Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a Experimental strategy to suppress β-catenin in vitro by employing adipocytes differentiated from SVFs of 4-week-old wild-type mouse iWAT. b Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), MSAB, Rosiglitazone, or MSAB plus Rosiglitazone ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). c mRNA expression of thermogenic genes in ( b ) ( n = 3 biological replicates). d Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( b ) ( n = 3 biological replicates). e Experimental strategy to suppress β-catenin while inhibiting the Ca²⁺ pathway in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. f Microscopy images of Rosiglitazone-differentiated adipocytes treated with vehicle (0.1% DMSO), MSAB, or MSAB plus BAPTA-AM ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). g mRNA expression of thermogenic genes in ( f ) ( n = 3 biological replicates). h Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( f ) ( n = 3 biological replicates). i OCR plots and measured maximal respiration levels across the three treatment groups (Vehicle, MSAB, MSAB and BAPTA-AM) ( n = 3 cells). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were unpaired two-sided Student’s t -tests or two-way ANOVA followed by Tukey’s multiple-comparisons test or one-way ANOVA with Tukey’s correction for multiple comparisons.

Article Snippet: For inhibition and activation experiments, once full adipogenic differentiation was confirmed, cells were treated with each of the following molecules for 4 days: MSAB (S6901,Selleck, 10 uM), BAPTA-AM (S7534, Selleck, 5 uM), Box5 (P1216, Selleck, 100 uM), Wnt5a recombinant protein (HY-P704313, MCE, 100 ng/mL) with the exception of Rosiglitazone (S2556, Selleck, 2 uM), which was added on the first day of adipogenic induction in SVFs.

Techniques: In Vitro, Microscopy, Staining, Labeling, Expressing, Western Blot

Journal: bioRxiv

Article Title: Wnts are endothelial cell-derived PKD1/PKD2-dependent autocrine/paracrine vasodilators

doi: 10.64898/2026.03.17.712518

Figure Lengend Snippet: (A) Representative RT-PCR showing that mRNA for PKD1 is present in isolated endothelial cells from tamoxifen-treated Pkd1 fl/fl but absent in isolated endothelial cells from tamoxifen-treated Pkd1 fl/fl :Cdh5(PAC)-CreERT2 mice. Representative of 5 independent experiments for each genotype. (B) Representative Western blots illustrating PKD1, PKD2, AT1, eNOS, Fzd-7 and actin proteins in mesenteric arteries of Pkd1 fl/fl and Pkd1 ecKO mice. (C) Mean data from experiments shown in panel B. Significance was assessed using Student t-tests, n=5 independent mesenteric arterial lysates for each genotype and each protein. (D) Immunofluorescence images (representative of three mesenteric arteries from three mice for each genotype) illustrating that PKD1 (Alexa Fluor 546) is abolished in endothelial cells of en face mesenteric arteries from Pkd1 ecKO mice. CD31 (Alexa Fluor 488) and DAPI are also shown. Scale bars, 50 μm. (E) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular Wnt9b (3 μg/ml) applied in continuous flow in pressurized (80 mmHg) mesenteric arteries from Pkd1 fl/fl and Pkd1 ecKO mice. (F) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular boiled Wnt9b (3 µg/ml) applied under continuous flow (10 dyn/cm 2 ) in pressurized (80 mmHg) mesenteric arteries from Pkd1 fl/fl and Pkd1 ecKO mice. (G) Mean data from experiments shown in panels E and F, and the modulation of flow and Wnt9b-mediated vasodilation by SRI37892 (Fzd-7 Inh, 5 µM). Significance was assessed using a two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=10 arteries from 7 mice of each genotype for flow, flow+ Wnt9b and Wnt9b. n=7 arteries from 5 mice for Fzd-7 Inh+flow and Fzd-7 Inh+flow+Wnt9b. n=5 arteries from 5 mice for each genotype for flow + boiled Wnt9b. (H) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular Wnt5a (3 µg/ml). (I) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular boiled Wnt5a (3 µg/ml). (J) Mean data from experiments shown in panels H and I and the modulation of flow and Wnt5a-induced vasodilation by SRI37892 (Fzd-7 Inh, 5 μM). Significance was assessed using two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=8 arteries from 6 mice from Pkd1 fl/fl for flow, flow+ Wnt5a. n=5 arteries from 5 mice from Pkd1 ecKO for flow, flow+ Wnt5a. n=5 arteries from 5 mice of each genotype for flow + boiled Wnt5a. n=8 arteries from 6 mice from Pkd1 fl/fl for flow+ Fzd-7 Inh and flow+ Wnt5a+ Fzd-7 Inh.

Article Snippet: Cells were cultured until confluence and were first exposed for 30 min at 37°C to either vehicle, BAPTA-AM, SRI37892 (MedChemExpress), NSC668036, (MedChemExpress) or SP600125 (MedChemExpress).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Wnts are endothelial cell-derived PKD1/PKD2-dependent autocrine/paracrine vasodilators

doi: 10.64898/2026.03.17.712518

Figure Lengend Snippet: (A) Wnt9b stimulates dilation in pressurized (80 mmHg) mesenteric arteries of Pkd1 fl/fl mice through eNOS and SK channel activation. Low flow (10 dyn/cm 2 ) was applied and used to introduce Wnt9b (3 µg/ml) into the lumen, after which flow was stopped and L-NNA (100 µM) or apamin (300 nM) were applied abluminally. (B) Mean data for experiments shown in panel A. Significance was assessed using one-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=16 arteries from 10 Pkd1 fl/fl mice for flow, flow+ Wnt9b and Wnt9b. n=8 arteries from 5 Pkd1 fl/fl mice for Wnt9b+L-NNA, and Wnt9b+Apamin.(C) Western blot illustrating p-eNOS (serine1176), total eNOS, and actin in segments of first- to fifth-order mesenteric arteries of Pkd1 fl/fl and Pkd1 ecKO mice. Representative of 5 independent experiments. (D) Mean data for p-eNOS/total eNOS from experiments in panel C. Significance was assessed using one-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=5 independent mesenteric arterial lysates from each genotype (E) Mean data illustrating the effect of Wnt9b (3 µg/ml) on total eNOS from experiments in panel C. Significance was assessed using Student t-tests. n=5 independent mesenteric arterial lysates from each genotype. (F) Western blot illustrating p-eNOS (serine 1176), total eNOS, and actin in endothelial cells and modulation by Wnt9b (900 ng/ml), BAPTA-AM (BAPTA, 5 µM), SRI37892 (Fzd-7 Inh, 2.5 µM) and NSC668036 (Dvl Inh, 10 µM). Representative of 8 independent experiments. (G) Mean data from experiments shown in panel F. Significance was assessed using one-way ANOVA with Holm-Sidak post hoc multiple comparisons test. (H) Mean data illustrating nitric oxide generation from endothelial cells and modulation by Wnt9b (900 ng/ml), BAPTA-AM (BAPTA, 5 µM), SRI37892 (Fzd-7 Inh, 2.5 µM) and NSC668036 (Dvl Inh, 10 µM). Representative of 7 independent experiments. (I) Western blot illustrating p-eNOS (serine 1176), total eNOS, and actin in endothelial cells and their modulation by Wnt5a (900 ng/ml), BAPTA-AM (BAPTA, 5 µM), SRI37892 (Fzd-7 Inh, 2.5 µM), NSC668036 (Dvl Inh, 10 µM), and SP600125 (JNK Inh, 100 nM). Representative of 8 independent experiments. (J) Mean data from experiments shown in panel I. Significance was assessed using one-way ANOVA with Holm-Sidak post hoc multiple comparisons test. (K) Mean data illustrating nitric oxide generation by endothelial cells and modulation by Wnt5a (900 ng/ml), BAPTA-AM (BAPTA, 5 µM), SRI37892 (Fzd-7 Inh, 2.5 µM), NSC668036 (Dvl Inh, 10 µM), and SP600125 (JNK Inh, 100 nM). Representative of 8 independent experiments. (L) Mean data illustrating plasma Wnt9b at baseline and 5 min post Wnt9b intravascular infusion (30 μg/kg) in Pkd2 fl/fl and Pkd2 ecKO mice. n=6 mice for each genotype. Significance was assessed using two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. (M) Mean data illustrating plasma nitric oxide at baseline and 5 min post Wnt9b infusion (30 μg/kg) in Pkd1 fl/fl , Pkd1 ecKO, Pkd2 fl/fl and Pkd2 ecKO mice. n=8 for Pkd1 fl/fl and Pkd1 ecKO mice. n=7 for Pkd2 fl/fl and n=7 Pkd2 ecKO mice. Significance was assessed using two-way ANOVA with Holm-Sidak post hoc multiple comparisons test.

Article Snippet: Cells were cultured until confluence and were first exposed for 30 min at 37°C to either vehicle, BAPTA-AM, SRI37892 (MedChemExpress), NSC668036, (MedChemExpress) or SP600125 (MedChemExpress).

Techniques: Activation Assay, Introduce, Western Blot, Clinical Proteomics

Journal: The FASEB Journal

Article Title: ALOX15‐Derived Oxylipins Attenuate Macrophage Inflammatory Signaling Via a Gα q –PLC–PKC Pathway

doi: 10.1096/fj.202600272R

Figure Lengend Snippet: Contribution of intracellular calcium and PKCε to 12‐HETE‐ and 13‐HODE‐mediated suppression of cytokine production in RAW264.7 cells. RAW264.7 cells were stimulated with LPS (1 ng/mL) for 24 h. TNF‐α (A, C, E, G) and IL‐6 (B, D, F, H) levels in the culture medium were quantified by ELISA. Cells were pretreated with 12‐HETE (A, B, E, F) or 13‐HODE (C, D, G, H) at 1 μmol/L for 1 h prior to LPS stimulation. Inhibitors targeting intracellular calcium chelation (BAPTA‐AM; A–D) or PKCε (Epsilon V1‐2; E–H) were added 30 min before oxylipin treatment. Data are presented as mean ± SEM. Statistical significance was assessed using the Tukey–Kramer post hoc test or Steel–Dwass test. Groups not sharing a common letter differ significantly ( p < 0.05).

Article Snippet: To examine cellular signaling pathways involved in 12‐HETE‐ and 13‐HODE‐mediated anti‐inflammatory effects, signaling inhibitors targeting Gα q/11 (YM‐254890; FOCUS Biomolecules, Plymouth Meeting PA, USA), Protein kinase C (PKC; Ro‐31‐8425; Merck KGaA), BAPTA‐AM (Nacalai Tesque, Kyoto, Japan), phospholipase C (PLC; Cayman Chemical), PKCε (Epsilon‐V1‐2; Selleck Chemicals, Houston, TX, USA), peroxisome proliferator‐activated receptor γ (PPARγ; GW9662; FUJIFILM Wako Pure Chemicals, Osaka, Japan), TRPV1 (AMG517; Merck KGaA), Gα i/o (NF023; Abcam, Waltham, MA, USA), adenylate cyclase (SQ22536; AdooQ BioScience, Irvine, CA, USA), and AMPK (Dorsomorphi; FUJIFILM Wako Pure Chemicals) were administered 30 min prior to 12‐HETE and 13‐HODE treatments.

Techniques: Enzyme-linked Immunosorbent Assay