Journal: EMBO Reports

Article Title: An insulin receptor activity surge in follicle cells drives vitellogenesis by upregulating CrebA

doi: 10.1038/s44319-025-00672-6

Figure Lengend Snippet: ( A ) Transcriptional regulons that were specifically switched off in follicle cells at State #3. Black and white bars represent cells in which the corresponding regulons were on or off (Fig. for all the active regulons in stages 8–10B follicle cells). ( B ) Single-follicle cell mRNA expression of the four HSD-deactivated transcription factors identified in ( A ). ( C ) Immunostaining with anti-CrebA (left) or anti-β-Gal (right) in egg chambers from females of w1118 or l(3)3576 (a lacZ reporter inserted in the first intron of CrebA ), respectively. This reporter line expresses β-gal in the majority of the tissues that normally express CrebA, including follicle cells at vitellogenic stages, and meanwhile disrupts the expression of CrebA from its locus (Andrew et al, ; Rose et al, ). While the line might not fully report all aspects of CrebA in wild-type ovaries, the immunostaining patterns of CrebA and β-Gal resembled each other in the ovaries. Scale bars = 50 µm. ( D ) Western blot analysis with anti-β-Gal in ovaries from l(3)3576 females under ND and HSD, respectively. The result was reproduced in three independent replicate experiments. The anti-CrebA antibody failed to work in Western blot experiments. ( E ) Single-follicle cell mRNA expression of four target genes in the CrebA-mediated regulon: Yp1 , Yp2 , Vm26Aa , and Vm26Ab . ( F ) Ovarian bulk RNA-seq confirms reduced expression of the 629 target genes (with DESeq2-computed Wald test p values <0.05) in the CrebA-mediated regulon by HSD, including CrebA , Yp1 , Yp2 , Vm26Aa , and Vm26Ab . ( G ) RT-qPCR confirms the reduced ovarian levels of CrebA , Yp1 , and Vm26Aa mRNA by HSD. For each experimental group, seven pairs of ovaries were pooled as one sample and three biological samples were used. Error bars represent mean ± SEM. Student’s t -test p = 0.04 (denoted as *), 0.002 (**), and 0.002 (**), respectively. .

Article Snippet: CrebA Rbt-PC , Developmental Studies Hybridoma Bank , AB_10805295.

Techniques: Expressing, Immunostaining, Western Blot, RNA Sequencing, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: An insulin receptor activity surge in follicle cells drives vitellogenesis by upregulating CrebA

doi: 10.1038/s44319-025-00672-6

Figure Lengend Snippet: ( A ) Top ten GO terms enriched with CrebA-regulon genes. ( B ) RT-qPCR analysis of the ovarian mRNA levels of CrebA , Yp1 , and Vm26Aa in tj > CrebA RNAi-1 and tj > CrebA RNAi-2 females. For each genotype, seven pairs of ovaries were sampled. Data were presented as mean ± SEM ( N = 3 biological replicates for each measurement). Error bars represent mean ± SEM. One-way ANOVA was performed to compare either RNAi line with the control line, and all p values <0.0001 (denoted as ****). .

Article Snippet: CrebA Rbt-PC , Developmental Studies Hybridoma Bank , AB_10805295.

Techniques: Quantitative RT-PCR, Control

Journal: EMBO Reports

Article Title: An insulin receptor activity surge in follicle cells drives vitellogenesis by upregulating CrebA

doi: 10.1038/s44319-025-00672-6

Figure Lengend Snippet: ( A ) Fecundity of tj > +, tj > CrebA RNAi-1 , tj > CrebA RNAi-2 , and tj > CrebB RNAi females on days 4–6 after eclosion. N = 4 biological replicates for each genotype. Error bars represent mean ± SEM. One-way ANOVA p < 0.0001 (denoted as ****), < 0.0001 (****), and =0.73 (denoted as ns) from left to right, respectively. ( B ) Distributions of egg chambers at different stages from tj > +, tj > CrebA RNAi-1 , and tj > CrebA RNAi-2 females. N = 6, 4, and 5 females, respectively. ( C ) A scatter plot showing good correlation of transcriptomic changes between tj > CrebA RNAi-1 and tj > CrebA RNAi-2 ovaries. Pearson’s correlation was computed based on 9436 genes that were expressed in all the samples: correlation coefficient R = 0.855 and p value is approaching 0. CrebA , Yp1 , Yp2 , Vm26Aa , and Vm26Ab were highlighted. ( D ) Top 10 GO enriched with genes downregulated by tj>CrebA RNAi-1 . ( E ) Representative images showing reduced levels of Yp1-GFP (green; DAPI, magenta) in follicle cells at stage 9 and oocytes at stage 12 from tj > Yp1-GFP ; CrebA RNAi-1 ovaries. Scale bars = 50 µm. ( F ) Representative transmission electron microscope (TEM) images showing aberrance in eggshell structure of the most developed egg chambers from tj > CrebA RNAi-1 and tj > CrebA RNAi-2 females. Scale bars = 2 µm. The major layers from the exterior of the egg to the interior are follicle cells (fc), chorionic layers (ch), vitelline membrane (vm), and the oocyte (oc). We note two common phenotypes: (1) missing periodic cavities (cv), and (2) thinner vitelline membrane (vm). For quantification of vm thickness, each genotype was measured in N = 5 samples prepared from different egg chambers. Error bars represent mean ± SEM. One-way ANOVA p values <0.0001 (denoted as ****) for both RNAi strains. .

Article Snippet: CrebA Rbt-PC , Developmental Studies Hybridoma Bank , AB_10805295.

Techniques: Transmission Assay, Microscopy, Membrane

Journal: EMBO Reports

Article Title: An insulin receptor activity surge in follicle cells drives vitellogenesis by upregulating CrebA

doi: 10.1038/s44319-025-00672-6

Figure Lengend Snippet: ( A ) Representative images of Yp1-GFP (green) in egg chambers at different stages from Yp1-GFP / CyO females. Nuclei were counterstained with DAPI (magenta). Scale bars = 50 µm. ( B ) Representative images of Yp1-GFP in egg chambers at different stages from Yp1-GFP / tj-Gal4;UAS-CrebA/+ females. Scale bars = 50 µm. ( C ) Representative images of Yp1-GFP in egg chambers at different stages from Yp1-GFP / tj-Gal4;UAS-CrebA/+ females. Scale bars = 50 µm. We note that, in stretched cells where both CrebA and Yp1-GFP are normally low, ectopic expression of CrebA significantly increased Yp1-GFP. .

Article Snippet: CrebA Rbt-PC , Developmental Studies Hybridoma Bank , AB_10805295.

Techniques: Expressing

Journal: EMBO Reports

Article Title: An insulin receptor activity surge in follicle cells drives vitellogenesis by upregulating CrebA

doi: 10.1038/s44319-025-00672-6

Figure Lengend Snippet: ( A ) Female fecundity measured for tj > + under ND, tj > + under HSD, tj > CrebA under ND and tj > CrebA under HSD on days 7–9 after eclosion. N = 4 biological replicates for each group. Error bars represent mean ± SEM. One-way ANOVA p values = 0.0001 (denoted as ***), <0.0001 (****) and =0.04 (*) for the marks from left to right, respectively. ( B ) Fecundity measured for tj > +, tj > InR DN , tj > CrebA and tj > InR DN ; CrebA females on days 4–6 after eclosion. N = 9, 10, 8, and 6 biological replicates, respectively. Error bars represent mean ± SEM. One-way ANOVA, all p values <0.0001 (denoted as ****). ( C ) A scatter plot shows a negative correlation between the HSD-to-ND fold changes and the rescue-to-control ( tj > CrebA vs. tj > +) fold changes in ovarian bulk RNA-seq. Pearson’s correlation was computed based on 9810 genes that were expressed in all the samples: R = −0.402, and p value is approaching 0. CrebA , Yp1 , Yp2 , Vm26Aa , Vm26Ab , and 6 outlier genes were highlighted. ( D ) RT-qPCR measurements of CrebA , Yp1 , and Vm26Aa mRNAs in ovaries from tj > +, tj > InR DN , tj > CrebA and tj > InR DN ; CrebA females. Yp1-GFP was also present in these flies. N = 3 biological replicates. Error bars represent mean ± SEM. One-way ANOVA p = 0.03 (*), 0.001 (**) and 0.02 (*) for CrebA ; p = 0.04 (*), <0.0001 (****), and 0.01 (*) for Yp1 ; p = 0.02 (*), 0.001 (**), and 0.04 (*) for Vm26Aa . ( E , F ) Representative images showing that the levels of Yp1-GFP (green; DAPI, magenta) in stage-9 follicle cells and oocytes are reduced by HSD ( E ) or by tj > InR DN ( F ) and can be recovered by tj > CrebA . Scale bars = 10 µm. ( G , H ) Representative TEM images showing that the vitelline membrane thickness of stage-14 egg chambers is reduced by HSD ( G ) or tj > InR DN ( H ) and can be recovered by tj > CrebA . The chorionic layer, the vitelline membrane and the oocyte are labeled as “ch”, “vm”, and “oc”. Scale bars = 2 µm. ( I ) Quantification of vitelline membrane thickness from TEM experiments in panels G and H. N = 5, 5, 3, 3, 3, and 4 biological replicates from left to right, respectively. Error bars represent mean ± SEM. One-way ANOVA p values = 0.0004 (***), 0.03 (*), 0.04 (*), 0.05 (*), <0.0001 (****), and 0.003 (**) for the significance marks, respectively. .

Article Snippet: CrebA Rbt-PC , Developmental Studies Hybridoma Bank , AB_10805295.

Techniques: Control, RNA Sequencing, Quantitative RT-PCR, Membrane, Labeling

Journal: EMBO Reports

Article Title: An insulin receptor activity surge in follicle cells drives vitellogenesis by upregulating CrebA

doi: 10.1038/s44319-025-00672-6

Figure Lengend Snippet: ( A ) Predicted FoxO binding sites, public ChIP-seq data and ChIP-qPCR primer designs at the CrebA gene locus. ( B ) Sequence logos of the consensus FoxO binding motif (JASPAR MA2236.1), and the two predicted 9mer sites (P1 and P1 in panel A ). ( C ) ChIP-qPCR fold enrichment measured using the two primer pairs of panel A in samples from tj > FoxO-GFP and tj > FoxO-GFP;InR DN ovaries. N = 3 biological replicates. Error bars represent mean ± SEM. Student’s t -test p = 0.02 (denoted as *) and 0.04 (*), respectively. ( D ) RT-qPCR measures the ovarian level of CrebA mRNA in tj > +, tj > FoxO-GFP and tj > FoxO-GFP;InR DN females. N = 3 biological replicates. Error bars represent mean ± SEM. One-way ANOVA p = 0.55 (denoted as ns) and < 0.0001 (****), respectively. ( E ) Representative images show the expression of FoxO-GFP (green) and CrebA (red) in main-body follicle cells at stages 8–11 from tj > FoxO-GFP females. Nuclei were counterstained with DAPI (blue). Scale bars = 20 µm. ( F ) Nuclear-to-cytoplasmic ratios of FoxO-GFP and CrebA proteins quantified from panel E. N = 15, 21, 20, and 15 follicle cells, respectively. Error bars represent mean ± SEM. One-way ANOVA was performed to compare each stage with stage 8. For CrebA, all p values <0.0001 (denoted as ****); for FoxO-GFP, p < 0.0001 (****), <0.0001 (****) and =0.58 (ns), respectively. ( G ) Representative images show the expression of FoxO-GFP (green) and CrebA (red) in follicle cells at stage 9 from tj > FoxO-GFP;InR DN females. Scale bar = 20 µm. .

Article Snippet: CrebA Rbt-PC , Developmental Studies Hybridoma Bank , AB_10805295.

Techniques: Binding Assay, ChIP-sequencing, ChIP-qPCR, Sequencing, Quantitative RT-PCR, Expressing

Journal: EMBO Reports

Article Title: An insulin receptor activity surge in follicle cells drives vitellogenesis by upregulating CrebA

doi: 10.1038/s44319-025-00672-6

Figure Lengend Snippet: ( A ) Alignment of the bZIP domains of CrebA, CREB3, and CREB3L1-4. Within this domain, CREB3 and CREB3L1-4 share 53.1, 60.9, 70.3, 54.7, and 51.6% identities with CrebA, respectively. ( B ) Phylogenetic tree based on the alignment of the bZIP domains. ( C ) RT-qPCR shows the increases of CREB3 , CREB3L1 and CREB3L2 mRNA in KGN cells treated with FOXO1 siRNAs. N = 3 biological replicates for each experimental group. Error bars represent mean ± SEM. One-way ANOVA p values = 0.005 (denoted as **), 0.01 (**), 0.02 (*), 0.047 (*), 0.93 (ns), 0.03 (*), 0.008 (**), and 0.0002 (***) for the significance marks from left to right respectively. ( D ) GO/KEGG terms enriched with genes that were downregulated in KGN cells treated with a CREB3L2 siRNA. ( E – G ) RT-qPCR, Western blot and ELISA analyses show that both CYP19A1 protein and 17-beta-estradiol (E2) were reduced by CREB3L2 siRNA treatments. N = 3 biological replicates for each experiment. Error bars represent mean ± SEM. One-way ANOVA p = 0.008 (**) and 0.009 (**) for qPCR, and p = 0.04 (*) and 0.04 (*) for ELISA, respectively. ( H – J ) RT-qPCR, Western blot and ELISA analyses show that knockdown of FOXO1 counteracted the effects of CREB3L2 siRNAs on CREB3L2 mRNA, CYP19A1 protein and E2 levels. N = 3 biological replicates for each experiment. Error bars represent mean ± SEM. One-way ANOVA p = 0.01 (*), 0.01 (*), 0.12 (ns), and 0.34 (ns) for qPCR, and p = 0.06 (ns) and 0.84 (ns) for ELISA, respectively. .

Article Snippet: CrebA Rbt-PC , Developmental Studies Hybridoma Bank , AB_10805295.

Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Journal of Dental Sciences

Article Title: Nuclear factor erythroid 2-related factor 2/heme oxygenase-1 activation by nattokinase reduces pro-inflammatory and matrix-degrading mediators in human gingival fibroblasts

doi: 10.1016/j.jds.2025.10.022

Figure Lengend Snippet: Nattokinase suppresses COX-2 and MMP-1 via Nrf2/HO-1 activation. Cells were pretreated with ML385 or Heme Oxygenase-1-IN-1 hydrochloride (HO-1i) for 1 h, with or without nattokinase, followed by PM exposure. (A) COX-2 protein expression, (B) PGE 2 release, and (C) MMP-1 release were examined. Cells were treated for different time points, and (D) HO-1 mRNA levels and (E) ARE-luciferase activity were examined. Data are presented as mean ± SD from at least three independent experiments. # P < 0.01 compared with cells treated with PM plus nattokinase (A–C). ∗ P < 0.05, # P < 0.01 compared with the control group (D and E). COX-2: cyclooxygenase-2; MMP-1: matrix metalloproteinase-1; Nrf2: nuclear factor erythroid 2-related factor 2; HO-1: heme oxygenase-1; PM: particulate matter; PGE 2 : prostaglandin E 2 ; HO-1i: heme oxygenase-1 inhibitor; ARE: antioxidant response element.

Article Snippet: The transcriptional activity of Nrf2 was assessed using the antioxidant response element (ARE) Reporter Kit for Nrf2 (BPS Bioscience, Cat. #60514).

Techniques: Activation Assay, Expressing, Luciferase, Activity Assay, Control

Journal: Journal of Dental Sciences

Article Title: Nuclear factor erythroid 2-related factor 2/heme oxygenase-1 activation by nattokinase reduces pro-inflammatory and matrix-degrading mediators in human gingival fibroblasts

doi: 10.1016/j.jds.2025.10.022

Figure Lengend Snippet: Schematic illustration of the proposed mechanism. PM stimulation activates multiple signaling pathways, including ROS generation, NADPH oxidase activation, and PI3K/Akt as well as MAPK cascades, leading to the upregulation of COX-2 expression and PGE 2 release, which in turn promote MMP-1 mRNA expression and protein release. Pretreatment with nattokinase attenuates these PM-induced responses through the induction of the Nrf2/HO-1 signaling pathway, thereby suppressing downstream inflammatory and matrix-degrading processes.

Article Snippet: The transcriptional activity of Nrf2 was assessed using the antioxidant response element (ARE) Reporter Kit for Nrf2 (BPS Bioscience, Cat. #60514).

Techniques: Protein-Protein interactions, Activation Assay, Expressing