Journal: Redox biology

Article Title: The absence of thioredoxin-interacting protein in alveolar cells exacerbates asthma during obesity.

doi: 10.1016/j.redox.2024.103193

Figure Lengend Snippet: Fig. 3. Thioredoxin-interacting protein (TXNIP) expression in asthma pathogenesis. (A) Immunohistochemical staining for TXNIP in lung sections from human healthy control and asthma patient (Scale bar = 80 μm) and quantification graph of TXNIP positive area. TXNIP expression is increased in asthma patients, con firming its involvement in the pathogenesis of asthma. Values: mean ± standard deviation (SD) (n = 5 field per group). Significance: **p < 0.01 vs Control by two- tailed t-test. (B) Immunohistochemical staining (Scale bar = 30 μm) and quantification graph of TXNIP positive area and (C) immunofluorescence staining (Scale bar = 30 μm) for TXNIP in lung sections from normal diet (ND)-fed mice and high fat diet (HFD)-fed mice induced with asthma using a protease from Aspergillus oryzae. Lung tissues were stained with TXNIP (green) and DAPI (blue). TXNIP expression in lung tissue was observed to be higher in HFD asthmatic mice than ND asthmatic mice. Values: mean ± SD (n = 7 per group). Significance: *p < 0.05 vs ND Asthma by two-tailed t-test. (D), (E) Western blot analysis of the indicated proteins in the lung tissue and graphs representing the densitometric values of protein expression. NLRP3, NOD-like receptor family pyrin domain containing 3; ASC, apoptosis- associated speck-like protein containing a caspase-recruitment domain; IL-1β, interleukin-1β. Values: mean ± SD (n = 4 per group). Significance: *,**p < 0.05 and 0.01 vs ND Asthma by two-tailed t-test, respectively.

Article Snippet: The primary antibodies used were as follows: TXNIP (Novus Biologicals, Littleton, CO, USA, 1:1000 dilution), NOD-like receptor family pyrin domain containing 3 (NLRP3; Bioss Inc., Woburn, MA, USA, 1:1000 dilution), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC; Novus Biologicals, 1:1000 dilution), Caspase 1 (full length) (Novus Biologicals, 1:1000 dilution), Cleaved Caspase 1 (Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), Pro IL-1β (Novus Biologicals, 1:1000 dilution), and Cleaved IL-1β (Cell Signaling Technology, 1:1000 dilution).

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Standard Deviation, Two Tailed Test, Immunofluorescence, Western Blot

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Schematic graph of animal sensitization and challenge. B Histological analysis of lung tissues stained with HE staining or PAS staining. C Histology score of HE staining and PAS staining. Statistical significance was determined with Mann-Whitney test. D mRNA expression of Bhlhe40 was assayed by RT-qPCR. Statistical analysis was performed by two-tailed Student’s t-test. E Western blot analysis of BHLBE40 in lung tissue. Statistical analysis was performed by two-tailed Student’s t-test. F Immunohistochemistry staining was performed to detected the level of BHLHE40 in lung tissue. Statistical analysis was performed by two-tailed Student’s t-test. G Double immunofluorescence staining for BHLHE40 (red) and F4/80 (green) in lung tissues. The number of double positive (F4/80 + BHLHE40 + ) cells was quantified. Statistical analysis was performed by two-tailed Student’s t-test. Error bars represent standard deviation. N = 8 biological replicates.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Staining, MANN-WHITNEY, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Immunohistochemistry, Double Immunofluorescence Staining, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Mice in asthma group received lentiviral delivery of LV- shBhlhe40 or LV-shNC. B RT-qPCR tested the mRNA expression of Bhlhe40 in lung tissues. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. C Representative western blot and quantitative analysis of BHLHE40 expression in lung tissues. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. D Representative HE images in lung tissue sections and quantitative analysis of histologic scoring. Statistical analysis was performed by Kruskal-Wallis test. E Representative PAS images in lung tissue sections and quantitative analysis of histologic scoring. Statistical analysis was performed by Kruskal-Wallis test. F Total protein concentrations in BALF in OVA-induced asthma. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation. N = 8 biological replicates.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A ELISA forYM1 in BALF. B – E RT-qPCR assay showed the mRNA expression of Ym1 , Fizz1 , Cd206 , Arg1 in lung tissues. F The protein level of CD206 and ARG1 in lung tissues. G Quantitative analysis of CD206 protein level. H Quantitative analysis of ARG1 protein level. I Western blot assessed the level of BHLHE40, CD206, and ARG1 in monocyte-derived alveolar macrophages (Mo-AMs). J Quantitative analysis of BHLHE40 protein level in Mo-AMs. K Quantitative analysis of CD206 protein level in Mo-AMs. L Quantitative analysis of ARG1 protein level in Mo-AMs. N = 8 biological replicates. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot, Derivative Assay, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Bone marrow derived macrophages (BMDMs) were isolated from BALB/c mice, and then stimulated with IL-4 to induce M1 polarization. Lentivirus infection was conducted before IL-4 treatment. B The expression level of Bhlhe40 was determined by RT-qPCR. C Western blot analysis of BHLBE40 in BMDMs. D – G The mRNA expression level of Ym1 , Fizz1 , Cd206 , and Arg1 in BMDMs. H The protein level of CD206 and ARG1 in BMDMs. I Immunofluorescence analysis of ARG1 expression in BMDMs, and the quantification of ARG1 positive cell count. N = 4 biological replicates. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Derivative Assay, Isolation, Infection, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Cell Counting, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A , B Nrtn expression level was determined by RT-qPCR and western blot. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. C Dual-luciferase reporter assay. N = 3 biological replicates. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. D Validation by ChIP-qPCR analysis of BHLHE40 binding to Nrtn in BMDMs. Statistical analysis was performed by two-tailed Student’s t-test. E RT-qPCR showed the mRNA expression of Fizz1 , Ym1 , Arg1 , and Cd206 . Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. F The protein level of CD206 and ARG1 was assayed by western blot. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. G Immunofluorescence analysis of ARG1 expression in BMDMs, and the quantification of ARG1 positive cell count. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation. N = 3-4 biological replicates.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay, Biomarker Discovery, ChIP-qPCR, Binding Assay, Two Tailed Test, Immunofluorescence, Cell Counting, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Mice were stimulated with thioglycolate and IL-4 by intraperitoneal injection, and then physiological peritoneal macrophages were obtained. B Validation by ChIP-qPCR analysis of BHLHE40 binding to Nrtn in peritoneal macrophages. Statistical analysis was performed by two-tailed Student’s t-test. C RT-qPCR examined the mRNA level of Bhlhe40 in peritoneal macrophages. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. D Representative immunoblot and quantitative analysis of BHLHE40 expression. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. E Immunofluorescence analysis of ARG1 expression in peritoneal macrophages, and the quantification of ARG1 positive cell count. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. F Peritoneal macrophages were infected with LV- shBhlhe40 and LV- shNrtn , and then were subjected to immunofluorescence staining for ARG1. The number of ARG1 positive cells was quantified. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation. N = 4 biological replicates.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Injection, Biomarker Discovery, ChIP-qPCR, Binding Assay, Two Tailed Test, Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Cell Counting, Infection, Staining, Standard Deviation

Journal: medRxiv

Article Title: Inactivating PLEKHA6 Mutations Cause Idiopathic Hypogonadotropic Hypogonadism Through Impaired Kisspeptin Secretion

doi: 10.64898/2026.04.10.26349358

Figure Lengend Snippet: Upper panel: RT-PCR of fresh-frozen human hypothalamic tissue demonstrates expression of PLEKHA6 alongside established KN Dy neuronal markers, including kisspeptin (KISS1), neurokinin B (TAC3), neurokinin B receptor (TACR3), and prodynorphin (PDYN), confirming transcriptional expression of PLEKHA6 within the KN Dy neuronal population. Lower panel: Representative confocal immunofluorescence images of fresh-frozen postmortem human hypothalamic tissue acquired using an Olympus FV3000 confocal microscope (60× objective). (A) DAPI nuclear counterstain, (B) Neurokinin B, (C) PLEKHA6, and (D) Merged image. The merged image demonstrates colocalization of PLEKHA6 and Neurokinin B signals. Images are representative of n = 3 subjects.

Article Snippet: Sections were fixed, permeabilized, blocked, and incubated with primary antibodies against PLEKHA6 (Rat RtSzr127, a gift from Dr. Sandra Citi) and neurokinin B (Rabbit NB300-201SS, Novus).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence, Microscopy