Journal: Molecular Metabolism

Article Title: Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice

doi: 10.1016/j.molmet.2023.101717

Figure Lengend Snippet: Direct regulation of Bnip3 transcription by AhR. (A) The mRNA level of Bnip3 and Ahr in the GEO dataset (GSE46495) during fed and fasted conditions. (n = 5, each). (B) Expression levels of Bnip3, Ahr, and Cyp1a1 in fed and fasted livers of WT or AhR KO mice (n = 3 or 4). Data were shown as box and whisker plots. Box, interquartile range (IQR); whiskers, min to the max; and horizontal line within box, median, ∗p < 0.05, ∗∗p < 0.01. (C) Pearson's correlation between Ahr and Bnip3 mRNA levels. (D) BNIP3 protein levels in fed and fasted livers of WT and AhR KO mice (upper) and IHC for BNIP3 in fasted livers (lower, left). Representative images were shown (n = 3 each). Scale bar, 100 μm. The quantification of BNIP3 expression (lower, right) was measured by Image J. (E) Increased BNIP3 expression by endogenous AhR ligand, kynurenine (Kyn) treatment (100 μM, 24 h) in mouse primary hepatocyte (left) and AML12 cells (middle and right). (F) AhR recruitment to the Bnip3 genomic locus in TCDD-treated liver (GSE97634). (G) ChIP-PCR analysis of AhR binding to Bnip3 genomic locus. (H) Reporter assays using the pGL3-basic vector containing WT ARE or Mutated ARE. HEK293 cells transfected with mock or AhR overexpression vector together with the reporter vector and treated Kyn for 24h. Results was normalized to WT control. (D), (E), (G) and (H), Data represented the mean ± SEM, ∗p < 0.05, ∗∗p < 0.01. (H), ## was compared to AhR overexpressed group.

Article Snippet: Antibodies against BNIP3 (nbp1-77683s), LC3A (NB100-2331), and LC3B (NB600-1384) were obtained from Novus Biologicals (Littleton, CO).

Techniques: Expressing, Whisker Assay, Binding Assay, Plasmid Preparation, Transfection, Over Expression, Control

Journal: Molecular Metabolism

Article Title: Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice

doi: 10.1016/j.molmet.2023.101717

Figure Lengend Snippet: Restored mitophagy by BNIP3 overexpression. (A) Changes of LC3A protein levels by Bnip3 overexpression in AhR knockdown cells. AML12 cells were co-transfected with siCon, siAhR, or siAhR with Bnip3-overexpressing plasmids. Band intensities represent values relative to siCon. (B) Changes of mitochondria-associated LC3A levels. Cytosolic and mitochondrial fractions were subjected to Western blotting. Cox IV was used for the control of mitochondrial fraction. (C) and (D) Visualization of mitophagy using mt-Keima assay and quantification. (E) Dual staining of mitochondria (Mitotracker) and autophagy marker (LC3A). Representative images are presented. Scale bar, 50 μm. (F) Quantification of colocalization of LC3A with mitotracker. (G) Mitochondrial superoxide levels measured by MitoSOX™ in AML12 cells transfected with siCon, siAhR, or siAhR with Bnip3 overexpression plasmids. (H) Mitoplate S-1 assay to measure mitochondrial substrate utilization. AML12 cells were transfected with siCon, siAhR, or siAhR with Bnip3-overexpressing plasmids for 48 h. The change in metabolic rate for each substrate/intermediate of mitochondrial/glycolytic pathways was shown in a heatmap (left) and change of substrate utilization in TCA cycle were shown as a bar graph (right). (A),(B), (D–H), Data represented the mean ± SEM, ∗p < 0.05, ∗∗p < 0.01. PPP; pentose phosphate pathway.

Article Snippet: Antibodies against BNIP3 (nbp1-77683s), LC3A (NB100-2331), and LC3B (NB600-1384) were obtained from Novus Biologicals (Littleton, CO).

Techniques: Over Expression, Knockdown, Transfection, Western Blot, Control, Staining, Marker

Journal: Scientific Reports

Article Title: Species generalization and differences in Hedgehog pathway regulation of fungiform and circumvallate papilla taste function and somatosensation demonstrated with sonidegib

doi: 10.1038/s41598-018-34399-3

Figure Lengend Snippet: Sonidegib treatment reduces taste buds (TB), SHH ligand and proliferation in rat fungiform papilla (FP) while innervation is retained. ( a ) Immunofluorescent antibody detection of SHH ligand (red) and K19 (green) for TB cells; K18 (red) for TB cells and Ki67 (green) for cell proliferation, after Vehicle, 16d or 28d Sonidegib treatments. SHH is reduced in association with TB, K19+ cell loss. Asterisks (*) indicate nonspecific SHH immunoproduct in cornified surface cells in 16d Sonidegib image. The Vehicle, K18/Ki67 image shows 3 regions positive for Ki67+ cells (Apical, Basal and Perigemmal). Proliferating cells are lost in Apical FP region after 16–28d Sonidegib. ( b ) Number of Ki67+ cells in Apical and Basal regions of FP in Vehicle- and Sonidegib-treated mice. Numbers of tongues analyzed are in parentheses. For each tongue 8–10 FP were analyzed. Sonidegib treatment reduces apical epithelial cell proliferation in FP compared to Vehicle. Statistical analysis was one-way ANOVA with Tukey HSD posthoc comparisons (*p ≤ 0.05, compared to Vehicle, APICAL). ( c ) Immunofluorescent antibody detection of K19 or K18 (red) for TB cells and NF (green) for lingual and CT innervation or P2X3 (green) for CT nerve fibers. Innervation was retained after Sonidegib exposure. Asterisks (*) indicate nonspecific P2X3 immunoproduct in surface layer in Vehicle image. ( d ) Enlarged images from 28d Sonidegib papillae. Arrows point to NF+ or P2X3+ fibers in the FP epithelium. Throughout, white dotted lines indicate the basal lamina. Yellow dotted lines indicate surface of epithelium. ( a , c ) Scale bar: 50 μm, applies to all images. ( d ) Scale bar: 25 μm, applies to both images.

Article Snippet: Antibodies included: SHH ligand: goat anti-SHH (AF464, 0.1 μg/ml, R&D Systems for mice), mouse anti-SHH (sc-365112, 1:500, Santa Cruz Biotechnology for rat); taste cell markers K8, K18, K19 – , with different cytokeratins for optimal immunoreactions in double labeling, K8 (rat TROMA-1, 1:1000, Developmental Studies Hybridoma Bank) for mice, K18 (mouse anti-cytokeratin 18, sc-51582, 1:100, Santa Cruz Biotechnology) or K19 (rabbit anti-keratin 19, ab52625, 1:5000, Abcam) for rat; cell proliferation, Ki67 (rabbit anti-Ki67, NB110-89717SS, 1:5000, Novus Biologicals); innervation, NF (chicken anti-neurofilament heavy, ab4680, 1:5000, Abcam), P2X3 (rabbit anti-P2X3, APR-016, 1:1000, Alomone Labs).

Techniques:

Journal: Scientific Reports

Article Title: Species generalization and differences in Hedgehog pathway regulation of fungiform and circumvallate papilla taste function and somatosensation demonstrated with sonidegib

doi: 10.1038/s41598-018-34399-3

Figure Lengend Snippet: Sonidegib treatment in rat reduces taste buds (TB) and SHH ligand in circumvallate papilla (CV) whereas cell proliferation and GL innervation are retained. Immunofluorescent antibody detection of SHH ligand (red) and K19 (green) for TB cells; K18 (red) for TB cells and Ki67 (green) for cell proliferation; K19 (red) for TB cells and NF (green) for innervation; and, K18 (red) for TB cells and P2X3 (green) for taste nerve fibers, after Vehicle or 36d Sonidegib treatment. White dotted lines outline the epithelium. Asterisk in SHH/K19, 36d Sonidegib indicates nonspecific K19 immunostaining. Arrow points to the P2X3+ nerves extending into CV epithelium after Sonidegib treatment. SHH is reduced in association with TB cells. Cell proliferation is maintained and nerves fibers are retained after Sonidegib treatment. Scale bar: 50μm, applies to all images, except K19/NF.

Article Snippet: Antibodies included: SHH ligand: goat anti-SHH (AF464, 0.1 μg/ml, R&D Systems for mice), mouse anti-SHH (sc-365112, 1:500, Santa Cruz Biotechnology for rat); taste cell markers K8, K18, K19 – , with different cytokeratins for optimal immunoreactions in double labeling, K8 (rat TROMA-1, 1:1000, Developmental Studies Hybridoma Bank) for mice, K18 (mouse anti-cytokeratin 18, sc-51582, 1:100, Santa Cruz Biotechnology) or K19 (rabbit anti-keratin 19, ab52625, 1:5000, Abcam) for rat; cell proliferation, Ki67 (rabbit anti-Ki67, NB110-89717SS, 1:5000, Novus Biologicals); innervation, NF (chicken anti-neurofilament heavy, ab4680, 1:5000, Abcam), P2X3 (rabbit anti-P2X3, APR-016, 1:1000, Alomone Labs).

Techniques: Immunostaining

Journal: Scientific Reports

Article Title: Species generalization and differences in Hedgehog pathway regulation of fungiform and circumvallate papilla taste function and somatosensation demonstrated with sonidegib

doi: 10.1038/s41598-018-34399-3

Figure Lengend Snippet: Long term Sonidegib treatment in mouse reduces taste buds (TB) and SHH ligand in circumvallate papilla (CV) whereas proliferation and innervation are retained. Immunofluorescent antibody detection of SHH ligand (red) and K8 (green) for TB cells; K8 (red) for TB cells and Ki67 (green) for cell proliferation; and K8 (red) with NF (green) for GL innervation or P2X3 (green) for GL taste fibers, after Vehicle or 48d Sonidegib treatment. For SHH/K8, large dotted lines indicate the basal lamina. Small dotted lines outline the surface epithelium. Inset (K8/P2X3) shows an image of nerves extending into CV epithelial basal lamina (arrow). Scale bar: 50 μm, applies to all images. Inset at 2×.

Article Snippet: Antibodies included: SHH ligand: goat anti-SHH (AF464, 0.1 μg/ml, R&D Systems for mice), mouse anti-SHH (sc-365112, 1:500, Santa Cruz Biotechnology for rat); taste cell markers K8, K18, K19 – , with different cytokeratins for optimal immunoreactions in double labeling, K8 (rat TROMA-1, 1:1000, Developmental Studies Hybridoma Bank) for mice, K18 (mouse anti-cytokeratin 18, sc-51582, 1:100, Santa Cruz Biotechnology) or K19 (rabbit anti-keratin 19, ab52625, 1:5000, Abcam) for rat; cell proliferation, Ki67 (rabbit anti-Ki67, NB110-89717SS, 1:5000, Novus Biologicals); innervation, NF (chicken anti-neurofilament heavy, ab4680, 1:5000, Abcam), P2X3 (rabbit anti-P2X3, APR-016, 1:1000, Alomone Labs).

Techniques: