Journal: Frontiers in Endocrinology

Article Title: Activation of estrogen-related receptor γ by calcium and cadmium

doi: 10.3389/fendo.2024.1400022

Figure Lengend Snippet: Primer sequence and taqman probe catalog number.

Article Snippet: H6PD , Hs00188728_m1 , .

Techniques: Sequencing, Mutagenesis

Journal: Frontiers in Endocrinology

Article Title: Activation of estrogen-related receptor γ by calcium and cadmium

doi: 10.3389/fendo.2024.1400022

Figure Lengend Snippet: Effects of calcium and cadmium on ERRγ-regulated metabolic genes in HEK293T cells transfected with ERRγ1 or ERRγ3. Cells were treated as described in Figure 3 and the expression of H6PD, HK2, and SLC2A2 was measured using a quantitative real-time qPCR assay as described in Figure 1 . Cells were treated with calcium (1, 3, 5, and 10 mM) and intracellular calcium was measured as described in Figure 1 . (A–F) Effects of calcium and cadmium on the induction of H6PD, HK2, and SLC2A2 mRNA in HEK293T cells transfected with ERRγ1. (G, H, J, K) Effects of calcium and cadmium on the induction of H6PD and HK2 mRNA in HEK293T cells transfected with ERRγ3. (I) Effects of extracellular calcium on the concentration of intracellular calcium. (L) Illustration of ERRγ1 and ERRγ3 structure. Data are expressed as fold change (mean ± SEM); n ≥ 3. Statistical significance is defined as a p -value of ≤0.05. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: H6PD , Hs00188728_m1 , .

Techniques: Transfection, Expressing, Concentration Assay

Journal: Nature

Article Title: A missing enzyme-rescue metabolite as cause of a rare skeletal dysplasia

doi: 10.1038/s41586-025-09397-x

Figure Lengend Snippet: a-f , UDP-xylose ( a , c ), UDP-glucuronate (UDP-GlcA, b , d ), UDP-4-ketoxylose ( e ) and UDP-arabinose ( f ) levels were determined by LC-MS in parental 293 T ( a , b ) and HAP1 ( c -f) cells and in a TGDS KO or a UXS 1 KO clone upon transduction with a recombinant lentivirus driving expression of ArnA (‘+’) or an empty vector control (‘-’). To allow comparison of the signal intensities obtained for different metabolites, data were only normalized to total ion current but not to the mean of the values observed in the parental cells within each experiment. g-h , Extracted ion chromatograms of the m/z of UDP-pentose (m/z 535.037) corresponding to panel a ( g ) and panels c , f ( h ). i-j , Extracted ion chromatograms of the m/z of UDP-GlcA ( i , 579.027) or UDP-xylose ( j , 535.037) in reactions containing 500 µM UDP-GlcA in the presence of 0.8 µM TGDS or 0.65 µM UXS1 for 24 h. k-l , Western blot for H6PD in wild-type and TGDS -deficient HAP1 ( k ) or 293 T ( l ) cell lines, transduced with lentiviral CRISPR/Cas9 constructs targeting H6PD (each ‘+‘ representing a different guide RNA) or a control gene (‘-‘), performed twice. See Supplementary Fig. for uncropped images. m-n , UDP-xylose ( m ) and UDP-GlcA ( n ) were quantified in 293 T cell lines described in panel l. o-p , Western blot for H6PD in wild-type and a TGDS -deficient U2OS ( o ) or HCT116 ( p ) cell lines, transduced with recombinant lentiviruses driving expression of human H6PD cDNA (‘+‘) or an empty control (‘-‘), performed twice. q-r , UDP-xylose ( q ) and UDP-GlcA ( r ) were quantified in HCT116 cell lines described in panel p . Data are means ± SD of 3 independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 in paired Sidak post-hoc testing of log-transformed data after one-way ANOVA. For exact p-values see source data file.

Article Snippet: Immunofluorescence staining was performed overnight in PBS containing 3% BSA at 4 °C using the following antibodies: TGDS (rabbit anti-TGDS; HPA040857, Atlas, 1:500); PDIA1 (mouse anti-PDIA1 (P4HB); ab2792, Abcam, 1:200); GM130 (mouse anti-GM130; 610823, BD Transduction Laboratories, 1:500); FLAG (mouse anti-FLAG M2; F1804, Sigma, 1:500); CALR (rabbit anti-Calreticulin; ab92516, Abcam, 1:500); V5 (rabbit anti-V5; V8137, Sigma, 1:400); H6PD (mouse anti-H6PD; TA501257, OriGene, 1:100) and GIANTIN (rabbit anti-Giantin; 621352, BioLegend, 1:1,000).

Techniques: Liquid Chromatography with Mass Spectroscopy, Transduction, Recombinant, Expressing, Plasmid Preparation, Control, Comparison, Western Blot, CRISPR, Construct, Transformation Assay

Journal: Nature

Article Title: A missing enzyme-rescue metabolite as cause of a rare skeletal dysplasia

doi: 10.1038/s41586-025-09397-x

Figure Lengend Snippet: a , Working hypothesis: an abortive catalytic cycle of UXS1 leads to the inactivation of the enzyme, which is counteracted by UDP-4-keto sugars. The decarboxylation of UDP-glucuronate by UXS1 depends on the oxidation of the C4 hydroxyl group using a tightly bound NAD + . This generates NADH and a UDP-4-ketoxylose intermediate. Normally, formation of UDP-xylose regenerates NAD + , preparing the enzyme for another cycle. Infrequently, the intermediate dissociates from the catalytic pocket, leaving UXS1 bound to NADH and inactive. We hypothesized that UDP-4-keto sugars restore activity by facilitating the oxidation of enzyme-bound NADH to NAD + . Loss of the cofactor can also yield an inactive apoenzyme, but reactivation by NAD + binding is limited by the low NAD + /NADH ratio in the endoplasmic reticulum maintained by H6PD. b , Schematic of the experiment to test whether UDP-4-ketoxylose produced by ArnA can reactivate UXS1. c – f , ArnA expression rescues UDP-xylose synthesis in TGDS- KO cells, but not in UXS1 -KO cells. UDP-xylose ( c , e ), UDP-glucuronate ( d , f ) were quantified by LC–MS in parental 293T ( c , d ) and HAP1 ( e , f ) cells, and in TGDS- KO or UXS1 -KO clones transduced with a lentivirus expressing ArnA or an empty vector. ND, not determined. g , Experiment exploring the role of H6PD in UXS1 dependency on TGDS. h – k , H6PD modulates UXS1 dependency on TGDS. In HAP1 cells, CRISPR–Cas9-mediated H6PD knockdown (using two different guide RNAs (gRNAs) versus control gRNA (−)) reduces the effect of TGDS deficiency on UXS1 function ( h , i ). In U2OS cells, H6PD overexpression (+) makes UXS1 activity dependent on TGDS ( j , k ). −, empty vector control. UDP-xylose ( h , j ) and UDP-glucuronate ( i , k ) were quantified by LC–MS. Data are mean ± s.d. of 3 independent experiments, each containing 3 biological replicates. Paired two-tailed Dunnett ( c , d ) or Sidak ( e , f , i – k ) post hoc testing of log-transformed data after one-way ANOVA. For exact P values see .

Article Snippet: Immunofluorescence staining was performed overnight in PBS containing 3% BSA at 4 °C using the following antibodies: TGDS (rabbit anti-TGDS; HPA040857, Atlas, 1:500); PDIA1 (mouse anti-PDIA1 (P4HB); ab2792, Abcam, 1:200); GM130 (mouse anti-GM130; 610823, BD Transduction Laboratories, 1:500); FLAG (mouse anti-FLAG M2; F1804, Sigma, 1:500); CALR (rabbit anti-Calreticulin; ab92516, Abcam, 1:500); V5 (rabbit anti-V5; V8137, Sigma, 1:400); H6PD (mouse anti-H6PD; TA501257, OriGene, 1:100) and GIANTIN (rabbit anti-Giantin; 621352, BioLegend, 1:1,000).

Techniques: Activity Assay, Binding Assay, Produced, Expressing, Liquid Chromatography with Mass Spectroscopy, Clone Assay, Transduction, Plasmid Preparation, CRISPR, Knockdown, Control, Over Expression, Two Tailed Test, Transformation Assay

Journal: Nature

Article Title: A missing enzyme-rescue metabolite as cause of a rare skeletal dysplasia

doi: 10.1038/s41586-025-09397-x

Figure Lengend Snippet: Immunofluorescence images of U2OS cells after transfection with pCMV-TGDS, pCMV-UXS1-FLAG and pCMV-H6PD-V5. TGDS localization was detected using anti-TGDS, UXS1 using anti-FLAG, and H6PD using either anti-H6PD or anti-V5 antibodies. Endogenous markers for the ER (PDIA1; CALR) and Golgi (GM130; GIANTIN) were used to determine the proteins localization. All panels except the costaining with GM130 (i.e. bottom three images in panel a ) were performed with methanol-fixed cells. In the latter case, paraformaldehyde fixation was needed, which explains the slightly different morphology of the TGDS expression pattern. Scale bars, 10 µm.

Article Snippet: Immunofluorescence staining was performed overnight in PBS containing 3% BSA at 4 °C using the following antibodies: TGDS (rabbit anti-TGDS; HPA040857, Atlas, 1:500); PDIA1 (mouse anti-PDIA1 (P4HB); ab2792, Abcam, 1:200); GM130 (mouse anti-GM130; 610823, BD Transduction Laboratories, 1:500); FLAG (mouse anti-FLAG M2; F1804, Sigma, 1:500); CALR (rabbit anti-Calreticulin; ab92516, Abcam, 1:500); V5 (rabbit anti-V5; V8137, Sigma, 1:400); H6PD (mouse anti-H6PD; TA501257, OriGene, 1:100) and GIANTIN (rabbit anti-Giantin; 621352, BioLegend, 1:1,000).

Techniques: Immunofluorescence, Transfection, Expressing

Journal: Nature

Article Title: A missing enzyme-rescue metabolite as cause of a rare skeletal dysplasia

doi: 10.1038/s41586-025-09397-x

Figure Lengend Snippet: a , Schematic of the suggested UDP-glucose-4,6-dehydratase activity of TGDS (bottom), and UDP-rhamnose or dTDP-rhamnose synthesis in bacteria (top). UDP-6dH, UDP-6-deoxyhexose. 4k6dG, 4-keto-6-deoxyglucose. b , TGDS activity was assessed at the indicated concentrations of UDP-glucose for 4 h at 30 °C. Formation of UDP-4-keto-6-deoxyglucose is presented in arbitrary units. c – f , Expression of human TGDS or B. cinerea UG46DH rescue the phenotype of TGDS- KO cells. UDP-4-keto-6-deoxyglucose ( c ), UDP-6-deoxyhexose ( d ), UDP-xylose ( e ) and UDP-glucuronate ( f ) were quantified in parental and TGDS- KO 293T cells upon transduction with recombinant lentiviruses driving expression of human TGDS or B. cinerea UG46DH. Metabolite levels in TGDS- KO cells in c , d were close to background. Levels of UDP-4-keto-6-deoxyglucose and UDP-6-deoxyhexose in TGDS -KO cells were unaffected by the inactivation of H6PD (Extended Data Fig. ). g , Experimental setup to assess the reactivation of UXS1 via the product of TGDS. h , The TGDS product rescues functionally inactivated UXS1. UDP-xylose, UDP-4-keto-6-deoxyglucose, UDP-6-deoxyhexose, NAD + and NADH were quantified by LC–MS in reactions where UDP-glucuronate was incubated with 0.56 µM UXS1, sodium borohydride-inactivated UXS1 or inactivated UXS1 in the presence of a 1.5-fold excess of the product of TGDS (TGDS-P). Data are mean ± s.d. from three independent experiments, each containing three biological replicates in c – f , h . Paired two-tailed Sidak ( c – f ) or Dunnett ( h ) post hoc testing of log-transformed data after one-way ANOVA. For exact P values see .

Article Snippet: Immunofluorescence staining was performed overnight in PBS containing 3% BSA at 4 °C using the following antibodies: TGDS (rabbit anti-TGDS; HPA040857, Atlas, 1:500); PDIA1 (mouse anti-PDIA1 (P4HB); ab2792, Abcam, 1:200); GM130 (mouse anti-GM130; 610823, BD Transduction Laboratories, 1:500); FLAG (mouse anti-FLAG M2; F1804, Sigma, 1:500); CALR (rabbit anti-Calreticulin; ab92516, Abcam, 1:500); V5 (rabbit anti-V5; V8137, Sigma, 1:400); H6PD (mouse anti-H6PD; TA501257, OriGene, 1:100) and GIANTIN (rabbit anti-Giantin; 621352, BioLegend, 1:1,000).

Techniques: Activity Assay, Bacteria, Expressing, Transduction, Recombinant, Liquid Chromatography with Mass Spectroscopy, Incubation, Two Tailed Test, Transformation Assay

Journal: Nature

Article Title: A missing enzyme-rescue metabolite as cause of a rare skeletal dysplasia

doi: 10.1038/s41586-025-09397-x

Figure Lengend Snippet: a , Recombinant purified human TGDS, B. cinerea UG46DH, E. coli ArnA and human UXS1 analysed by SDS-PAGE and Coomassie staining to demonstrate purity. b , Extracted ion chromatogram (EIC) of the m/z corresponding to UDP-4-keto-6-deoxyglucose (i.e., 547.037) in reactions of recombinant fungal UG46DH or human TGDS with UDP-glucose. c , UDP-4-keto-6-deoxyglucose, the reaction product of UG46DH and TGDS, is partially hydrated in a non-enzymatic reaction to form UDP-4,4-dihydroxy-6-deoxyglucose, which has the same mass as UDP-glucose . d-e , Extracted ion chromatograms of the m/z of UDP-glucose ( d , 565.048) and UDP-4-keto-6-deoxyglucose ( e , UDP-4k6dG, 547.037) in reactions containing 500 µM UDP-glucose in the presence of 0.8 µM TGDS or 0.65 µM UXS1 for 24 h. The trailing peak (arrow, d ) in the ‘UDP-glucose’ chromatogram corresponds to the hydrated form of UDP-4k6dG previously reported in reactions for fungal orthologues . f-g , UDP 6-deoxyhexose (UDP-6dH, f) and UDP-4-keto-6-deoxyglucose (UDP-4k6dG, g ) levels were determined by LC-MS in parental cells and 2 TGDS KO clones from U2OS, HCT116, HAP1 and 293 T. h , Tentative identification of UDP-6-deoxyhexose as UDP-6-deoxyglucose. Extracted ion chromatogram (EIC) of the m/z corresponding to UDP-6-deoxyhexose (i.e., 549.053) in a reaction where the TGDS product (TGDS-P) was chemically reduced with NaBH 4 , leading to the formation of UDP-6-deoxyglucose and UDP-6-deoxygalactose (upper panel). Based on the catalytic cycle of UXS1, only UDP-6-deoxyglucose formation is expected when the TGDS product is incubated with UXS1 that has been inactivated using NaBH 4 beforehand (second panel). The UDP-6-deoxyhexose peak in cells coelutes with the one corresponding to UDP-6-deoxyglucose (third panel). Coelution is corroborated (lower panel) by the fact that the peak increases upon addition of a small amount of the sample presented in the upper panel. i-l , Inactivation of H6PD in TGDS -deficient cells does not lead to an increase in the rescue metabolite UDP-4k6dG ( i , j ) nor in UDP-6dH ( k , l ). LC-MS analysis for the indicated metabolites was performed in wild-type and a TGDS -deficient HAP1 ( i , k ) or 293 T ( j , l ) cell line, transduced with lentiviral CRISPR/Cas9 constructs targeting H6PD (each ‘+‘ representing a different guide RNA) or a control gene (‘-‘). Data are means ± SD of the means obtained in three ( f , g ) and two ( i - l ) independent experiments performed with three biological replicates. * p < 0.05; ** p < 0.01; *** p < 0.001 in paired two-tailed Dunnett (f-g) post-hoc testing of log-transformed data after one-way ANOVA. For exact p-values see source data file.

Article Snippet: Immunofluorescence staining was performed overnight in PBS containing 3% BSA at 4 °C using the following antibodies: TGDS (rabbit anti-TGDS; HPA040857, Atlas, 1:500); PDIA1 (mouse anti-PDIA1 (P4HB); ab2792, Abcam, 1:200); GM130 (mouse anti-GM130; 610823, BD Transduction Laboratories, 1:500); FLAG (mouse anti-FLAG M2; F1804, Sigma, 1:500); CALR (rabbit anti-Calreticulin; ab92516, Abcam, 1:500); V5 (rabbit anti-V5; V8137, Sigma, 1:400); H6PD (mouse anti-H6PD; TA501257, OriGene, 1:100) and GIANTIN (rabbit anti-Giantin; 621352, BioLegend, 1:1,000).

Techniques: Recombinant, Purification, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Clone Assay, Incubation, Transduction, CRISPR, Construct, Control, Two Tailed Test, Transformation Assay

Journal: Heliyon

Article Title: Mandatory role of endoplasmic reticulum in preserving NADPH regeneration in starved MDA-MB-231 breast cancer cells

doi: 10.1016/j.heliyon.2024.e38718

Figure Lengend Snippet: Role of PPP in cell under glucose shortage. (A) H6PD and G6PD mRNA expression in MDA-MB-231grown under HG (blue) or LG (orange) condition (n = 3). (B and C) H6PD (B) and G6PD (C) mRNA expression under control (Ctrl), control siRNA (siCtrl), siRNA H6PD (siH6PD) or siRNA G6PD (siG6PD) in HG (blue) or LG (orange) condition, expressed as percentual of relative control (n = 3). (D) Cell numbers at 24 and 48 h of HG (left) and LG (right) condition under control (Ctrl, blue), control siRNA (siCtrl, purple), siRNA H6PD (siH6PD, brown) or siRNA G6PD (siG6PD, orange) (n = 3). (E) Percent of cell in cell cycle phase in HG (blue) or LG (orange) medium: G1 phase (empty column) and S phase (dashed column) (n = 3). (F–J) Percent of PI positive cells (n = 4) (F), glucose consumption (n = 3) (G), lactate release (n = 3) (H), intracellular pyruvate (n = 3) (I) and intracellular lactate (n = 3) (J) in MDA-MB-231 cells silenced for H6PD and G6PD in all experimental conditions under HG (blue) or LG (orange) medium, expressed as percentual of relative control. Data are represented as mean ± SD. ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001; ∗∗∗∗ = p < 0.0001 versus corresponding control when alone, or between H6PD and G6PD siRNA when combined with the comparison line. # = p < 0.05; ### = p < 0.001; #### = p < 0.0001 versus corresponding condition in HG cultures. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: G6PD-siRNA (Silencer Select ID s5447, Thermo Fisher Scientific), H6PD-siRNA (Silencer ID s97551, Thermo Fisher Scientific) or Silencer Select Negative Control #2-siRNA (Thermo Fisher Scientific) were used.

Techniques: Expressing, Control, Comparison

Journal: Heliyon

Article Title: Mandatory role of endoplasmic reticulum in preserving NADPH regeneration in starved MDA-MB-231 breast cancer cells

doi: 10.1016/j.heliyon.2024.e38718

Figure Lengend Snippet: Effect of H6PD and G6PD siRNA on energy asset, redox stress and NADPH regeneration. (A–D) NADPH/NADP ratio (n = 3) (A), ATP intracellular concentration (n = 3) (B), total antioxidant capacity (n = 3) (C) and malondialdehyde levels (n = 4) (D) under control (Ctrl), control siRNA (siCtrl), siRNA H6PD (siH6PD) or siRNA G6PD (siG6PD) in HG (blue) or LG (orange) medium, data are expressed as percentual of relative control. (E and F) mRNA ME1 expression (n = 3) (E) and Pyruvate/Malate ratio (n = 3) (F) under the same experimental conditions as A. Data are represented as mean ± SD. ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001; ∗∗∗∗ = p < 0.0001 versus corresponding control. ## = p < 0.001; ### = p < 0.001; #### = p < 0.0001 versus corresponding condition in HG cultures. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: G6PD-siRNA (Silencer Select ID s5447, Thermo Fisher Scientific), H6PD-siRNA (Silencer ID s97551, Thermo Fisher Scientific) or Silencer Select Negative Control #2-siRNA (Thermo Fisher Scientific) were used.

Techniques: Concentration Assay, Control, Expressing