Journal: EMBO Reports

Article Title: LncRNA Ctcflos orchestrates transcription and alternative splicing in thermogenic adipogenesis

doi: 10.15252/embr.202051289

Figure Lengend Snippet: A GO term analysis of genes upregulated in response to Ctcflos knockdown. In dark gray, negative log10 P ‐values of overrepresented GO terms 24 h after KD and percentages of genes belonging to the respective GO terms among upregulated and among all genes. Small numbers next to the bars show rank positions of GO terms. In light gray, corresponding values for the same GO terms after 72 h, hypergeometric test. B–D Impact of splicing inhibition by different concentrations of general splicing inhibitor isoginkgetin on brite adipogenesis, (B) expressed as Ucp1 relative to Fabp4 mRNA levels, (C) Ucp1 relative to Gtf2b and (D) Fabp4 relative to Gtf2b . Mean values ± SD, n = 3 (biological replicates), one‐way ANOVA (Šídák‐test), n.s. P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. E–I Effect of Ctcflos KD on serine/arginine‐rich splicing factor 2 (SC35/SRSF2) and nuclear speckle abundance in Ctcflos tr1 (ASO 1) KD and control cells (KD day 1 of differentiation, analysis after 48h). (E) Immunocytochemistry of SC35. SC35 signal (blue) alone (middle images) and SC35 (blue), DNA staining by DAPI (green) and phase contrast (PC) (gray) combined (outer images). (F) Quantification of SC35 fluorescence signal as whole image fluorescence intensity subtracted by background and normalized to total cell number. Mean and individual values, one representative replicate of three experiments with comparative results (see Fig ), unpaired t ‐test, ** P < 0.01. (G) Quantification of nuclear speckle number as mean number of SC35 signals per nucleus. Mean and individual values, one representative replicate of three experiments with comparative results (see Fig ), unpaired t ‐test, ** P < 0.01. (H, I) Correlation of SC35 and UCP1 fluorescence signals across individual cells in (H) control and (I) Ctcflos tr1 (ASO 1) KD cells. Extreme values were excluded to avoid distortion of correlation analysis. One representative replicate of four experiments with comparable results (see Fig ). Pearson correlation, **** P < 0.0001. J–X Influence of Ctcflos KD on Prdm16 alternative splicing. (J) Splice graph of Prdm16 and resulting Prdm16 long and short isoforms. Primers used to amplify both Prdm16 isoforms at once by PCR are displayed in orange; primers used to amplify specifically Prdm16 short and Prdm16 long, respectively, are displayed in blue. (K) Gel picture of PCR products using exon 16 spanning primers to detect Prdm16 long (upper band (348 bp)) and Prdm16 short (lower band (173 bp)) comparing Ctcflos tr1 (ASO 1) KD and control samples. (L) Time course of relative expression of Prdm16 total, long and short isoforms during brite adipogenesis, assessed by qPCR using isoform‐specific primers. Mean values ± SD, one representative replicate of two experiments with comparable results, two‐way ANOVA (Šídák‐test), *** P < 0.001, **** P < 0.0001. (M) Levels of Prdm16 long and short isoforms in Ctcflos tr1 (ASO1) KD and control cells. Signal intensities quantified from PCR gel picture subtracted by background and normalized to overall mean signal intensity of the respective biological replicate. Mean and individual values. Two‐way ANOVA (Šídák‐test), * P < 0.05, **** P < 0.0001. (N) Correlation of Prdm16 short and Ucp1 expression levels across Ctcflos tr1 (ASO1) KD and control samples and within both groups. Pearson correlation, n.s. P > 0.05, ** P < 0.01, **** P < 0.0001.(O) Relative Prdm16 total, (P) relative Prdm16 long and (Q) relative Prdm16 short transcript levels 24 h after Ctcflos KD, comparing Ctcflos tr1 (ASO1) and tr3, tr4 (ASO2) KD and control samples, assessed by qPCR, Mean values ± SD, n = 3–4 (biological replicates), unpaired t ‐tests, n.s. P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001. (R, S) Fold change of Prdm16 short and Prdm16 long (R) ASO1 to NC and (S) ASO2 to NC 24 h after Ctcflos KD, n = 3–4 (biological replicates), unpaired t ‐tests, n.s. P > 0.05, ** P < 0.01. (T) Relative Prdm16 total, (U) relative Prdm16 long and (V) relative Prdm16 short transcript levels 72 h after Ctcflos KD, comparing Ctcflos tr1 (ASO 1) KD and control samples, assessed by qPCR, mean values ± SD, n = 3–4 (biological replicates), unpaired t ‐tests, * P < 0.05, *** P < 0.001. (W, X) Fold change of Prdm16 short and Prdm16 long (W) ASO1 to NC and (X) ASO2 to NC 72 h after Ctcflos KD, n = 3–4 (biological replicates), unpaired t ‐tests, * P < 0.05. Y, Z Global transcriptome analysis of Ctcflos KD impact on alternative splicing (KD day 1 of differentiation, analysis after 24 h). (Y) MA‐plot of differential alternative splicing analysis using SGSeq algorithm comparing Ctcflos tr1 (ASO 1) KD with controls. Each data point depicts a splice event. Splice events of differential abundance between Ctcflos tr1 (ASO 1) KD and controls ( P ‐adj. < 0.05) are colored in red. (Z) Overview of selected genes involved in adipogenesis, lipid metabolism, mitochondria, or splicing, with differential splicing profiles comparing Ctcflos tr1 (ASO 1) KD and control samples. Genes previously described to be spliced into alternative isoforms of varying function in brown adipogenesis are marked in red. AA Schematic summary of Ctcflos mechanistic functions in brite adipogenesis. Source data are available online for this figure.

Article Snippet: On the first day of differentiation, cells were treated with 10, 15, or 20 nM isoginkgetin (Tocris) diluted in DMSO or a corresponding amount of DMSO as control.

Techniques: Knockdown, Inhibition, Control, Immunocytochemistry, Staining, Fluorescence, Alternative Splicing, Expressing

Journal: Nucleic Acids Research

Article Title: Tracing the molecular basis of transcriptional dynamics in noisy data by using an experiment-based mathematical model

doi: 10.1093/nar/gku1272

Figure Lengend Snippet: Re-fitting the model can indicate the perturbation in the system. Human HepG2 cells were pre-incubated with 100 μM isoginkgetin for 16 h and subsequently treated with PPARδ ligand for the indicated time periods. qPCR was performed in order to measure pre-mRNA ( A ) and mature mRNA levels ( B ) and copy numbers were calculated. Small filled circles represent all data points from at least four biological experiments corrected for outlier and empty circles indicate their mean. All the individual data points are reported in Supplementary Tables S9. The mathematical model (adjusted for apparent fold induction) was fitted to the data by changing a single constant at a time and the quality of the fit was compared for pre-mRNA (grey) and mature mRNA (green) time courses ( C ). The best model obtained by lowering the splicing rate constant was further tested for fit improvement by changing the remaining constants ( D ). The best fit given by adjusting both splicing and degradation constants is also shown for the pre-mRNA (A) and mature mRNA (B) time courses.

Article Snippet: The splicing inhibitor isoginkgetin (kindly provided by the Dr Willmar, Schwabe GmbH & Co. KG, Karlsruhe, Germany) was added after 24 h to a final concentration of 50 μM.

Techniques: Incubation

Journal: ACS Omega

Article Title: Effects of U1 Small Nuclear Ribonucleoprotein Inhibition on the Expression of Genes Involved in Alzheimer’s Disease

doi: 10.1021/acsomega.0c03568

Figure Lengend Snippet: Effects of U1 knockdown on the protein expression of APP, Nicastrin, and GSK-3β. (A) Western blot to demonstrate the expression of these proteins under U1 knockdown. Antibodies used: APP, Y188. (B–D) Quantitative and correlation analyses of the protein expression levels of APP, Nicastrin, and GSK-3β under the knockdown of U1-70K, U1A, and U1C. Overexpression and knockdown were performed in HEK293T cells cultured in 24-well plates. Blank: no siRNA were added; control: scrambled siRNA. 1–4: four different siRNAs that target distinct sequences of U1-70K mRNA. Doses used for the titration: 10, 3, and 1 pmol, respectively, in which the mixture of four siRNAs was used for the U1-70K knockdown. Ctl: control. Three repeats of each treatments were used in a separate experiment for each protein in which siRNA 1 was used for U1-70K knockdown. Image J was used for quantification of the intensity of the Western blot bands. The dose effect data in (C) were from the quantiation results of blots in (A). Student’s t test was used for statistical analysis. The asterisk (*) indicates p < 0.05, and “N.S.” means insignificant.

Article Snippet: In the chemical RNA splicing inhibitor treatment, HEK293T cells were first treated by 33 μM Isoginkgetin (416154, Merck Millipore) for 12 h. After the U1 knockdown or the chemical inhibition, the Tau minigene construct prepared in lipofectamine 2000 (ThermoFisher Sicentific) was added to the medium for 24 h before the enhanced green fluorescent protein (EGFP) expression was examined and the cells were harvested for qPCR.

Techniques: Knockdown, Expressing, Western Blot, Over Expression, Cell Culture, Control, Titration

Journal: Molecules

Article Title: 3′,8″-Dimerization Enhances the Antioxidant Capacity of Flavonoids: Evidence from Acacetin and Isoginkgetin

doi: 10.3390/molecules24112039

Figure Lengend Snippet: The structures and molecular models of acacetin and isoginkgetin: ( A ) structure of acacetin; ( B ) structure of isoginkgetin; ( C ) molecular model of acacetin; ( D ) molecular model of isoginkgetin. The molecular model was created based on the preferential conformation by using the Chem3D Pro 14.0 program (PerkinElmer, Waltham, MA, USA). The molecular model D has a dihedral angle of 45°, see .

Article Snippet: Acacetin (C 16 H 12 O 5 , CAS number 480-44-4, MW 284.2, purity 98%; ) and isoginkgetin (C 32 H 22 O 10 , CAS number 548-19-6, MW 566.5, purity 98%; ) were obtained from Chengdu Biopurify Phytochemicals, Ltd. (Chengdu, China).

Techniques:

Journal: Molecules

Article Title: 3′,8″-Dimerization Enhances the Antioxidant Capacity of Flavonoids: Evidence from Acacetin and Isoginkgetin

doi: 10.3390/molecules24112039

Figure Lengend Snippet: IC 50 values of acacetin and isoginkgetin in various antioxidant assays (μM).

Article Snippet: Acacetin (C 16 H 12 O 5 , CAS number 480-44-4, MW 284.2, purity 98%; ) and isoginkgetin (C 32 H 22 O 10 , CAS number 548-19-6, MW 566.5, purity 98%; ) were obtained from Chengdu Biopurify Phytochemicals, Ltd. (Chengdu, China).

Techniques:

Journal: Molecules

Article Title: 3′,8″-Dimerization Enhances the Antioxidant Capacity of Flavonoids: Evidence from Acacetin and Isoginkgetin

doi: 10.3390/molecules24112039

Figure Lengend Snippet: Main results of the ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC−ESI−Q−TOF−MS) analysis of acacetin and isoginkgetin mixed with 4-methoxy-TEMPO radicals (negative ion model). ( A ) Chromatographic peaks of acacetin when the formula [C 16 H 12 O 5 -H] − was extracted; ( B ) MS spectra of acacetin; ( C ) MS/MS spectra of acacetin (from the precursor m / z 283.0610); ( D ) chromatographic peaks of dimeric acacetin when the formula [C 32 H 22 O 10 -H] − was extracted; ( E ) MS spectra of dimeric acacetin; ( F ) MS/MS spectra of dimeric acacetin (from the precursor m / z 565.1129); ( G ) chromatographic peaks of authentic isoginkgetin when the formula [C 32 H 22 O 10 -H] − was extracted; ( H ) MS spectra of authentic isoginkgetin; and ( I ) MS/MS spectra of authentic isoginkgetin (from the precursor m / z 565.1125).

Article Snippet: Acacetin (C 16 H 12 O 5 , CAS number 480-44-4, MW 284.2, purity 98%; ) and isoginkgetin (C 32 H 22 O 10 , CAS number 548-19-6, MW 566.5, purity 98%; ) were obtained from Chengdu Biopurify Phytochemicals, Ltd. (Chengdu, China).

Techniques: Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: Molecules

Article Title: 3′,8″-Dimerization Enhances the Antioxidant Capacity of Flavonoids: Evidence from Acacetin and Isoginkgetin

doi: 10.3390/molecules24112039

Figure Lengend Snippet: Proposed MS elucidation of acacetin dimer ( A ) and isoginkgetin ( B ).

Article Snippet: Acacetin (C 16 H 12 O 5 , CAS number 480-44-4, MW 284.2, purity 98%; ) and isoginkgetin (C 32 H 22 O 10 , CAS number 548-19-6, MW 566.5, purity 98%; ) were obtained from Chengdu Biopurify Phytochemicals, Ltd. (Chengdu, China).

Techniques:

Journal: bioRxiv

Article Title: Unraveling antiviral efficacy of multifunctional immunomodulatory triterpenoids against SARS-COV-2 targeting virus-specific enzymes

doi: 10.1101/2023.06.24.546363

Figure Lengend Snippet: (a) Chemical structures of triterpenoids. (b, c, d) The inhibitory potencies of azadirachtin, withanolide_A, and isoginkgetin against the enzymatic activity of Mpro. (e, f, g) The inhibitory potencies of azadirachtin, withanolide_A, and isoginkgetin against the enzymatic activity of PLpro. Dose–response curves for IC 50 values were determined by nonlinear regression fit. The calculated enzymatic activity with each compound was normalized to control. All data are shown as mean ± standard error, n = 2 biological replicates.

Article Snippet: Isoginkgetin was dissolved in Dimethyl sulfoxide (DMSO; Himedia, India) at 10 mM concentration.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Unraveling antiviral efficacy of multifunctional immunomodulatory triterpenoids against SARS-COV-2 targeting virus-specific enzymes

doi: 10.1101/2023.06.24.546363

Figure Lengend Snippet: Binding analysis of azadirachtin, withanolide_A, and isoginkgetin with SARS-CoV-2 Mpro and PLpro characterized by ITC. The top of each ITC titration is its raw data and the bottom represents the binding isotherm fitted to one sets of site model. K D values are inset.

Article Snippet: Isoginkgetin was dissolved in Dimethyl sulfoxide (DMSO; Himedia, India) at 10 mM concentration.

Techniques: Binding Assay, Titration