation, leading to monoubiquitination, which triggers hERG endocytosis through a caveolin-dependent pathway. Endocytosed hERG channels transit through early endosomes into MVBs. While Vps24 is critical for the formation and sorting of monoubiquitinated hERG channels into MVBs, STAMBP deubiq- uitinates the monoubiquitinated hERG to facilitate hERG sorting in the MVBs. The sorted hERG is deposited in the lysosomes for degradation. Lactacystin prevents deubiquitination, leading to accumulation of monoubiquitinated hERG channels which are primarily localized in the plasma membrane. Bafilo- mycin A1 prevents hERG degradation in lysosomes, leading to accumulation of deubiquitinated hERG in lysosomes.

Article Snippet: Vps24 human cDNA open reading frame (ORF) clone with Myc tag in pCMV6-entry vector (hVps24; plasmid number RC220006) was obtained from OriGene Technologies (Rockville, MD). hVps24 siRNA and STAMBP siRNA were purchased from Sigma.

Techniques: Clinical Proteomics, Membrane

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: High glucose stress activates the mitochondrial repressor MTERF3. A) Quantification of the MTERF3 (Mitochondrial Transcription Termination Factor 3) RNA transcript by taqman quantitative PCR in HDF grown in 5.55 mM or 12 mM glucose. Normalization of the data was performed using GusB (β-glucuronidase) (N = 3). B) Determination by Simple WES of MTERF3 protein expression in HDF cultivated 48H in DMEM with 5.55 mM or 12 mM glucose. Total protein was used for normalization. (N = 3). C) Human Reconstructed Skin (HRS) generation process. HDF were seeded on bovine collagen matrix. After 21 days, Human Equivalent Dermis (HED) was produced. Human Epidermal Keratinocytes were seeded on HED. After 38 days, HRS was produced. D) Immunofluorescence analysis of MTERF3 expression in HRS epidermis grown in 5.55 mM or 12 mM glucose. Signal intensity was quantified by measuring MTERF3 fluorescence (purple color) normalized to the number of cells, as determined by the number of nuclei (DAPI staining; blue color). 10 images were acquired per HRS (N = 3). E) Similar analysis was performed in HRS dermis. F) Determination by Simple WES of the protein expression level of various mitochondrial respiratory chain subunits in human dermal fibroblasts (HDF): complex I (NADH:Ubiquinone Oxidoreductase Subunit B8), complex II (Succinate dehydrogenase [ubiquinone] iron-sulfur subunit), complex III (Ubiquinol-Cyt C Reductase Core Protein 2), complex IV (Cyt C Oxidase Subunit 4) and complex V (ATP synthase alpha subunit) in HDF cultivated 48 h in DMEM with 5.55 mM or 12 mM of glucose. Total protein loading was used for normalization (N = 3). Similar analysis was performed in G) Human Equivalent Dermis (HED, N = 3) and H) Human Reconstructed Skin (HRS, N = 3). All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Unpaired t -test was for all panels.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Produced, Immunofluorescence, Fluorescence, Staining

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: MTERF3 ectopic overexpression represses OXPHOS. A) Proteomic study of HDF overexpressing human MTERF3 (MTERF3OE) as compared to wild-type control expressing the empty plasmid p -lenti (N = 4). B) Schematic representation of the mitochondrial proteins reduced in expression (with Adjp<0.05) as a result of the MTERF3 overexpression (MTERF3OE) in HDF. C) Ingenuity Pathway Analysis (Z-score) of the proteins significantly altered in expression, in response to MTERF3 overexpression. D) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration, non-phosphorylating respiration (oligomycin) and uncoupled respiration (CCCP) were determined in human skin fibroblasts with MTERF3 overexpression, as compared to control cells grown in 5.55 mM or 12 mM of glucose (N = 6). E) Mitochondrial morphology study of HDF overexpressing MTERF3 and control cells grown in DMEM with 5.55 mM. Mitochondrial staining was performed using 50 nM of MitoTracker Red (N = 15). Three parameters of the mitochondrial network were analyzed using Image J: particles count, tubules length and interconnections. F) Quantification of TFAM (Transcription Factor A, Mitochondrial) RNA transcript by Taqman quantitative PCR in HDF overexpressing MTERF3, as compared to plenti-control cells. Normalization of the RT-QPCR data was performed to GusB (β-glucuronidase), N = 3. G) Growth curves of HDF lenti-control and MTERF3 overexpression grown during 7 days in DMEM with 5.55 mM or 12 mM of glucose (N = 12). H) Determination by Simple WES of the protein expression level of mitochondrial respiratory chain subunits: complex I (NADH:Ubiquinone Oxidoreductase Subunit B8), complex II (Succinate dehydrogenase [ubiquinone] iron-sulfur subunit), complex III (Ubiquinol-Cyt C Reductase Core Protein 2), complex IV (Cyt C Oxidase Subunit 4) and complex V (ATP synthase alpha subunit) in HDF control and MTERF3 overexpression cultivated 48 h in DMEM with 5.55 mM or 12 mM of glucose. Protein normalization was performed using total proteins (N = 3). All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Unpaired t -test was used for panels A,E,F and H.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Over Expression, Control, Expressing, Plasmid Preparation, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: NR2F2/Coup-TFII glucose-dependent transcription factor inhibition upregulates MTERF3. A) Promoter region of the human MTERF3 gene and regulatory sites for transcription factors binding. Identification of NR2F2 binding site using Swiss Regulon (Expasy). B) Quantification of NR2F2 (Nuclear Receptor Subfamily 2 Group F Member 2) RNA transcript by taqman quantitative PCR in HDF cultivated in DMEM with 5.55 mM, 12 mM or 25 mM of glucose. Normalization of the data was performed to GusB (β-glucuronidase) (N = 3). C) MTERF3 promoter reporter activity assay using a Gaussia-luciferase (Gluc) lentiviral approach. D) Determination by bioluminescence of the MTERF3 promoter activity in HDF with lentiviral transduction of the reporter. Fibroblasts were grown 24 h in DMEM with 5.55 mM,12 mM or 25 mM of glucose, or 5.55 mM of galactose (N = 4). Data were normalized to the number of cells. E) Dose-dependent relationships between MTERF3 promoter activation and glucose concentration in the cell culture medium. F) Quantification of NR2F2 mRNA transcript by taqman quantitative PCR in HDF transfected with esiControl and esiNR2F2. Normalization of the data to GusB (β-glucuronidase), N = 3. G) MTERF3 promoter activity determination in HDF transfected with esiNR2F2 (N = 4). H) Quantification of MTERF3 mRNA transcript by taqman quantitative PCR in HDF transfected with esiNR2R2. Normalization of data to GusB (β-glucuronidase), N = 3. I) Determination by Simple WES of MTERF3 protein expression level in HDF transfected with esiNR2F2. Protein normalization was performed to total protein loading (N = 3). J) Schematic representation of lentiviral CRISPR guide RNA for the generation of NR2F2 knock-out. K) Quantification of NR2F2 mRNA transcripts by taqman quantitative PCR in stable HDF using sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Normalization of the data to GusB (β-glucuronidase), N = 3. L) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration, non-phosphorylating respiration (oligo) and uncoupled respiration (CCCP) were determined in HDF expressing sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. M) Quantification of RNA transcripts by taqman quantitative PCR of COQ9 (coenzyme Q9), TFAM (Transcription Factor A, Mitochondrial) and MT-RNR1 (Mitochondrially Encoded 12S RRNA), in stable HDF sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Normalization of data to GusB (β-glucuronidase), N = 3. N) Mitochondrial morphology of HDF expressing sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Imaging was performed using 50 nM of MitoTracker Red (N = 40). O) Schematic representation of the impact of high(12 mM)-glucose medium on NR2F2, MYCN, MTERF3 and OXPHOS. P) Quantification of MTERF3 mRNA transcript by taqman quantitative PCR in HDF transfected with esiMYCN. Normalization of the data to GusB (β-glucuronidase), N = 3. Q) MTERF3 promoter activity assay in HDF transfected with esiMYCN (N = 4). R) Determination by Simple WES of the MTERF3 protein expression level in HDF transfected with esiMYCN (N = 4). 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 B, D, K, L, M and N. Unpaired t -test was used for panels F, G, H, I, P, Q and R.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Inhibition, Binding Assay, Real-time Polymerase Chain Reaction, Activity Assay, Luciferase, Transduction, Activation Assay, Concentration Assay, Cell Culture, Transfection, Expressing, CRISPR, Knock-Out, Imaging

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: 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.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture, Activity Assay, Activation Assay, Concentration Assay, Sequencing, Binding Assay, Transfection, Control

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: 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.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Inhibition, shRNA, Expressing, Real-time Polymerase Chain Reaction, Transfection

Journal: bioRxiv

Article Title: Barth syndrome cellular models have dysregulated respiratory chain complex I and mitochondrial quality control due to abnormal cardiolipin

doi: 10.1101/2021.01.06.425502

Figure Lengend Snippet: (A) The enrichment of the CI-associated terms is driven by 7 genes; MT-ND3, NDUFAF1, NDUFA5, NDUFAB1, NDUFB2, NDUFB4, OXA1L . (B) Relative mRNA expression of NUDFAF1 , determined by qRT-PCR and ΔΔCT quantification. Data plotted relative to WT/CTRL expression; WT n=6, TAZ Δ45 n=6, CTRL n=10, BTHS n=15. (C) Whole cell lysate (45 ug) of the indicated lines were immunoblotted for the indicated proteins. (D) Band intensities, relative to the loading control GRP75, were quantified and plotted relative to WT/CTRL abundance; WT n=27, TAZ Δ45 n=26, CTRL n=49, BTHS n=48. (E) BN-PAGE of HEK293 WT and TAZ Δ45 or CTRL and BTHS LCL cells (100-250K cells) solubilized in 1% Triton X-100 immunoblotted for the indicated proteins. (F) Band intensities were quantified, and CI abundance was represented as the ratio of CI band intensity to CIV (COXIV) or CII (SDHA). Abundance was plotted relative to WT/CTRL abundance; CI:CIV(WT n=13, TAZ Δ45 n=16, CTRL n=12, BTHS n=12), CI:CII (WT n=10, TAZ Δ45 n=13, CTRL n=9, BTHS n=9). (G) CI activity measured in HEK293 WT and TAZ Δ45 or CTRL and BTHS LCLs mitochondria (200 ug total protein) on a microplate reader (450nm) by following the oxidation of NADH to oxidized nicotinamide adenine dinucleotide (NAD+). Activity plotted relative to WT/CTRL abundance; WT n=25, TAZ Δ45 n=26, CTRL n=36, BTHS n=42. (H) CII activity measured in HEK293 WT and TAZ Δ45 or CTRL and BTHS LCLs mitochondria (200 ug total protein) on a microplate reader by following the production of ubiquinol by CII coupled to the reduction of the dye diclorophenolindophenol (DCPIP, 600nm). Activity plotted relative to WT/CTRL abundance; WT n=12, TAZ Δ45 n=12, CTRL n=10, BTHS n=12. Targeted metabolomics was used to measured (I) NADH, NAD+, and (J) cellular AMP via mass spectrometry in HEK293 WT (n=3), HEK293 TAZ Δ45 (n=3), CTRL LCL (n=5) and BTHS LCL (n=5) cells. Significant differences are indicated; * ≤ 0.05, ** ≤ 0.005, *** ≤ 0.0005, **** ≤ 0.00005.

Article Snippet: When cells reached ∼80% confluency cells were transiently transfected with NDUFAF1 (NM_016013) C-Myc/DDK-tagged plasmid (Origene #RC200029) with Lipofectamine 3000 (Thermo #L3000001) according to manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Control, Activity Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: Barth syndrome cellular models have dysregulated respiratory chain complex I and mitochondrial quality control due to abnormal cardiolipin

doi: 10.1101/2021.01.06.425502

Figure Lengend Snippet: (A) HEK293 WT and TAZ Δ45 cells were treated for 48 hours with 2.5uM bromoenol lactone (BEL) and 7 days with 100nM SS-31. Whole cell lysate (40-45 ug) of the indicated lines were immunoblotted for the indicated proteins. (B) Band intensities, relative to the loading control GRP75, were quantified and plotted relative to WT abundance; WT n=27, TAZ Δ45 n=26, WT-BEL n=9, TAZ Δ45 -BEL n=9, WT-SS-31 n=9, TAZ Δ45 -SS-31 n=9. (C) Relative mRNA expression of NDUFAF1 determined by qRT-PCR and ΔΔCT quantification using each respective control; WT n=6, TAZ Δ45 n=3, WT-BEL n=3, TAZ Δ45 -BEL n=3, WT-SS-31 n=3, TAZ Δ45 -SS-31 n=3 per gene. (D) BN-PAGE of HEK293 WT and TAZ Δ45 cells treated for 48 hrs with 2.5uM BEL (120 ug total protein) and 7 days with 100nM SS-31 solubilized in 1% Triton X-100 immunoblotted for the indicated proteins. All samples were resolved on a single gel and exposed for the same duration. The WT lane indicated with the asterisk was not used for quantification due to air bubbles. (E) Band intensities were quantified, and CI abundance was represented as the ratio of CI band intensity to CIV or CII. Abundance was plotted relative to respective control; CI:CIV(WT n=13, TAZ Δ45 n=16, WT-BEL n=9, TAZ Δ45 -BEL n=11, WT-SS-31 n=4, TAZ Δ45 -SS-31 n=6), CI:CII (WT n=10, TAZ Δ45 n=13, WT-BEL n=9, TAZ Δ45 -BEL n=10, WT-SS-31 n=4, TAZ Δ45 -SS-31 n=6). (F) Whole cell lysate (40-45 ug) of the indicated lines and treatment conditions were immunoblotted for the indicated proteins. (G) Band intensities, relative to the loading control GRP75, for both full-length and cleaved PGAM5 were individually quantified and plotted as the percent of cleaved PGAM5 (cleaved/full+cleaved); WT n=41, TAZ Δ45 n=41, WT-BEL n=16, TAZ Δ45 -BEL n=16, WT-SS-31 n=9, TAZ Δ45 -SS-31 n=8. (H) Whole cell lysate (40-45 ug) of the indicated lines and treatment conditions were immunoblotted for the indicated proteins. (I) Band intensities, relative to the loading control GRP75, were quantified and plotted relative to WT abundance; WT n=54, TAZ Δ45 n=48, WT-BEL n=11, TAZ Δ45 -BEL n=11, WT-SS-31 n=10, TAZ Δ45 -SS-31 n=12. Significant differences are indicated; * ≤ 0.05, ** ≤ 0.005, *** ≤ 0.0005, **** ≤ 0.00005.

Article Snippet: When cells reached ∼80% confluency cells were transiently transfected with NDUFAF1 (NM_016013) C-Myc/DDK-tagged plasmid (Origene #RC200029) with Lipofectamine 3000 (Thermo #L3000001) according to manufacturer’s instructions.

Techniques: Control, Expressing, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: The depletion of USP39 suppresses proliferation and induces cell death in OPM2 myeloma cell line

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 is overexpressed in MM patients compared to healthy donors and its high expression is correlated with shorter survival. A Kaplan–Meier of overall survival in patients with MM with high (red line) or low (black line) USP39 mRNA expression (P = 0.038) (GEO dataset GSE9782). B USP39 mRNA expression in normal donor, MGUS, Smoldering and MM patients (GEO dataset GSE6477). C Left, Representative USP39 staining of bone marrow samples from healthy individuals. Right, Representative USP39 staining of bone marrow samples from MM patients. “NR” denotes a non-relevant antibody. D Representative USP39 immunostaining of bone marrow samples from MM patients. Staining intensity was interpreted by a pathologist using visual scoring: "– “ undetectable, “ + ” denotes low intensities, “ + + ” denotes medium intensities, and “ + + + ” denotes high intensities. E Table representing USP39 staining of 12 bone marrows from MM patients and 6 bone marrows from healthy individuals. The percentage of USP39 positive cells was determined by ImageJ quantification and confirmed by pathologist visual scoring. Staining intensity were interpreted by pathologist visual scoring: "– “ denotes undetectable, “ + ” denotes low intensities, “ + + ” denotes medium intensities, and “ + + + ” denotes high intensities

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Expressing, Staining, Immunostaining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 Depletion Suppresses Cell Proliferation, Induces Apoptosis, and decreases Clonogenicity in OPM2 and KMM1 Multiple Myeloma Cells. A OPM2 cells were transfected with either control or two different single USP39 siRNA (siUSP39 #1 and siUSP39 #2) for 96 h. Then, lysates from these cells were subjected to immunoblots using GAPDH and USP39 antibodies (upper part). In parallel, the percentage of cell death was measured by flow cytometry after IP staining (left lower part) and cell metabolism was assessed by XTT assay (right lower part). B KMM1 were treated as described for OPM2 cells and subjected to the same analysis. C OPM2 cells were transfected with either control or single USP39 siRNAs for 96 h or stimulated with BTZ for 48 h. Lysates from these cells were subjected to immunoblots using GAPDH and USP39 antibodies (upper left). In parallel, the clonogenic capacity of the cells was measured after 10 days within a semi-solid medium. The quantification of the clonogenic assay is reported in the upper right part of the figure. Representative pictures were shown in the lower part. D KMM1 were treated as described for OPM2 cells and subjected to the same analysis. E KMM1 cells were either transfected with Control or USP39 siRNAs for 24 h. Then cells were transfected with Myc-Tag or Myc-USP39 vectors. After 72 h, lysates from these cells were subjected to immunoblots using GAPDH, myc-Tag or USP39 antibodies (left part). After 96 h of transfections, cell metabolism was measured in each condition (right part)

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Transfection, Control, Western Blot, Flow Cytometry, Staining, XTT Assay, Clonogenic Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: Inhibition of USP39 Triggers G2/M Cell Cycle Arrest and Apoptosis in Multiple Myeloma Cells. A OPM2 cells were transfected with control or USP39 siRNAs for 72 h, 96 h or 120 h. In parallel, cells were stimulated with nocodazole (1 µg/ml) for 24 h to block the cells in G2/M phase. Cell cycle distribution was examined by flow cytometry, and percentage of cells in each phase is indicated (top left and right). A representative flow cytometry profile of cells transfected with control (blue area) or USP39 siRNAs (red area) for 96 h (bottom left). B In parallel, OPM2 cells were transfected with either control or single USP39 siRNAs for 72 h. Then, lysates from these cells were subjected to immunoblots using GAPDH, USP39, CDK4 and CyclinB1 antibodies. C OPM2 cells were transfected with either control or single USP39 siRNAs for 96 h or stimulated with BTZ for 48 h. Then, cells were stained by Annexin and PI and analyzed by flow cytometry. % of apoptotic cells (annexin V + /DAPI-) and dead cells (annexin V + /DAPI +) are represented in grey and black respectively. D Lysates from these cells were subjected to immunoblots using USP39, PARP, cleaved caspase 3 and GAPDH antibodies as a loading control. E , F OPM2 cells were transfected with either control or single USP39 siRNAs for 72 h and 96 h, or stimulated with BTZ for 48 h. Then, cells lysates were subjected to caspase 3 (E) and caspase 9 (F) assays

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Inhibition, Transfection, Control, Blocking Assay, Flow Cytometry, Western Blot, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 Inhibition Overcomes Bortezomib Resistance in MM Cells. A U266 cells and its BTZ-resistant counterpart U266R were stimulated with increased concentrations of BTZ (1, 3, 10, 30 and 100 ng/ml) for 24 h, and cell metabolism was measured by XTT assay. B U266 and U266R cells were transfected with either control or three different single USP39 siRNAs (#1, #2 and #3) for 72 h. USP39 silencing was confirmed by immunoblots using USP39 and GAPDH antibodies (top). In parallel, cell metabolism was measured by XTT assay (bottom). C U266 and U266R cells were transfected with either control or two different single USP39 siRNAs (#1 and #2) for 72 h. Then both cells were stimulated with increased concentrations of BTZ (1, 3, 10, 30 and 100 ng/ml) for 24 h and cell metabolism was measured by XTT assay

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Inhibition, XTT Assay, Transfection, Control, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 Stabilizes and Deubiquitinates ZEB1 Protein in Multiple Myeloma Cells . A OPM2 cells were transfected with either control or USP39 siRNAs for 48 h, 72 h or 96 h. Lysates were subjected to immunoblots using USP39, ZEB1, SP1, CHK2, STAT1 and GAPDH antibodies. B USP39 was transiently silenced or overexpressed in OPM2 and KMM1 cells respectively. Then, immunoblots were performed using USP39, ZEB1 and GAPDH antibodies (left) and protein quantifications were determined (right). C KMM1 cells were transfected with plasmids encoding either the Myc-tag or the USP39-Myc-tag proteins. After 48 h, cells were stimulated with cycloheximide (CHX) at 10 µM for 24 h. Lysates were subjected to immunoblots using USP39, ZEB1, and GAPDH antibodies and protein quantification was determined. D KMM1 cells were transfected with either control or USP39 siRNAs. After 72 h, cells were stimulated with cycloheximide (CHX) at 10 µM for 24 h. Lysates were subjected to immunoblots using USP39, ZEB1, and GAPDH antibodies and protein quantification was determined. E OPM2 cells were transfected with either control or USP39 siRNAs for 72 h. Then cells were stimulated for 2 h, 4 h or 8 h with the proteasome inhibitor MG132 at 1 µM. Lysates were subjected to immunoblots using USP39, ZEB1 and GAPDH antibodies. Protein quantification was determined. F Lysates from OPM2 cells were subjected to co-immunoprecipitation experiments using either non relevant (NR), USP39 or ZEB1 antibodies. Immunoblots was performed to visualize complexes using USP39 and ZEB1 antibodies. G KMM1 cells were transfected for 48 h with HA-ub plasmid in the presence or in the absence of Myc-USP39 plasmid. Then cells were treated with MG132 at 1 µM for 8 h and lysates were subjected to immunoprecipitation using non relevant IgG or ZEB1 antibodies. Inputs were immunoblotted with HA and Myc antibodies to visualize poly-HA-Ub and USP39 respectively. IPs products were immunoblotted with HA and ZEB1 antibodies to visualize Ub-ZEB1 complex and immunoprecipitated ZEB1. The graph represents Ub-ZEB1 quantification. H The complementary deubiquitination experiment was performed in presence or absence of USP39 siRNA (72 h)

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Transfection, Control, Western Blot, Immunoprecipitation, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 Promotes In Vitro Transmigration of MM Cells. A OPM2 cells were transfected with control, USP39 or ZEB1 siRNAs for 48 h. Then, immunoblots were performed using USP39, ZEB1, β-Catenin, N-Cadherin, Vimentin and GAPDH antibodies (left) and protein quantifications were determined (right). In parallel, the metabolic activity (lower left) and the migration of the cells (lower right) were measured under the same conditions. B KMM1 cells were either transfected with Control or USP39 siRNAs for 24 h. Then cells were transfected with pcDNA3-Flag or pcDNA3-Flag-USP39 vectors. After 72 h, lysates from these cells were subjected to immunoblots using USP39, Flag-Tag or GAPDH, antibodies (left part). After 96 h of transfections, the migration capacity of cells was measured by Boyden chamber assays (right part). C and D ) U266 ( C ) and OPM2 ( D ) cells were stably transduced with lentiviral particles encoding GFP or GFP-USP39 and were subjected to immunoblots using USP39, ZEB1 or GAPDH antibodies. E , F U266 ( E ) and OPM2 ( F ) cells were stably transfected with lentiviral particles encoding Myc or Myc-USP39 and were subjected to immunoblots using USP39, ZEB1 or GAPDH antibodies. In parallel, the migration capacity of corresponding cells was measured by Boyden chamber assays

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: In Vitro, Transmigration Assay, Transfection, Control, Western Blot, Activity Assay, Migration, FLAG-tag, Stable Transfection, Transduction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Disrupting USP39 deubiquitinase function impairs the survival and migration of multiple myeloma cells through ZEB1 degradation

doi: 10.1186/s13046-024-03241-2

Figure Lengend Snippet: USP39 Enhances Metastasis in Zebrafish: Implications for MM Progression. A-D Zebrafish embryos ( N = 36) were injected with U266 cells stably infected with lentiviral particles encoding either Myc-tag or Myc-USP39 proteins (labeled with red DiD) into the perivitelline space. Zebrafish embryos were monitored on Day 0 and Day 2 for tumor metastases using a fluorescent microscope. A (Left) Representative images of local and distant metastases are shown. (Right) Magnification of images representing distant metastases. Arrows indicate metastases. Quantification of the area of local metastases at Day 0 ( B ) and Day 2 ( C ) of Myc-tag and Myc-USP39 embryos. D Quantification of the number of distant metastases at Day 2 of Myc-tag and Myc-USP39 embryos

Article Snippet: USP39 Myc-DDK plasmid (NM_006590) Human Tagged ORF Clone (RC209551) was purchased from OriGene.

Techniques: Injection, Stable Transfection, Infection, Labeling, Microscopy