Journal: International journal of molecular sciences

Article Title: Isocitrate Dehydrogenase Alpha-1 Modulates Lifespan and Oxidative Stress Tolerance in Caenorhabditis elegans .

doi: 10.3390/ijms24010612

Figure Lengend Snippet: Figure 6. Expression of idha-1 negatively modulates the phosphorylated S6K levels. The level of phosphorylated S6K (p-S6K), a TOR activity indicator, in each strain was determined and normal- ized with β-actin level. (a,b) Long-lived idha-1 overexpression strain (red bar, idha-1 o/e) displays significantly reduced p-S6K levels compared with the control (grey bar) (** p < 0.01, t test). Each bar represents mean ± SD, n = 4. (c,d) Short-lived idha-1 knockdown strains (blue bar, N2 idha-1 (RNAi) 5′; and yellow bar, N2 idha-1 (RNAi) 3′) exhibit significantly increased p-S6K levels compared with the control (black bar, N2 EV) (* p < 0.05, t test). Each bar represents mean ± SD, n = 3.

Article Snippet: The primary antibodies used were listed below: IDHA-3A (Genetex, GTX114486), beta-actin (GeneTex, GTX109639), and p-S6K (Cell Signaling, Cat# 9209).

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

Journal: bioRxiv

Article Title: Functional characterization of eicosanoid signaling in Drosophila development

doi: 10.1101/2025.01.13.632770

Figure Lengend Snippet: (A) Unrooted maximum-likelihood phylogenetic tree of class A G protein-coupled receptors (GPCRs) in H. sapiens and D. melanogaster . GPCRs in D. melanogaster are indicated by blue branches, whereas branches for H. sapiens GPCRs are color-coded based on different classes of ligands. The scale bar indicates an evolutionary distance of 0.5 amino acid substitutions per site. Opsins and leucine-rich repeat containing GPCRs are not included in the phylogenetic tree. Accession numbers of the receptors analyzed are listed in S1 Table. (B) List of lipid mediator receptors in H. sapiens and their Drosophila orthologs. (C) Luminescence responses of S2 cells co-expressing Aequorin and G α 15 , along with CG7497/PGR or H. sapiens PGE2 receptor EP2 ( HsEP2 ), to prostanoids or their synthetic analogs. Relative luminescence normalized by luminescence in ligand-free controls is shown. Cells expressing CG7497/PGR were significantly activated by all ligands, whereas cells expressing EP2 were activated only by PGE2. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 (Dunnett’s test vs mock treatment control).

Article Snippet: Early pupae (up to 24 h APF) were collected and kept in Drosophila narrow vials (Genesee Scientific) containing wet tissue at 25°C under 12 h-light and 12 h-dark photoperiod.

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Functional characterization of eicosanoid signaling in Drosophila development

doi: 10.1101/2025.01.13.632770

Figure Lengend Snippet: (A) Schematic diagram of prostanoid synthetic pathways. Multiple prostanoids are synthesized from arachidonic acid (AA) by different enzymes. (B) Drosophila enzymes orthologous to human prostanoid synthases were identified by phylogenetic analyses. Phylogenetic trees are shown in S4–S10 Figs. PGD, PGE, and PGF synthases are highly conserved in insects, although D. melanogaster lacks some of them. (C) Prostanoids detected in whole body extracts of pupae by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics. AA was detected in intact pupae at 0–4 hours after pupation, whereas prostanoids were undetectable. PGD2, PGE2, and PGF2α were detected in pupae that were raised in AA-supplemented diet during larval development. Data shown are mean total amount of eicosanoids in the whole body per animal ± SEM. n = 4–5.

Article Snippet: Early pupae (up to 24 h APF) were collected and kept in Drosophila narrow vials (Genesee Scientific) containing wet tissue at 25°C under 12 h-light and 12 h-dark photoperiod.

Techniques: Synthesized, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Functional characterization of eicosanoid signaling in Drosophila development

doi: 10.1101/2025.01.13.632770

Figure Lengend Snippet: (A) Developmental phenotype of PGD and PGE synthase ortholog mutants. Null mutant of cPges / p23 and triple mutants of Mgst - , CG33177 , and CG33178 died during pupa-adult development. Multiple comparison Chi-square test with Bonferroni correction was applied among cPges / p23 mutants including the heterozygous control. *** p < 0.001. (B) Representative images of cPges / p23 knockout flies. About 70% of flies died before wing pigmentation, whereas ∼30% of flies died as pharate adults. Scale bar, 200 µm. (C, D) Relative expression levels of hypoxia response genes, Ldh (C) and Hph (D), in the PGD and PGE synthase ortholog mutants. The cPges / p23 mutant showed high expression of hypoxia response genes at 24 and 48 hours after puparium formation (APF). Expression levels are normalized by the levels of a reference gene, rp49 , in the same cDNA samples and shown as relative to 0 hours APF of GstS1 - . n = 3–4. p < 0.001 (Tukey’s test). (E) Rescue of the cPges / p23 mutant by Gal4 -driven expression of either cPges / p23 or human prostaglandin E synthase 3 ( HsPTGES3 ). The lethal phenotype was rescued by overexpressing cPges / p23 or HsPTGES3 either ubiquitously ( tubP-Gal4 ), in PGR expression sites ( PGR-Gal4 ), in tracheal cells ( btl-Gal4 ), or in the epidermis ( Eip71CD-Gal4 ). ** p < 0.01, *** p < 0.001 (Chi-square test vs + > cPges / p23 or + > HsPTGES3 with Bonferroni correction). (F) Developmental deficiency caused by cPges / p23 RNAi. cPges / p23 knockdown flies died during pupa-adult development when induced using either a ubiquitous driver (tubP-Gal4 ) or a combination of the tracheal cell ( btl-Gal4 ) and epidermis ( Eip71CD-Gal4 ) drivers. Lethality caused by cPges / p23 RNAi in tracheal cells and epidermis was significantly rescued by 40% O 2 . *** p < 0.001 (multiple comparison Chi-square test vs + > cPges / p23 RNAi with Bonferroni correction, or 20% O 2 vs 40% O 2 ). (G) PGE2 conversion assay using Drosophila S2 cells. Arachidonic acid (AA) was added to the culture medium of S2 cells transfected with cPges / p23 or HsPTGES3 along with a H. sapiens cyclooxygenase ( HsCOX1 or HsCOX2 ), and PGE2 amount in the medium was determined. The basal levels of PGE2 immunoactivity in all samples is likely due to the cross-reactivity of the antibody used in the assay. Increased PGE2 immunoreactivity was detected in cyclooxygenase -expressing cells regardless of cPges / p23 or HsPTGES3 transfection. n = 4. (H) Reduction of the PGE2 synthetic activity of S2 cells by knocking down endogenous cPges / p23 expression. AA was added to the culture medium of S2 cells transfected with HsCOX2 and treated with cPges / p23 dsRNAs. PGE2 amount in the medium was determined. n = 2 (AA 0 µM) or n = 4 (AA 10 µM). *** p < 0.001 (Dunnett’s test vs EGFP dsRNA-treated cells).

Article Snippet: Early pupae (up to 24 h APF) were collected and kept in Drosophila narrow vials (Genesee Scientific) containing wet tissue at 25°C under 12 h-light and 12 h-dark photoperiod.

Techniques: Mutagenesis, Comparison, Control, Knock-Out, Expressing, Knockdown, Transfection, Activity Assay

Journal: Nature Communications

Article Title: Colonic epithelial-derived FGF1 drives intestinal stem cell commitment toward goblet cells to suppress inflammatory bowel disease

doi: 10.1038/s41467-025-58644-2

Figure Lengend Snippet: A Heatmap of differentially expressed Atoh1 and its downstream target genes in PBS and rFGF1-treated UC mice ( n = 4). The mRNA level of Atoh1 in PBS or rFGF1-treated UC mice (upper panel, n = 4) and CD mice (lower panel, n = 5) was detected by qRT-PCR. B , C IF staining of ATOH1 in distal colon sections of Fgf1 fl/fl and VilCre Fgf1 fl/fl mice challenged with DSS ( B ) or TNBS ( C ), and its semi-quantitation of IF intensity ( n = 4, the mean value of 2 fields in each mouse). D , E IF staining of ATOH1 in distal colon sections of DSS ( D ) or TNBS ( E )-induced two IBD mouse models, followed by PBS or rFGF1 treatment and its semi-quantitation of IF intensity ( n = 4, the mean value of 2 fields in each mouse). F Representative images (upper panel) and IF staining of Muc2 (lower panel) in sh Atoh1 or shNC-transfected colonic organoids stimulated with vehicle or rFGF1 ( n = 6). G , H Deletion of Atoh1 in VilCre ERT2 Atoh1 fl/fl mice was confirmed by qRT-PCR ( G ) and IF staining of colon tissues ( H ) ( n = 4). I – M VilCre ERT2 Atoh1 fl/fl mice were injected with tamoxifen for five consecutive days and then given drinking water containing 1.5% DSS to induce acute colitis, followed by vehicle or rFGF1 administration for 7 days. At end of the experiment, the distal colon tissues were harvested and examined. The schematic diagram shows the strategy of tamoxifen injection, DSS challenge and rFGF1 administration ( n = 4). J – L Weight loss ( J ), disease activity index ( K ), colonic length ( L ) were monitored (n = 4). M H&E (upper panel), PAS-AB staining (middle panel) and Muc2 IF staining (lower panel) of distal colon sections ( n = 4). Data was presented as mean ± SEM. ( A , D – E , H ) two-tailed unpaired t -test; ( B , C , F , J – M ) ordinary two-way ANOVA, followed by Sidak; ( G ) Non-parametric statistical method, two-tailed Mann-Whitney test. ns, not significance; nd, not detectable.

Article Snippet: The primary antibodies are listed below: rabbit anti-FGF1 (1:200, 17400, Proteintech), mouse anti-FGF1 (1:100, sc-55520, Santa Cruz Biotechnology), EpCAM (1:800, CST42515, Cell Signaling Technology), Col1α1 (1:100, CST72026, Cell Signaling Technology), ATOH1 (1:400, 21215-1-AP, Proteintech), TCF4 (1:100, ab217668, Abcam), Muc2 (1:400, ab272692, Abcam), Dclk1 (1:300, CST62257, Cell Signaling Technology), ChgA (1:200, sc-393941, Santa Cruz Biotechnology), Fabp1 (1:50, CST13368, Cell Signaling Technology), Ki67 (1:400, ab15580, Abcam), FGFR2 (1:200, CST23328S, Cell Signaling Technology) and Lgr5 (1:100, UM870104, ORIGENE).

Techniques: Quantitative RT-PCR, Staining, Quantitation Assay, Transfection, Injection, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Colonic epithelial-derived FGF1 drives intestinal stem cell commitment toward goblet cells to suppress inflammatory bowel disease

doi: 10.1038/s41467-025-58644-2

Figure Lengend Snippet: A Workflow to find the rFGF1-regulated potential transcription factors of Atoh1 , and heatmap of expression level of potential transcription factors ( n = 4). B The expression levels of potential transcription factors of Atoh1 ( Tcf4 , Nr4a1 , Nfya , Tec , Hoxa13 , Ncaph2 , Kdm1a ) in distal colon sections of PBS or rFGF1-treated UC mice were analyzed by qRT-PCR ( n = 4). C , D IF staining of TCF4 in distal colon sections of Fgf1 fl/fl and VilCre Fgf1 fl/fl mice challenged with DSS ( C ) or TNBS ( D ) and its semi-quantitation of IF intensity ( n = 4, the mean value of 2 fields in each mouse). E , F The expression levels of TCF4 in distal colon sections of DSS-treated UC mice ( E ) and TNBS-treated CD mice ( F ) after PBS or rFGF1 administration and IF staining ( n = 8). G Predicted TCF4 binding sites on the promoter of Atoh1 by the JASPAR database (left panel). Six binding sites with the most significant prediction scores were selected for pairwise deletion mutation analysis (right panel; M1, in purple; M2, in green; M3, in red). H , I Luciferase activity in HT-29 cells transfected with a human Atoh1 promoter reporter plasmid or the predicted TCF4 binding site mutant reporter along with a Tcf4 overexpression plasmid ( n = 4). J ChIP experiments. The data were normalized to the corresponding IgG ( n = 4). K Images of murine colonic organoids transfected small hairpin RNA targeting Tcf4 (sh Tcf4 ) or negative control hairpin (shNT) followed by treated with rFGF1 or vehicle ( n = 6). L IF staining of ATOH1 (upper panel) and Muc2 (lower panel) in colonic organoids in different groups and its semi-quantitation of fluorescent intensity ( n = 6). Data was presented as mean ± SEM. ( B , right panel of E – F , J ) two-tailed unpaired t -test ( Tec and Ncaph2 in penal B using non-parametric statistical method, two-tailed Mann-Whitney Test); ( H – I ) ordinary one-way ANOVA, followed by Dunnett; (right panel of C – D and L , lower panel of K ) ordinary two-way ANOVA, followed by Sidak. ns, no significance.

Article Snippet: The primary antibodies are listed below: rabbit anti-FGF1 (1:200, 17400, Proteintech), mouse anti-FGF1 (1:100, sc-55520, Santa Cruz Biotechnology), EpCAM (1:800, CST42515, Cell Signaling Technology), Col1α1 (1:100, CST72026, Cell Signaling Technology), ATOH1 (1:400, 21215-1-AP, Proteintech), TCF4 (1:100, ab217668, Abcam), Muc2 (1:400, ab272692, Abcam), Dclk1 (1:300, CST62257, Cell Signaling Technology), ChgA (1:200, sc-393941, Santa Cruz Biotechnology), Fabp1 (1:50, CST13368, Cell Signaling Technology), Ki67 (1:400, ab15580, Abcam), FGFR2 (1:200, CST23328S, Cell Signaling Technology) and Lgr5 (1:100, UM870104, ORIGENE).

Techniques: Expressing, Quantitative RT-PCR, Staining, Quantitation Assay, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression, Negative Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Colonic epithelial-derived FGF1 drives intestinal stem cell commitment toward goblet cells to suppress inflammatory bowel disease

doi: 10.1038/s41467-025-58644-2

Figure Lengend Snippet: A Single-cell RNA sequencing analysis (GSM4983265) showed that Fgfr2 is highly expressed in stem cells. B Representative image of colon distal stained with Lgr5 (green), FGFR2 (red) and DAPI (blue) from the C57BL/6 J mice ( n = 3). C Deletion of Fgfr2 in Lgr5-EGFP-Cre ERT2 Fgfr2 fl/fl mice treated with vehicle or tamoxifen was confirmed by IF staining ( n = 5). D Murine colonic organoids derived from vehicle or tamoxifen-treated Lgr5-EGFP-Cre ERT2 Fgfr2 fl/fl mice were stimulated with vehicle or rFGF1. Representative images, IF staining of TCF4, ATOH1 and Muc2 in colonic organoids were shown (n = 6). E – I Vehicle or tamoxifen-treated Lgr5-EGFP-Cre ERT2 Fgfr2 fl/fl mice were challenged with drinking water containing 2.5% DSS to induce UC, followed by colon tissues were harvested and examined (n = 4 for TAM + PBS group, n = 5 for other groups). Weight loss ( E ), disease activity index ( F ) and colon length ( G ) of different groups were monitored. H H&E staining of distal colon sections and histology scores (upper panel), PAS-AB staining of distal colon sections and mucin granules-positive cells per crypt (lower panel). I IF staining of TCF4 (upper panel), ATOH1 (middle panel) and Muc2 (lower panel) in distal colon sections, and its semi-quantification of IF intensity. Data was presented as mean ± SEM. (right panel of C ) two-tailed unpaired t -test; (right panel of D – F , right panel of G – I ) ordinary two-way ANOVA, followed by Sidak. ns, no significance.

Article Snippet: The primary antibodies are listed below: rabbit anti-FGF1 (1:200, 17400, Proteintech), mouse anti-FGF1 (1:100, sc-55520, Santa Cruz Biotechnology), EpCAM (1:800, CST42515, Cell Signaling Technology), Col1α1 (1:100, CST72026, Cell Signaling Technology), ATOH1 (1:400, 21215-1-AP, Proteintech), TCF4 (1:100, ab217668, Abcam), Muc2 (1:400, ab272692, Abcam), Dclk1 (1:300, CST62257, Cell Signaling Technology), ChgA (1:200, sc-393941, Santa Cruz Biotechnology), Fabp1 (1:50, CST13368, Cell Signaling Technology), Ki67 (1:400, ab15580, Abcam), FGFR2 (1:200, CST23328S, Cell Signaling Technology) and Lgr5 (1:100, UM870104, ORIGENE).

Techniques: RNA Sequencing, Staining, Derivative Assay, Activity Assay, Two Tailed Test