Journal: PLoS Genetics

Article Title: Aldh inhibitor restores auditory function in a mouse model of human deafness

doi: 10.1371/journal.pgen.1009040

Figure Lengend Snippet: ( A ) Upregulation of Pou4f3 mRNA expression by DEAB and BMS195614 in P3 wildtype cochlear explants. DEAB and BMS195614 (BMS) concentrations were 100 and 5 μM, respectively. DMSO was used as control (-). * P < 0.05 and ** P < 0.01 by unpaired student’s t-test, n = 13–15 explants for DEAB, n = 4–8 explants for BMS195614. ( B ) Effects of DEAB (red bar) on mRNA expressions of Tmem237 and Sra1 . * P < 0.05 by unpaired student’s t-test, n = 4 from each treatment. ( C ) Effects of DEAB (red bar) on mRNA expressions of known Pou4f3 targets. * P < 0.05 and *** P < 0.001 by unpaired student’s t-test, n = 4 from each treatment. ( D-F ) Effect of DEAB (100 μM) and RA (0.3 μM) treatment on mRNA expressions of ( D ) Cyp26a1 , ( E ) Pou4f3 and ( F ) Lhx3 in P3 wildtype cochlear explants. DMSO was used as control (-). * P < 0.05, ** P < 0.01 and *** P < 0.001 by one-way ANOVA, n = 4 explants for each treatment. ( G ) Upregulation of Pou4f3 mRNA expression by DEAB in P3 Pou4f3(Δ/+) cochlear explants. ** P < 0.01 by unpaired student’s t-test, n = 6 explants for each treatment. ( H ) Western blots and ( I ) densitometrical measurements show upregulation of wildtype Pou4f3 protein expression by DEAB in Pou4f3(Δ/+) cochlear explants. The black open arrow indicates the wildtype Pou4f3 and red open arrow shows the truncated mutant Pou4f3 protein. * P < 0.05 by unpaired student’s t-test, n = 4 explants from each treatment.

Article Snippet: After 24 h, 100 μM 4-Diethylaminobenzaldehyde (DEAB, Sigma, USA), 5 μM BMS195614 (Tocris Bioscience, UK) or 0.3 μM all-trans retinoic acid (RA, Sigma, USA) or DMSO vehicle control were added to the culture medium for 5 days with fresh drug-containing media replaced every 2 days.

Techniques: Expressing, Control, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Central Role for Glycolysis and Fatty Acids in LH-responsive Progesterone Synthesis

doi: 10.1101/2024.02.14.580329

Figure Lengend Snippet: ( A-D ) Progesterone production by the small luteal cells pretreated with inhibitor of ACSS2 (ACSS2 i.; 10 µM), HMGCR (Fluvastatin; 1-50 µM), ACLY (BMS303141; 5-50 µM) or Etomoxir for 60 min and then treated with LH (10 ng/ml) for 240 min. Data are presented as a fold change (FC) and mean±SEM (n=2-5). Data were analyzed using a one-way ANOVA test. Asterisks *, ** and **** mean significant change with P < 0.05, P < 0.01 and P < 0.0001, respectively. ( E ) Representative blots showing phosphorylation of ACLY Ser455 and ACACA Ser89 in the small luteal cells treated with LH (1-100 ng/ml) for 30 min or cAMP/PKA activator forskolin (FSK; 10 μM) for 2-30 min as well as cells pretreated with PKA inhibitor (H89; 20 µM) and then treated with LH (10 ng/ml) for 10 min.

Article Snippet: H89, UK5099, Shikonin, CPI613, BMS303141, 6AN, Fluvastatin, Lonidamine were purchased from Tocris Bioscience (Minneapolis, MN).

Techniques: Phospho-proteomics

Journal: bioRxiv

Article Title: Central Role for Glycolysis and Fatty Acids in LH-responsive Progesterone Synthesis

doi: 10.1101/2024.02.14.580329

Figure Lengend Snippet: ( A ) Phosphorylation of ACC1 Ser79 and ACLY Ser455 in the small luteal cells treated with different concentrations of LH (1-100 ng/ml) for 30 min. Data are presented as fold change and mean±SEM (n=3-5). Data were analyzed using a one-way ANOVA test. Asterisks ** and *** mean significant change with P < 0.01 and P < 0.001, respectively. ( B ) Phosphorylation of ACC1 Ser79 and ACLY Ser455 in the small luteal cells treated with cAMP/PKA activator-forskolin (FSK; 10 μM) for 2-30 min. Data are presented as a fold change (FC) and mean±SEM (n=3-4). Asterisks * and ** mean significant change with P < 0.05 and P < 0.01, respectively. ( C ) ATP production measured by luminescence method in the small luteal cells pretreated with ACLY inhibitor (BMS303141; 10-50 µm) and then treated with LH (10 ng/ml) for 240 min. Data presented as a fold change (FC) and mean±SEM (n=3-5). Data were analyzed by using a two-way ANOVA test. Asterisks **, *** and **** mean significant change with P < 0.05, P < 0.01 and P < 0.001, respectively. ( D ) cyclic AMP (cAMP) production by the small luteal cells pretreated with ACLY inhibitor (BMS303141; 25 µm) and then treated with LH (10 ng/ml) for 240 min. Data presented as fold change (FC) and mean±SEM (n=3-5). Data were analyzed by using a one-way ANOVA test. Asterisks * and **mean significant change with P < 0.05 and P < 0.01, respectively. ( E - F ) Representative blots showing phosphorylation of PKA substrates and content of steroidogenic proteins (STAR, CYP11A1), electron transport chain proteins or marker of mitochondria (TOM20) and PKA catalytic subunits in the small luteal cells pretreated with ACLY inhibitor (BMS303141; 25 µM) and then treated with LH (10 ng/ml) for 240 min. ( G ) cyclic AMP (cAMP) production by the small luteal cells with knockdown CPT1A (si CPT1A ; 50 nM) and then treated with LH (10 ng/ml) for 240 min. Data are presented as fold change (FC) and mean±SEM (n=3-5). Data were analyzed by using one-way ANOVA. Asterisks ** and *** mean significant change with P < 0.05 and P < 0.01, respectively. ( H ) Representative blots showing phosphorylation of PKA substrates and content of PKA catalytic subunits in the small luteal cells with knockdown CPT1A (si CPT1A ; 50 nM) and then treated with LH (10 ng/ml) for 240 min.

Article Snippet: H89, UK5099, Shikonin, CPI613, BMS303141, 6AN, Fluvastatin, Lonidamine were purchased from Tocris Bioscience (Minneapolis, MN).

Techniques: Phospho-proteomics, Marker, Knockdown

Journal: bioRxiv

Article Title: Central Role for Glycolysis and Fatty Acids in LH-responsive Progesterone Synthesis

doi: 10.1101/2024.02.14.580329

Figure Lengend Snippet: ( A ) Representative blots showing phosphorylation of PKA substrates and content of STAR in the small luteal cells pretreated with ACLY inhibitor (BMS303141; 5-50 µm) and then treated with LH (10 ng/ml) for 240 min. ( B ) cyclic AMP (cAMP) production by small luteal cells pretreated with CPT1A inhibitor (Teglicar; 25 µM) and then treated with LH (10 ng/ml) for 240 min. Data are presented as a fold change (FC) and mean±SEM (n=2-5). Data were analyzed using a one-way ANOVA test. Asterisks ** mean significant change with P < 0.01. ( C-D ) Representative blots showing phosphorylation of PKA substrates and content of STAR in the small luteal cells pretreated with CPT1A inhibitor (Teglicar; 5-50 µm) and then treated with LH (10 ng/ml) or PKA activator Forskolin (FSK; 10 µM) for 240 min; ( E ) Progesterone production by small luteal cells pretreated with Teglicar (5-50 µM) and then treated with PKA activator Forskolin (FSK; 10 µM) for 240 min. ( F ) Small luteal cells were pretreated with inhibitor of ACLY (BMS303141; 5-50 µM) and then treated with PKA activator forskolin (FSK; 10 µM) for 240 min. Representative blots of phosphorylation of PKA substrates in the small luteal cells pretreated with ACLY inhibitor (BMS303141) and then treated with forskolin (FSK). ( G ) Progesterone production by the small luteal cells pretreated with ACLY inhibitor (BMS303141) and then treated with PKA activator forskolin (FSK). Data are presented as a fold change (FC) and mean±SEM (n=2-5). Data were analyzed using a two-way ANOVA test. Asterisks *, P < 0.05.

Article Snippet: H89, UK5099, Shikonin, CPI613, BMS303141, 6AN, Fluvastatin, Lonidamine were purchased from Tocris Bioscience (Minneapolis, MN).

Techniques: Phospho-proteomics

Journal: Nucleic Acids Research

Article Title: New structural insights into the control of the retinoic acid receptors RAR/RXR by DNA, ligands, and transcriptional coregulators

doi: 10.1093/nar/gkaf967

Figure Lengend Snippet: The interaction of RR–BMS493 bound to DNA with the NCoR NID reveals new features. ( A – D ) The differential interdomain distances in RAR/RXR upon NCoR NID interaction (NRR–DNAs versus RR–DNA). Light green indicates differences in distances for RAR–DBD–LBD, purple represents RXR, dark green corresponds to RAR–DBD/RXR–LBD, and red denotes RXR–DBD/RAR–LBD. Panels are organized by the bound element (panel A: DR0L, panel B: DR1, panel C: DR5, panel D: IR0). ( E and F ) Differential plots of normalized weighted contact densities (Δρ) calculated as Δρ = ρNRR-DNA – ρRR-DNA. Positive Δρ values (blue) represent regions where the contact density increases in the presence of NCoR NID , while negative (red) indicate regions where it decreases. ( E ) The lower-left quadrant corresponds to NRR–DR0L, while the upper-right quadrant represents NRR–DR1. ( F ) The lower-left quadrant corresponds to NRR–DR5, and the upper-right quadrant represents NRR–IR0. ( G ) Illustration comparing apo RR–DNA–BMS493 versus RR–DNA–BMS493 bound to NCoR NID . RAR is depicted in green, RXR is shown in purple, and NCoR NID is represented in gray. The RAR inverse agonist (BMS493) is drawn as a light green circle, and the DNA is shown as brown bars. ( H ) Differential heatmap highlighting structural changes in RAR (apo-NB and DNA bound forms) upon BMS493 and NCoR NID binding. Red and blue shades indicate increased and decreased deuterium incorporation, respectively. Secondary structural elements are shown above each heatmap, where α-helices are represented in lighter colors and labeled with their respective numbering, and β-strands are shown in orange. ( I ) Same structural effects as in panel (H) but on the RXR part of the RAR/RXR heterodimer. ( J ) Illustration comparing NRR with NRR–DNA–BMS493. ( K and L ) Differential heatmaps showing structural changes in RAR and RXR, respectively, based on the conditions illustrated in panel (J). ( M ) HDX differential protections showing NCoRNID-binding effect plotted on the model generated from the crystallographic structure of RARβ/RXR–DR1 . The data correspond to exchange times of 3 h and 10 s (inset)—highlighted on the right side of the heatmaps by colored arrowheads. ( N ) Illustration of the NCoR NID versus the NCoR NID bound to RR–DNAs. ( O ) Differential HDX-MS heatmap of NCoR NID , showing the impact of the RR–DNA–BMS493 interaction on the corepressor protection.

Article Snippet: The ligands AM580, BMS493, and CD3254 were purchased from Tocris Bioscience.

Techniques: Binding Assay, Labeling, Generated