Journal: The Journal of Biological Chemistry

Article Title: Lipopolysaccharide induces HIF-1α accumulation via MAPK p38–mediated mRNA stabilization and dexamethasone-sensitive protein stabilization

doi: 10.1016/j.jbc.2025.111094

Figure Lengend Snippet: MAPK p38 regulates HIF-α accumulation in LPS-activated mouse BMDMs . Here and throughout, treatment conditions are indicated by different colors, and genotypes (where appropriate) are indicated by different shadings. A , mouse BMDMs were treated for 8 h with 10 ng/ml LPS alone or in combination with 100 nM dexamethasone (Dex), 1 μM SB202190 (SB), or 1 μM VX-745 (VX). HIF-1α and α-tubulin were detected by Western blotting. B , quantification of HIF-1α expression (mean ± SD, normalized to α-tubulin and expressed relative to untreated control; five independent experiments as in A ; one-way ANOVA with Dunnett’s correction for multiple comparison). C , Dusp1 +/+ and Dusp1 −/− BMDMs were treated with 10 ng/ml LPS for the indicated times, and DUSP1, phospho-p38, HIF-1α, and α-tubulin were detected by Western blotting. Representative blots from one of three independent experiments. D , quantification of HIF-1α expression in Dusp1 +/+ and Dusp1 −/− BMDMs 8 h after addition of LPS (mean ± SD, normalized to α-tubulin; six independent experiments; unpaired t test). E , Dusp1 +/+ and Dusp1 −/− BMDMs were treated with LPS for 8 h in the absence or the presence of 1 μM SB. Hif1a mRNA was measured by quantitative PCR (normalized to unstimulated control; mean ± SD of four independent experiments; two-way ANOVA). F , Dusp1 +/+ and Dusp1 −/− BMDMs were treated with LPS for 8 h, and Hif1a primary transcript was measured by quantitative PCR (normalized to unstimulated control; mean ± SD of five independent experiments; unpaired two-tailed t test). No PCR product was detected if the reverse transcriptase step was omitted, indicating the absence of contaminating genomic DNA. G , Dusp1 +/+ and Dusp1 −/− BMDMs were treated with LPS for 8 h, and then cycloheximide (CHX; 5 μg/ml) was added at t = 0, and protein was harvested after the indicated times. HIF-1α and α-tubulin were detected by Western blotting. Loading was adjusted for approximately equal HIF-1α band intensity. Representative blot of one of three independent experiments. ns, p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.005; and ∗∗∗∗ p < 0.001. BMDM, bone marrow–derived macrophage; DUSP1, dual specificity phosphatase 1; HIF-α, hypoxia-inducible transcription factor 1α; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; ns, not significant.

Article Snippet: The following primary antibodies were used: mouse HIF-1α (Cell Signaling catalog no.: 14179); human HIF-1α (BD Biosciences; catalog no.: 610959); phospho-p38(Thr180/Tyr182) (Cell Signaling; catalog no.: 9211); DUSP1 (Cell Signaling; catalog no.: 35217); TTP (Cell Signaling; catalog no.: 71632); GLUT1 (Abcam; catalog no.: ERP3915); MCT4 (Novus Biologicals; catalog no.: NBP1-81251); and α-tubulin (Sigma–Aldrich; catalog no.: T9026).

Techniques: Western Blot, Expressing, Control, Comparison, Real-time Polymerase Chain Reaction, Two Tailed Test, Reverse Transcription, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipopolysaccharide induces HIF-1α accumulation via MAPK p38–mediated mRNA stabilization and dexamethasone-sensitive protein stabilization

doi: 10.1016/j.jbc.2025.111094

Figure Lengend Snippet: DUSP1 controls HIF-1α expression by modulating the phosphorylation of TTP . A , evolutionary conservation of the HIF1A 3′ UTR between 100 vertebrate species (from UCSC Genome Browser). Conservation of consensus TTP binding sites in mouse Hif1a and human HIF1A 3′UTRs. B , Dusp1 +/+ and Dusp1 −/− BMDMs were treated with 10 ng/ml LPS for the indicated times. TTP and α-tubulin were detected by Western blotting. Representative of two independent experiments. C , Zfp36 +/+ and Zfp36 aa/aa BMDMs were treated with 10 ng/ml LPS for the indicated times, and Hif1a mRNA abundance was measured by quantitative PCR (mean ± SD fold change relative to untreated control; three independent experiments; two-way ANOVA with Dunnett’s correction; asterisk indicates significant effect of genotype). D , Zfp36 +/+ and Zfp36 aa/aa BMDMs were left untreated or stimulated with 10 ng/ml LPS for 8 h. HIF-1α and α-tubulin were detected by Western blotting. Representative of three independent experiments. Vertical line represents splicing of different sections of a single gel image. E , wildtype ( Dusp1 +/+ : Zfp36 +/+ ), DUSP1 knockout ( Dusp1 −/− : Zfp36 +/+ ), and double-modified ( Dusp1 −/− : Zfp36 aa/aa ) BMDMs were stimulated with 10 ng/ml LPS for 4 h. Then Hif1a mRNA abundance was measured by quantitative PCR (mean ± SD fold change relative to untreated control; three independent experiments; one-way ANOVA with Dunnett’s correction). ∗ p < 0.05; ∗∗ p < 0.01. BMDM, bone marrow–derived macrophage; DUSP1, dual specificity phosphatase 1; HIF-1α, hypoxia-inducible transcription factor 1α; LPS, lipopolysaccharide; TTP, tristetraprolin; UCSC, University of California, Santa Cruz.

Article Snippet: The following primary antibodies were used: mouse HIF-1α (Cell Signaling catalog no.: 14179); human HIF-1α (BD Biosciences; catalog no.: 610959); phospho-p38(Thr180/Tyr182) (Cell Signaling; catalog no.: 9211); DUSP1 (Cell Signaling; catalog no.: 35217); TTP (Cell Signaling; catalog no.: 71632); GLUT1 (Abcam; catalog no.: ERP3915); MCT4 (Novus Biologicals; catalog no.: NBP1-81251); and α-tubulin (Sigma–Aldrich; catalog no.: T9026).

Techniques: Expressing, Phospho-proteomics, Binding Assay, Western Blot, Real-time Polymerase Chain Reaction, Control, Knock-Out, Modification, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipopolysaccharide induces HIF-1α accumulation via MAPK p38–mediated mRNA stabilization and dexamethasone-sensitive protein stabilization

doi: 10.1016/j.jbc.2025.111094

Figure Lengend Snippet: Dexamethasone impairs HIF-1α expression independently of MAPK p38 and TTP phosphorylation . A , Zfp36 +/+ and Zfp36 aa/aa BMDMs were treated for 8 h with combinations of 10 ng/ml LPS, 100 nM dexamethasone (Dex), and 1 μM SB202190 (SB) as shown. HIF-1α and α-tubulin were detected by Western blotting. The graph shows the mean ± SD of HIF-1α/α-tubulin ratio from three independent experiments (two-way ANOVA with Tukey’s correction). B , Dusp1 −/− BMDMs were treated for 8 h with combinations of 10 ng/ml LPS, 100 nM Dex, and 1 μM SB as shown. HIF-1α and α-tubulin were detected by Western blotting. C , mean ± SD HIF-1α protein expression (normalized against α-tubulin) in four independent experiments using Dusp1 −/− BMDMs treated as in ( B ; one-way ANOVA with Dunnett's corection ). ns p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.005. BMDM, bone marrow–derived macrophage; HIF-1α, hypoxia-inducible transcription factor 1α; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; TTP, tristetraprolin.

Article Snippet: The following primary antibodies were used: mouse HIF-1α (Cell Signaling catalog no.: 14179); human HIF-1α (BD Biosciences; catalog no.: 610959); phospho-p38(Thr180/Tyr182) (Cell Signaling; catalog no.: 9211); DUSP1 (Cell Signaling; catalog no.: 35217); TTP (Cell Signaling; catalog no.: 71632); GLUT1 (Abcam; catalog no.: ERP3915); MCT4 (Novus Biologicals; catalog no.: NBP1-81251); and α-tubulin (Sigma–Aldrich; catalog no.: T9026).

Techniques: Expressing, Phospho-proteomics, Western Blot, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipopolysaccharide induces HIF-1α accumulation via MAPK p38–mediated mRNA stabilization and dexamethasone-sensitive protein stabilization

doi: 10.1016/j.jbc.2025.111094

Figure Lengend Snippet: Dexamethasone (Dex)-mediated inhibition of LPS-induced glycolysis is not dependent on DUSP1 . A , Dusp1 +/+ and Dusp1 −/− BMDMs were treated for 8 h with 10 ng/ml LPS alone or in combination with 100 nM Dex. HIF-1α and α-tubulin were detected by Western blotting. Representative of six independent experiments. B , Dusp1 +/+ and Dusp1 −/− BMDMs were treated for 24 h with 10 ng/ml LPS alone or in combination with 100 nM Dex. Change in extracellular concentration of lactate ( upper ) or glucose ( lower ) (mean ± SD; three independent experiments; two-way ANOVA with Sidak’s correction). C , Dusp1 +/+ and Dusp1 −/− BMDMs were treated as in ( B ), and whole-cell lysates were blotted for MCT4 ( upper ) or GLUT1 ( lower ), with actin as a loading control. Numbers above the blots are mean protein quantities from three independent experiments, normalized against actin and untreated Dusp1 +/+ control. Vertical line represents splicing of different sections of a single gel image. D , expression of Slc16a3 and Slc2a1 mRNA was measured by RT–quantitative PCR after 24 h of treatment as indicated (mean fold change relative to unstimulated control ± SD; five to six independent experiments; two-way ANOVA with Tukey’s correction). ns, p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.005; and ∗∗∗∗ p < 0.001. BMDM, bone marrow–derived macrophage; DUSP1, dual specificity phosphatase 1; GLUT1, glucose transporter 1; HIF-1α, hypoxia-inducible transcription factor 1α; LPS, lipopolysaccharide; MCT4, monocarboxylate transporter 4.

Article Snippet: The following primary antibodies were used: mouse HIF-1α (Cell Signaling catalog no.: 14179); human HIF-1α (BD Biosciences; catalog no.: 610959); phospho-p38(Thr180/Tyr182) (Cell Signaling; catalog no.: 9211); DUSP1 (Cell Signaling; catalog no.: 35217); TTP (Cell Signaling; catalog no.: 71632); GLUT1 (Abcam; catalog no.: ERP3915); MCT4 (Novus Biologicals; catalog no.: NBP1-81251); and α-tubulin (Sigma–Aldrich; catalog no.: T9026).

Techniques: Inhibition, Western Blot, Concentration Assay, Control, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipopolysaccharide induces HIF-1α accumulation via MAPK p38–mediated mRNA stabilization and dexamethasone-sensitive protein stabilization

doi: 10.1016/j.jbc.2025.111094

Figure Lengend Snippet: In human MDMs, MAPK p38 regulates the LPS-induced glycolytic response at the level of HIF1A mRNA expression . A , MDMs were treated for 24 h with combinations of 10 ng/ml LPS, 100 nM dexamethasone (Dex), and 1 μM SB202190 (SB) as shown, then glycolysis was measured by Seahorse XFe96 Mito + Glyco stress test (see the Experimental procedures section). B , mean ± SD fold change in glycolysis relative to unstimulated control from four independent experiments as in A ; two-way ANOVA with Dunnett’s correction. C and D , MDMs were treated for 8 h with combinations of 10 ng/ml LPS, 100 nM Dex, 1 μM SB, and 1 μM VX-475 (VX) as shown. HIF-1α and α-tubulin were detected by Western blotting. Representative of three ( C ) or four ( D ) independent experiments. E and F , MDMs were treated with 10 ng/ml LPS ± 100 nM Dex or 1 μM SB for 8 h (peak of HIF1A expression) or 12 h (peak of SLC2A1 expression). Fold change ± SD of HIF1A ( E ) or SLC2A1 ( F ) mRNA abundance relative to unstimulated control; three ( F ) or four ( E ) independent experiments; one-way ANOVA with Dunnett’s correction. G , MDMs were stimulated for 8 h with 10 ng/ml LPS in the absence or the presence of 100 nM Dex or 1 μM SB. Cycloheximide (CHX) was added at t = 0, and cells were harvested at the time points shown. HIF-1α and α-tubulin were detected by Western blotting. Representative of three independent experiments. ns, p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.005; and ∗∗∗∗ p < 0.001. HIF-1α, hypoxia-inducible transcription factor 1α; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MDM, monocyte-derived macrophage; ns, not significant.

Article Snippet: The following primary antibodies were used: mouse HIF-1α (Cell Signaling catalog no.: 14179); human HIF-1α (BD Biosciences; catalog no.: 610959); phospho-p38(Thr180/Tyr182) (Cell Signaling; catalog no.: 9211); DUSP1 (Cell Signaling; catalog no.: 35217); TTP (Cell Signaling; catalog no.: 71632); GLUT1 (Abcam; catalog no.: ERP3915); MCT4 (Novus Biologicals; catalog no.: NBP1-81251); and α-tubulin (Sigma–Aldrich; catalog no.: T9026).

Techniques: Expressing, Control, Western Blot, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipopolysaccharide induces HIF-1α accumulation via MAPK p38–mediated mRNA stabilization and dexamethasone-sensitive protein stabilization

doi: 10.1016/j.jbc.2025.111094

Figure Lengend Snippet: Regulation of LPS-induced HIF-1α expression by dexamethasone . LPS promotes accumulation of Hif1a mRNA by activating transcription ( e . g ., via NF- κB) and by inactivating the mRNA destabilizing factor TTP ( via p38-mediated activation of MK2 and MK2-mediated phosphorylation of TTP Ser52 and Ser178). DUSP1 negatively regulates Hif1a mRNA accumulation by preventing the phosphorylation and inactivation of TTP. (1) Although dexamethasone can induce DUSP1 expression, this mechanism is dispensable for the inhibition of LPS-induced HIF-1α accumulation. (2) Dexamethasone promotes destabilization of HIF-1α protein via a mechanism that remains to be identified. DUSP1, dual specificity phosphatase 1; HIF-1α, hypoxia-inducible transcription factor 1α; LPS, lipopolysaccharide; TTP, tristetraprolin.

Article Snippet: The following primary antibodies were used: mouse HIF-1α (Cell Signaling catalog no.: 14179); human HIF-1α (BD Biosciences; catalog no.: 610959); phospho-p38(Thr180/Tyr182) (Cell Signaling; catalog no.: 9211); DUSP1 (Cell Signaling; catalog no.: 35217); TTP (Cell Signaling; catalog no.: 71632); GLUT1 (Abcam; catalog no.: ERP3915); MCT4 (Novus Biologicals; catalog no.: NBP1-81251); and α-tubulin (Sigma–Aldrich; catalog no.: T9026).

Techniques: Expressing, Activation Assay, Phospho-proteomics, Inhibition

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: GDF15 preserves mitochondrial homeostasis in LPS-stimulated macrophages through dual regulation of SMAD7 and PKM2 pathways. (A) HIF-1α and SMAD7 expression in RAW264.7 macrophages across conditions: Untreated, LPS, LPS with rAAV8-mGdf15 overexpression (LPS+GDF15), and LPS with GDF15 knockdown (si-GDF15). β-actin: loading control.(HIF-1α suppression and SMAD7 induction by GDF15.) (B) Cytosolic and nuclear PKM2 protein levels. Lamin B1 (nuclear) and α-tubulin (cytosolic) markers validate fractionation efficiency. Study groups and individual replicates are identified in the figure key.(PKM2 subcellular redistribution modulated by GDF15.) (C) Immunofluorescence of PKM2 (red) and nuclei (DAPI, blue). Arrows indicate nuclear PKM2 accumulation. Scale bar: 15 μm.(Nuclear PKM2 enrichment upon LPS challenge mitigated by GDF15 and exacerbated by GDF15 knockdown.).

Article Snippet: Proteins (30 μg/lane) were separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, IPVH00010), and probed with the following primary antibodies: rabbit anti-mouse UQCRC1 (Proteintech, Cat. No. 21705-1-AP, 1:1000), rabbit anti-mouse GDF15 (Proteintech, Cat. No. 27455-1-AP, 1:1000), rabbit anti-mouse HIF-1α (Proteintech, Cat. No. 20960-1-AP, 1:1000), rabbit anti-mouse SMAD7 (Proteintech, Cat. No. 725840-1-AP, 1:1000), mouse anti-mouse PKM2 (Proteintech, Cat. No. 60268-1-Ig, 1:1000), rabbit anti-mouse H3 (Proteintech, Cat. No. 17168-1-AP, 1:5000) and mouse anti-mouse β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:5000).

Techniques: Expressing, Over Expression, Knockdown, Control, Fractionation, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: HIF-1α and PKM2 are critical effectors of GDF15-driven mitochondrial protection and anti-inflammatory responses. (A) HIF-1α inhibition by BAY 87-2243 (5 μM, 24 h). β-actin: loading control.(Pharmacological HIF-1α blockade.) (B) PKM2 inhibition by Shikonin (2 μM, 24 h). β-actin: loading control.(PKM2 activity suppression.) (C) UQCRC1 recovery in LPS-injured macrophages treated with: GDF15 overexpression, HIF-1α inhibitor (BAY), or PKM2 inhibitor (Shikonin). β-actin: loading control.(Mitochondrial complex III rescue via HIF-1α/PKM2 inhibition mirrors GDF15 effects.) (D) Inflammatory (TNF-α, IL-6) and metabolic (lactate) markers in cell supernatant (n = 5). Study groups and individual replicates are identified in the figure key. ***p < 0.001.(HIF-1α/PKM2 targeting replicates GDF15-mediated anti-inflammatory and metabolic homeostasis.) (E) UQCRC1 expression under GDF15 loss-of-function: si-GDF15 alone vs. combined with BAY 87–2243 or Shikonin. β-actin: loading control. Study groups and individual replicates are identified in the figure key.(Mitochondrial rescue in GDF15-deficient macrophages requires HIF-1α/PKM2 inhibition.) (F) Supernatant cytokines and lactate in si-GDF15 macrophages with/without inhibitors (n = 5). ***p < 0.001. (Inflammation reversal in GDF15-knockdown macrophages depends on HIF-1α/PKM2 blockade.).

Article Snippet: Proteins (30 μg/lane) were separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, IPVH00010), and probed with the following primary antibodies: rabbit anti-mouse UQCRC1 (Proteintech, Cat. No. 21705-1-AP, 1:1000), rabbit anti-mouse GDF15 (Proteintech, Cat. No. 27455-1-AP, 1:1000), rabbit anti-mouse HIF-1α (Proteintech, Cat. No. 20960-1-AP, 1:1000), rabbit anti-mouse SMAD7 (Proteintech, Cat. No. 725840-1-AP, 1:1000), mouse anti-mouse PKM2 (Proteintech, Cat. No. 60268-1-Ig, 1:1000), rabbit anti-mouse H3 (Proteintech, Cat. No. 17168-1-AP, 1:5000) and mouse anti-mouse β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:5000).

Techniques: Inhibition, Control, Activity Assay, Over Expression, Expressing, Knockdown

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: SMAD7 activation suppresses HIF-1α to mediate GDF15-dependent mitochondrial protection in LPS-challenged macrophages. (A) Pharmacological SMAD7 activation by Asiaticoside (20 μM, 48 h). β-actin: loading control. (B) HIF-1α expression under LPS challenge: LPS alone, LPS + AVV-GDF15, or LPS + SMAD7 activation (Asiaticoside). β-actin: loading control. (C) HIF-1α modulation across conditions: LPS, LPS + si-GDF15, LPS + Asiaticoside, or LPS + si-GDF15 + Asiaticoside. β-actin: loading control.

Article Snippet: Proteins (30 μg/lane) were separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, IPVH00010), and probed with the following primary antibodies: rabbit anti-mouse UQCRC1 (Proteintech, Cat. No. 21705-1-AP, 1:1000), rabbit anti-mouse GDF15 (Proteintech, Cat. No. 27455-1-AP, 1:1000), rabbit anti-mouse HIF-1α (Proteintech, Cat. No. 20960-1-AP, 1:1000), rabbit anti-mouse SMAD7 (Proteintech, Cat. No. 725840-1-AP, 1:1000), mouse anti-mouse PKM2 (Proteintech, Cat. No. 60268-1-Ig, 1:1000), rabbit anti-mouse H3 (Proteintech, Cat. No. 17168-1-AP, 1:5000) and mouse anti-mouse β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:5000).

Techniques: Activation Assay, Control, Expressing

Journal: Cells

Article Title: Hypoxia Affects Stem Cell Fate in Patient-Derived Ileum Enteroids in a HIF-1α-Dependent Manner

doi: 10.3390/cells15010031

Figure Lengend Snippet: Growth of human ileum-derived enteroids is reduced in hypoxia. ( a ) Ileum-derived enteroids were lysed after 24 h incubation under normoxia (N, red) or hypoxia (H, blue), and stabilization of HIF-1α was assessed by Western blot analysis. A representative image from enteroid donor 1 is shown, and β-actin was used as a loading control. ( b ) The expressions of the HIF-target genes CA9 , VEGF , and GLUT1 were quantified using qRT-PCR after 48 h incubation of enteroids derived from donor 1 in normoxia (red) or hypoxia (blue). Figures show the mean ± SEM ( n = 3 independent experiments), and an unpaired t-test with Welch’s correction was applied. p < 0.05 = *, <0.001 = ***. ( c ) Schematic depicting the experimental setup of enteroid seeding followed by incubation in normoxia (20% oxygen, red) or hypoxia (1% oxygen, blue) two days post-seeding. ( d – f ) Enteroids derived from donor 1 were cultured in normoxia or hypoxia according to ( c ), and enteroid growth was monitored over time. ( d ) Brightfield images were acquired each day using a ZEISS Celldiscoverer 7 microscope using a 5× 0.5× magnification. Representative images are shown, and yellow arrows point towards enteroids (not all enteroids were marked). Scale bar = 200 μm. ( e , f ) The number of enteroids per well ( e ) and enteroid size ( f ) were determined. Enteroids from ≥2 wells per independent experiment were counted and measured ( n = 3 independent experiments). ( e , f ) The graphs depict the mean ± 95% confidence interval. A 2-way ANOVA with multiple comparisons was applied. p ≥ 0.05 = ns (not significant), <0.05 = *, <0.0001 = ****.

Article Snippet: Proteins were separated on SDS-PAGE gels and wet-transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA #1620177) for 90 min at 100 V. For detection of HIF-1α, membranes were blocked in 5% milk in TBS-T and then incubated in 1:1000 mouse anti-HIF-1α antibody (Fisher Scientific, Waltham, MA, USA BDB610959) at 4 °C overnight.

Techniques: Derivative Assay, Incubation, Western Blot, Control, Quantitative RT-PCR, Cell Culture, Microscopy

Journal: Cells

Article Title: Hypoxia Affects Stem Cell Fate in Patient-Derived Ileum Enteroids in a HIF-1α-Dependent Manner

doi: 10.3390/cells15010031

Figure Lengend Snippet: HIF-1α stabilization by roxadustat in normoxia reduces stemness and proliferation in human ileum-derived enteroids. Enteroids were treated with 100 μM roxadustat (Rox, purple) to stabilize HIF-1α protein expression in normoxia and compared to solvent (DMSO)-treated enteroids incubated in normoxia (N, red) or hypoxia (H, blue). ( a ) To confirm HIF-1α stabilization, enteroids were lysed 24 h post-treatment, and HIF-1α protein expression was assessed via Western blotting, and β-actin was used as a loading control. A representative Western blot image of enteroids derived from donor 1 is shown. ( b , c ) Gene expression of the HIF-1α target gene CA9 ( b ) and the stem cell-associated gene OLFM4 ( c ) from all three donors was assessed using qRT-PCR 48 h post-treatment. The graphs depict the mean ± SEM ( n = 3 independent experiments), and a 1-way ANOVA with multiple comparisons was applied. p < 0.05 = *, <0.01 = **, <0.001 = ***, <0.0001 = ****. ( d ) Enteroids from all three donors were seeded into untreated high-Wnt media. After two days, media were exchanged and enteroids were incubated with 100 μM roxadustat (purple) or a solvent control (DMSO, red) in normoxia. Brightfield images were acquired at the indicated time points post-media change using a ZEISS Celldiscoverer 7 microscope using a 5× 1× magnification. Enteroid growth was quantified by counting the number of enteroids per field of view. Means ± 95% confidence intervals are depicted from 8 fields of view. A 2-way ANOVA with multiple comparisons was applied. p ≥ 0.05 = ns (not significant), <0.05 = *, <0.01 = **. ( e , f ) Enteroids from donor 1 were seeded into normoxia (red) into untreated high-Wnt media. After two days, media were exchanged, and enteroids were incubated with 100 μM roxadustat (purple) or a solvent control (DMSO, red) for the indicated incubation spans. On day 5, enteroids were split into untreated high-Wnt medium, and imaging was performed on day 7. Brightfield images were acquired two days post-splitting using a ZEISS Celldiscoverer 7 microscope using a 5× 0.5× magnification. ( e ) Schematic depicting the experimental setup to assess enteroid formation efficiencies. ( f ) Enteroid formation efficiency was determined by quantifying the number of enteroids. Means ± 95% confidence intervals are depicted from ≥7 fields of view per independent experiment ( n = 3 independent experiments). A 2-way ANOVA with multiple comparisons was applied. p < 0.01 = **, <0.0001 = ****.

Article Snippet: Proteins were separated on SDS-PAGE gels and wet-transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA #1620177) for 90 min at 100 V. For detection of HIF-1α, membranes were blocked in 5% milk in TBS-T and then incubated in 1:1000 mouse anti-HIF-1α antibody (Fisher Scientific, Waltham, MA, USA BDB610959) at 4 °C overnight.

Techniques: Derivative Assay, Expressing, Solvent, Incubation, Western Blot, Control, Gene Expression, Quantitative RT-PCR, Microscopy, Imaging