Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Impact of fibulin‐5 deficiency on the skin aging process. (A) Schematic representation of the interfollicular epidermis of mouse tail skin. Slow‐cycling epidermal stem cells (SCs) produce the K10 + interscale lineage (orange), and fast‐cycling epidermal SCs produce the K36 + scale lineage (blue). (B, C) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 2‐month‐old versus 30‐month‐old C57BL/6J mice and quantification (C). The white dashed line represents the epidermal–dermal boundary. Scale bars: 50 μm. (D, E) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 3‐month‐old Fbln5 WT versus KO mice and quantification (E). The white dashed line represents the epidermal–dermal boundary and hair follicles. Scale bars: 50 μm. (F) Images of 12‐month‐old Fbln5 WT and KO mice. (G) The body weights of 12‐month‐old Fbln5 WT and KO mice. (H–K) Hematoxylin and eosin staining of sagittal sections of the skin of 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (I, K). Scale bars: 150 μm. Epidermal thickness was measured in interscale and scale regions. (L–O) Whole‐mount staining of BrdU (green, a proliferation marker) and Hoechst (blue) in 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (M, O). Scale bars: 200 μm. (P–U) Whole‐mount staining of K10 (green, interscale lineage), K36 (red, scale lineage), and Hoechst (blue) in 2‐month‐old versus 30‐month‐old C57BL/6J mice and 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (Q, S, U). Scale bars: 200 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (C, E, G, I, K, M, O, Q, S, U). *, p < 0.05; **, p < 0.01; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Immunostaining, Staining, Marker, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Changes in integrin and extracellular matrix expression due to the loss of fibulin‐5. (A) The heatmap shows changes in integrins and ECM proteins in 12‐month‐old Fbln5 WT and KO epidermal stem cells. Genes with a ≥ 2‐fold change are used for analysis. (B) Schematic representation of the epidermal–dermal junction and its associated proteins. (C–V) Immunostaining and quantification of the indicated proteins: Collagen XVII (C–F; green), integrin β1 (G–J; green), integrin α6 (K–N; red) integrin β3 (O–R; green), nectin‐3 (S–V; green), K5 (S–V; gray), and K36 (S–V; red, scale lineage). The white dashed lines represent the epidermal–dermal boundary. Scale bars: 50 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (D, F, H, J, N, P, R, T, V) or Mann–Whitney U test (L). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. The schematic in panel B is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Immunostaining, Two Tailed Test, MANN-WHITNEY

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Extracellular fibulin‐5 enhances YAP activity and fast‐cycling stem cell‐associated gene expression in human keratinocytes. (A–H) Immunostaining of YAP (A, green), SLC1A3 (C, red), Ki‐67 (E, gray), and ASS1 (G, green) in human keratinocytes and quantification (B, D, F, H). Cells are seeded at 150,000, 50,000, and 25,000 cells per well in 12‐well plates and cultured for 48 h before analysis. Scale bars: 50 μm. (I, J) Immunostaining of YAP in primary human keratinocytes and quantification (J). Cells are seeded at 50,000 cells per well in 12‐well plates and cultured for 24 h and then treated with verteporfin or vehicle control for 8 h. Nuclear YAP (%) was calculated as the proportion of cells with nuclear YAP localization among all Hoechst + nuclei. Scale bars: 50 μm. (K–M) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following 8 h of verteporfin treatment. (N, O) Immunostaining of YAP in primary human keratinocytes and quantification (O). Cells are seeded at 300,000 cells per well on collagen IV–coated plates with or without recombinant human fibulin‐5 and cultured to ~80% confluence. The medium is then replaced, and cells are analyzed 8 h later. Scale bars: 50 μm. (P–R) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following culture on plates coated with collagen IV ± fibulin‐5. All data are presented as the mean ± SD. Each dot represents one independent biological replicate. Statistical significance is assessed using a two‐tailed unpaired t ‐test (K, L, P, Q, R), Welch's t ‐test (J, M, O), or one‐way ANOVA (B, D, F, H). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Activity Assay, Gene Expression, Immunostaining, Cell Culture, Control, Quantitative RT-PCR, Recombinant, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Proposed model of cellular and molecular alterations associated with fibulin‐5 deficiency during skin aging. In young skin, slow‐cycling and fast‐cycling epidermal stem cells (SCs) are spatially compartmentalized and give rise to their respective lineages. During aging, decreased fibulin‐5 expression is associated with altered integrin and extracellular matrix (ECM) protein expression, potentially affecting intracellular signaling through fibulin‐5–integrin interactions. Reduced YAP activity is associated with a decrease in the fast‐cycling epidermal stem cell compartment in aged skin and human keratinocytes. The schematic is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Activity Assay

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Effect of EFEMP1 knockdown by small interfering RNA (siRNA) on chondrocyte phenotype. A The human chondrocyte cell line C20A4 was transfected with either EFEMP1 siRNA (siEFEMP1) or scrambled siRNA (siCtrl). EFEMP1 and MMP-13 protein expression was analyzed by Western blotting. B Chondrocyte-related gene expression, including SOX9, aggrecan (ACAN), MMP13, type X collagen (COL10A1), and type II collagen (COL2A1), was assessed by quantitative PCR in siEFEMP1- and siCtrl-treated C20A4 cells. C Phosphokinase protein arrays were used to profile phosphorylation changes in intracellular proteins extracted from C20A4 chondrocytes. D Spot intensities exhibiting changes greater than ± 25% between groups were quantified using ImageJ software. Red lines indicate baseline levels from the siCtrl group. E Phosphorylation of p70S6K at threonine 421/serine 424 (T421/T424) was further examined by Western blot analysis. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined using an independent-samples t-test. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Knockdown, Small Interfering RNA, Transfection, Expressing, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Phospho-proteomics, Software

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Effect of EFEMP1 knockdown on osteoblast differentiation and mineralization in preosteoblastic cells. A The preosteoblastic MC3T3-E1 cell line was transfected with either EFEMP1 siRNA (siEFEMP1) or scrambled siRNA (siCtrl). EFEMP1 and RUNX2 protein expression was analyzed by Western blotting. B, C Alizarin Red staining was used to assess matrix mineralization in MC3T3-E1 cells cultured in osteogenic induction medium (OIM), with quantification performed on days 21, 28, and 35. D Alkaline phosphatase (ALP) staining was performed to evaluate osteogenic differentiation. E ALP enzymatic activity was measured in cell lysates on days 9 and 14 following siRNA transfection. F Osteopontin (OPN) expression was analyzed on days 3 and 7 during osteogenic induction. Data are presented as mean ± standard error of the mean (SEM). Statistical analyses: Data in A were analyzed using an independent-samples t -test, while data in C , E , and F were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Knockdown, Transfection, Expressing, Western Blot, Staining, Cell Culture, Activity Assay, Comparison

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: EFEMP1 antibody treatment and cartilage histological assessment in a mouse model of osteoarthritis (OA). A Schematic illustration of the EFEMP1 antibody treatment protocol in STR/ort mice from 16 to 21 weeks of age. B Representative Safranin O–stained knee joint sections, with magnified views of the non-calcified cartilage (NCC) and calcified cartilage (CC) regions. Matrix-producing chondrocytes (MPCs) and matrix-non-producing chondrocytes (MNCs) are indicated by arrows. C Quantification of MPC and MNC numbers in the NCC and CC regions of articular cartilage, with counts combined from medial and lateral compartments. D Osteoarthritis severity was evaluated using Osteoarthritis Research Society International (OARSI) scores across four joint compartments: lateral femoral condyle (LF), medial femoral condyle (MF), lateral tibial plateau (LT), and medial tibial plateau (MT). Data in C are presented as mean ± standard error of the mean (SEM), while data in D are presented as median and quartiles using a violin plot. Statistical analysis was performed using an independent-samples t-test. * P < 0.05 and ** P < 0.01

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Staining

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Immunofluorescence analysis of cartilage markers following EFEMP1 antibody treatment in STR/ort mice. A Representative immunofluorescence (IF) staining for EFEMP1, SOX9, MMP13, aggrecan fragments, COL2A1, and COL10A1 in the medial femoral condyle (MF) and medial tibial plateau (MT). Nuclei were counterstained with DAPI (blue), and articular cartilage regions are outlined by orange dashed lines. B Quantification of fluorescence intensity in articular cartilage regions of the femoral condyle and tibial plateau, with medial and lateral compartments combined. EFEMP1 and SOX9 signals were normalized to DAPI, whereas MMP13, aggrecan fragments, COL10A1, and COL2A1 signals were quantified as absolute FITC intensity. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using an independent-samples t -test. P < 0.05, P < 0.01, and ** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Cytokine profiling following EFEMP1 antibody treatment in STR/ort mice with osteoarthritis. A Heat-map representation of serum cytokine expression profiles measured using a multiplex assay. B Quantification of serum cytokine concentrations determined by Luminex xMAP analysis. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. P < 0.05, P < 0.01, and P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Expressing, Multiplex Assay, Luminex

Journal: The FASEB Journal

Article Title: ZNF384 ‐Driven Fibulin‐1 Exacerbates Vascular Stiffness via TGF ‐β/Smad3‐Mediated Senescence and Fibrosis

doi: 10.1096/fj.202501262RR

Figure Lengend Snippet: Fibulin‐1 (Fbln1) demonstrates a significant correlation with vascular stiffness. A: Genetic Pedigree of Familial Vascular Stiffness. B: Pulse wave velocity (PWV) in individuals with a familial predisposition to vascular stiffness. C: Differentially expressed plasma proteins between health control and individuals with a familial predisposition to vascular stiffness. D: Co‐expression correlation analysis of genes associated with vascular stiffness and Fbln1 in public aging databases. E: Western blot results showed the expression levels of Fbln1 in vascular tissues of naturally aged mice. F: Immunofluorescence analysis of vascular tissues in young and elderly cohorts. L, lumen; M, tunica media; A, tunica adventitia. *Significant differences. n ≥ 5, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then the membranes were incubated with the following primary antibodies at 4°C overnight: P53 (sc‐6246, Santa Cruz, 1:1000), P21 (R&D, MAB1355, 1:800), Fbln1 (sc‐374 539, Santa Cruz, 1:1000), osteopontin (OPN, sc‐73 631, Santa Cruz, 1;800), smooth muscle protein 22α (SM22α, 10 493–1‐AP, Proteintech, 1:1000), α‐smooth muscle actin (αSMA, #19245, CST, 1:1000), Collagen I (ab270993, Abcam, 1:1000), Collagen III (ab184993, Abcam, 1:1000), TGF‐β (#3709, CST, 1:1000), Samd3 (#9523, CST, 1:1000), P‐Samd3 (ab52903, Abcam, 1:1000) or GAPDH (#2118, CST, 1:5000).

Techniques: Clinical Proteomics, Control, Expressing, Western Blot, Immunofluorescence

Journal: The FASEB Journal

Article Title: ZNF384 ‐Driven Fibulin‐1 Exacerbates Vascular Stiffness via TGF ‐β/Smad3‐Mediated Senescence and Fibrosis

doi: 10.1096/fj.202501262RR

Figure Lengend Snippet: In vivo knockdown of Fbln1 improved vascular stiffness in nature aging mice. A: PWV measurements in aged mice. B–D: Effect of Fbln1 deficiency on systolic blood pressure (B), diastolic blood pressure (C) and mean arterial pressure (D) in normal WT and Fbln1 −/− mice is shown. E–L: Western blot results showed that Fbln1 knockdown could modulate the expression of p53, p21, αSMA, SM22α, OPN, collagenI and collagenIII induced by aging in mice. wild‐type mice: WT, Fbln1 knockout mice: Fbln1 −/− . *Significant differences. n ≥ 5, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then the membranes were incubated with the following primary antibodies at 4°C overnight: P53 (sc‐6246, Santa Cruz, 1:1000), P21 (R&D, MAB1355, 1:800), Fbln1 (sc‐374 539, Santa Cruz, 1:1000), osteopontin (OPN, sc‐73 631, Santa Cruz, 1;800), smooth muscle protein 22α (SM22α, 10 493–1‐AP, Proteintech, 1:1000), α‐smooth muscle actin (αSMA, #19245, CST, 1:1000), Collagen I (ab270993, Abcam, 1:1000), Collagen III (ab184993, Abcam, 1:1000), TGF‐β (#3709, CST, 1:1000), Samd3 (#9523, CST, 1:1000), P‐Samd3 (ab52903, Abcam, 1:1000) or GAPDH (#2118, CST, 1:5000).

Techniques: In Vivo, Knockdown, Western Blot, Expressing, Knock-Out

Journal: The FASEB Journal

Article Title: ZNF384 ‐Driven Fibulin‐1 Exacerbates Vascular Stiffness via TGF ‐β/Smad3‐Mediated Senescence and Fibrosis

doi: 10.1096/fj.202501262RR

Figure Lengend Snippet: In vivo knockdown of Fbln1 attenuates AngII‐induced vascular stiffness in mice. A: PWV measurements in AngII‐treated and PBS control mice. B–E: Western blot results showed that Fbln1 knockdown could modulate the expression of P53, P21 in mice. F: Assessment of collagen deposition in vascular tissues by Masson's trichrome staining in WT and Fbln1 −/− mice (scale = 50 μm). EVG of the vascular wall of the aortic in different groups (scale = 50 μm). The red arrows denote areas of morphological discontinuity in the elastic fibers. Transmission electron microscopy (TEM) of elastin in aortic wall (scale = 50 nm). wild‐type mice: WT, Fbln1 knockout mice: Fbln1 −/− . *Significant differences. n ≥ 5, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then the membranes were incubated with the following primary antibodies at 4°C overnight: P53 (sc‐6246, Santa Cruz, 1:1000), P21 (R&D, MAB1355, 1:800), Fbln1 (sc‐374 539, Santa Cruz, 1:1000), osteopontin (OPN, sc‐73 631, Santa Cruz, 1;800), smooth muscle protein 22α (SM22α, 10 493–1‐AP, Proteintech, 1:1000), α‐smooth muscle actin (αSMA, #19245, CST, 1:1000), Collagen I (ab270993, Abcam, 1:1000), Collagen III (ab184993, Abcam, 1:1000), TGF‐β (#3709, CST, 1:1000), Samd3 (#9523, CST, 1:1000), P‐Samd3 (ab52903, Abcam, 1:1000) or GAPDH (#2118, CST, 1:5000).

Techniques: In Vivo, Knockdown, Control, Western Blot, Expressing, Staining, Transmission Assay, Electron Microscopy, Knock-Out

Journal: The FASEB Journal

Article Title: ZNF384 ‐Driven Fibulin‐1 Exacerbates Vascular Stiffness via TGF ‐β/Smad3‐Mediated Senescence and Fibrosis

doi: 10.1096/fj.202501262RR

Figure Lengend Snippet: ZNF384 promotes the transcription of Fbln1 . A: Potential transcription factors regulating Fbln1 were identified through DNA pull‐down assays coupled with protein mass spectrometry. B. Western blot analysis demonstrated direct binding of ZNF384 to the Fbln1 promoter. C: Knockdown of ZNF384 resulted in decreased Fbln1 mRNA levels. D–F: Knockdown of ZNF384 led to decreased Fbln1 protein levels. G–H: Both ChIP‐seq and subsequent ChIP‐qPCR experiments confirm the specific binding of ZNF384 to the promoter region of the Fbln1 gene. I: Dual‐luciferase assay results demonstrated that ZNF384 activates transcription by binding to specific sequences within the Fbln1 promoter region. *Significant differences. n ≥ 3, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then the membranes were incubated with the following primary antibodies at 4°C overnight: P53 (sc‐6246, Santa Cruz, 1:1000), P21 (R&D, MAB1355, 1:800), Fbln1 (sc‐374 539, Santa Cruz, 1:1000), osteopontin (OPN, sc‐73 631, Santa Cruz, 1;800), smooth muscle protein 22α (SM22α, 10 493–1‐AP, Proteintech, 1:1000), α‐smooth muscle actin (αSMA, #19245, CST, 1:1000), Collagen I (ab270993, Abcam, 1:1000), Collagen III (ab184993, Abcam, 1:1000), TGF‐β (#3709, CST, 1:1000), Samd3 (#9523, CST, 1:1000), P‐Samd3 (ab52903, Abcam, 1:1000) or GAPDH (#2118, CST, 1:5000).

Techniques: Mass Spectrometry, Western Blot, Binding Assay, Knockdown, ChIP-sequencing, ChIP-qPCR, Luciferase

Journal: The FASEB Journal

Article Title: ZNF384 ‐Driven Fibulin‐1 Exacerbates Vascular Stiffness via TGF ‐β/Smad3‐Mediated Senescence and Fibrosis

doi: 10.1096/fj.202501262RR

Figure Lengend Snippet: Knockdown of Fbln1 suppressed the expression of the TGF‐β/Smad3 signaling pathway. A–B: Bulk RNA‐sequence was performed in AngII‐induced vascular aging mice: Differential gene expression heatmap analysis (A) and Gene Ontology (GO) enrichment analysis of DEGs (B). C–D: Western Blot analysis was performed to detect the expression levels of Fbln1, TGF‐β, p‐Smad3/Smad3 in AngII‐treated and PBS control mice. E: Immunofluorescence staining showed the expression changes of p‐Smad3 after knocking down Fbln1 (scale = 50 μm). F–G: Western Blot analysis was performed to detect the expression levels of Fbln1, TGF‐β, p‐Smad3 and Smad3 in primary VSMCs after knocking down Fbln1. n ≥ 5, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then the membranes were incubated with the following primary antibodies at 4°C overnight: P53 (sc‐6246, Santa Cruz, 1:1000), P21 (R&D, MAB1355, 1:800), Fbln1 (sc‐374 539, Santa Cruz, 1:1000), osteopontin (OPN, sc‐73 631, Santa Cruz, 1;800), smooth muscle protein 22α (SM22α, 10 493–1‐AP, Proteintech, 1:1000), α‐smooth muscle actin (αSMA, #19245, CST, 1:1000), Collagen I (ab270993, Abcam, 1:1000), Collagen III (ab184993, Abcam, 1:1000), TGF‐β (#3709, CST, 1:1000), Samd3 (#9523, CST, 1:1000), P‐Samd3 (ab52903, Abcam, 1:1000) or GAPDH (#2118, CST, 1:5000).

Techniques: Knockdown, Expressing, Sequencing, Gene Expression, Western Blot, Control, Immunofluorescence, Staining

Journal: The FASEB Journal

Article Title: ZNF384 ‐Driven Fibulin‐1 Exacerbates Vascular Stiffness via TGF ‐β/Smad3‐Mediated Senescence and Fibrosis

doi: 10.1096/fj.202501262RR

Figure Lengend Snippet: Inhibition of the TGF‐β/smad3 signaling pathway relieves the facilitating effect of Fbln1 on VSMCs. After treatment with recombinant Fibulin‐1 protein, the TGF‐β/Smad3 signaling pathway in VSMCs was blocked by adding the TGF‐β receptor inhibitor A83‐01 and the Smad3 phosphorylation inhibitor SIS3. A–B: Detection of VSMC senescence by senescence‐associated β‐galactosidase (SA‐β‐gal) staining. C–I: Western Blot was used to detect the expression levels of p53, p21, SM22α, OPN, collagenI and collagenIII. n ≥ 5, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then the membranes were incubated with the following primary antibodies at 4°C overnight: P53 (sc‐6246, Santa Cruz, 1:1000), P21 (R&D, MAB1355, 1:800), Fbln1 (sc‐374 539, Santa Cruz, 1:1000), osteopontin (OPN, sc‐73 631, Santa Cruz, 1;800), smooth muscle protein 22α (SM22α, 10 493–1‐AP, Proteintech, 1:1000), α‐smooth muscle actin (αSMA, #19245, CST, 1:1000), Collagen I (ab270993, Abcam, 1:1000), Collagen III (ab184993, Abcam, 1:1000), TGF‐β (#3709, CST, 1:1000), Samd3 (#9523, CST, 1:1000), P‐Samd3 (ab52903, Abcam, 1:1000) or GAPDH (#2118, CST, 1:5000).

Techniques: Inhibition, Recombinant, Phospho-proteomics, Staining, Western Blot, Expressing

Journal: bioRxiv

Article Title: Satellite Glial Cells Control Sensory Neuron Excitability via the Release of Fibulin-2

doi: 10.64898/2026.02.13.705760

Figure Lengend Snippet: A. Representative images of SGC cultures stained for the SGCs markers FABP7, and GS, the Schwann cell marker S100B, the neuron marker TUJ1 and the fibroblast marker PDGFRα. Scale bar 100μm. B. RT-qPCR analysis of SGC cultures for known SGCs markers ( Fabp7, Glul, GjaI, Ednrb, Gfap ), Schwann cells ( Scn7a, Mpz, Ncam ), macrophages ( Aif, Itgam, Cx3cr1 ), neurons ( Nfeh, Prph ), and fibroblasts ( Fgf13, Fgf9 ). N=2. C. Transcriptional similarity between cultured cells and primary tissue cell types. Heatmap showing Spearman correlation coefficients between bulk RNA-seq profiles from cultured samples (columns) and pseudobulk expression profiles from major cell classes in the single-cell atlas (rows). Correlation was computed using 5,000 highly variable genes following joint TMM normalization. Values within cells indicate correlation coefficients; color scale ranges from blue (low correlation) to red (high correlation). Hierarchical clustering was performed using Euclidean distance. D. Bar plot showing mRNA expression levels (counts per million, CPM) for a panel of cell-type-specific marker genes in bulk RNA-seq from primary SGC cultures. Bars are colored by cell type: Glial/SGC (blue), Schwann (green), Neuronal (black), Fibroblast (purple), Endothelial (orange), Mural (red), and Immune (yellow). Individual sample values are shown as points; circles indicate serum-free (24h) samples, triangles indicate serum-containing samples. Error bars represent standard deviation. E. Representative western blot (from 3 independent experiments) of DRG and SGC lysate from WT and Fibulin-2 KO mice, probed for Fibulin-2. GAPDH and Ponceau are used as loading controls. F. Representative western blot of SGC-CM from WT and Fibulin-2 KO mice, probed for Fibulin- 2. Ponceau staining is used as a loading control. G. Ponceau staining of western blot showed in for protein loading control.

Article Snippet: Fibulin-2 KO mice (B6.129S1-Fbln2tm1Chu/J) were obtained from The Jackson Laboratory.

Techniques: Staining, Marker, Quantitative RT-PCR, Cell Culture, RNA Sequencing, Expressing, Single Cell, Standard Deviation, Western Blot, Control

Journal: bioRxiv

Article Title: Satellite Glial Cells Control Sensory Neuron Excitability via the Release of Fibulin-2

doi: 10.64898/2026.02.13.705760

Figure Lengend Snippet: A. GO pathway analysis (cellular component) of the SGC-secreted proteins showing enrichment for extracellular proteins. B. SGC-secreted proteins were analyzed using the matrisome analyzer. C. Representative images of cultured SGCs immunostained for Fibulin-2, along with the indicated cellular markers. Lamp1-Lysosome, CD63-multivesicular bodies, VAMP1/2/3 (note that the antibody recgonizes VAMP1, VAMP2 and VAMP3) and VAMP5-secretory vesicles, and GM130-Golgi apparatus. 10-15 fields per group were imaged using confocal microscopy. D. Quantification of the Pearson correlation coefficient for the co-localization of Fibulin2 with the different cellular compartment markers. Each data point in the bar graph represents the colocalization coefficient from an individual cell (LAMP1 n=25 cells, CD63 n=25 cells, VAMP5 n=15cells, VAMP1/2/3 n=35 cells, GM130 n=23 cells). E. SGC-CM obtained from SGC cultures treated with Brefeldin-A was analyzed by western blot for Fibulin-2. CD9, a marker of EV, is used as a control. Ponceau staining is shown for loading control. n= 3 independent experiments. F. SGC cell pellet obtained from SGC cultures treated with Brefeldin-A was analyzed by western blot for Fibulin-2. GAPDH is used as a loading control. Ponceau staining is shown as another loading control. n= 3 independent experiments. G. Quantification of Fibulin-2 level in SGC-CM shown in E. n=3 T-test; *** P < 0.001. H. Quantification of Fibulin-2 level in SGC cell pellet shown in F. n=3, T-test; * P < 0.05. I. EVs were isolated from SGC-CM prepared from cultures from WT or Fibulin2-KO mice and analyzed by western blot alongside the SGC lysate for Fibulin2, CD9 (EVs marker) and GM130 (Golgi-associated vesicles, negative control for EV). GAPDH is used as a loading control. n= 3 independent experiments.

Article Snippet: Fibulin-2 KO mice (B6.129S1-Fbln2tm1Chu/J) were obtained from The Jackson Laboratory.

Techniques: Cell Culture, Confocal Microscopy, Western Blot, Marker, Control, Staining, Isolation, Negative Control

Journal: bioRxiv

Article Title: Satellite Glial Cells Control Sensory Neuron Excitability via the Release of Fibulin-2

doi: 10.64898/2026.02.13.705760

Figure Lengend Snippet: A. Representative images of DRG sections from WT and Fibulin-2 KO mice immunostained for Fibulin-2, FABP7, TUJ1, and DAPI. Scale bar 50μm. Two DRG sections from n=3 mice were imaged for each group. B. Super-resolution imaging of DRG tissue section highlighting the subcellular localization of Fibulin-2 in SGCs near the perinuclear region. Fibulin-2 (Red), FABP7 (White), TUJ1 (Green), and DAPI (Blue). Scale bar 2μm C. Electron micrograph of DRG sections showing the portion of an SGC covering the neuron soma. The SGC cytoplasm contains multivesicular bodies (white arrow), vesicle fusion/budding at the plasma membrane (red arrow), and a Golgi apparatus (asterisk). Scale bar 1μm D. Immunofluorescence of DRG section showing localization of Fibulin-2 in SGCs (labeled for Fabp7 or PDPN) surrounding neurons labeled with NeuN, NF200, TRPV1, TH, TRKA, and IB4. Scale bar 50μm. E. Quantification of the size distribution of the indicated DRG neurons subtypes. The size of neurons surrounded by Fibulin-2 positive SGC is indicated by a red dotted line. A total of 8956 neurons were analyzed from n=7 mice, with three DRG sections per mice. F. Quantification of the percentage of neuronal subtype surrounded by Fibulin-2-positive SGCs. Each data point in the bar graph represents an independent biological replicate. NF200 (n=5 mice), TRPV1 (n=5 mice), TRKA (n=3 mice), IB4 (n=4 mice), TH (n=4). Two sections from each biological replicate were imaged and used for quantification. Error bars represent SEM.

Article Snippet: Fibulin-2 KO mice (B6.129S1-Fbln2tm1Chu/J) were obtained from The Jackson Laboratory.

Techniques: Imaging, Clinical Proteomics, Membrane, Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: Satellite Glial Cells Control Sensory Neuron Excitability via the Release of Fibulin-2

doi: 10.64898/2026.02.13.705760

Figure Lengend Snippet: A . Voltage protocols for measurement of different types of K + currents: total ( I Total ), K-type ( I K ) and A-type ( I A ) K + currents. B . Sample traces of voltage-dependent K + currents I total (left), I K (middle) and I A (right) evoked by the protocols in ( A ) from Control (upper panel) and rFibulin-2 treated DRG cells (lower panel). C . rFibulin-2 increases voltage-dependent K + currents I Total (left), I K (middle) and I A (right) in DRG cells. Insert bar graphs are K + currents at membrane potential of -10 mV (around voltage threshold level), indicating that rFibulin-2 decreases excitability mainly mediated by enhancement of I A conductance, which reduces input resistance. Number of cells tested from 3 independent experiments: control n = 10; rFibulin-2: n = 8. D . Phrixotoxin-1 (PaTx1) was used to isolate Kv4 current evoked by voltage ramp (-100 to +20 mV, 100 mV/s). Sample traces of ramp-evoked K + currents before (a) and during (b) application of PaTx1, and the PaTx1-sensitive current (c, c = a - b). Currents were normalized to membrane capacitance for better comparison. E . I-V curves were constructed from the ramp-evoked Kv4 current (mean current value over 0.1 mV intervals from averages of five trials for each cell to approximate quasi-steady-state current). Note PaTx1 significantly increases the Kv4 current when the membrane potentials are depolarized to positive values greater than -25 mV. Number of cells tested from 3 independent experiments: control n = 6; rFibulin-2: n = 6; T-test; * P < 0.05; ** P < 0.01. F . Representative western blot of control and Fibulin-2 KO DRG lysate analyzed for Fibulin-2 and Kv4.2. GAPDH is used as a loading control. G . Quantification of Kv4.2 expression in control and Fibulin-2 KO mice. n=3 WT and n=3 Fibulin-2 KO mice. T-test; ** P < 0.01. H . Representative western blot of control and Fibulin-2 KO DRG lysate analyzed for Fibulin-2 and Kv4.3. GAPDH is used as a loading control. I . Quantification of Kv4.3 expression in control and Fibulin-2 KO mice. n=3 WT and n=3 Fibulin-2 KO mice. T-test; *** P < 0.001 J . Fibulin-2 KO mice show hypersensitivity to mechanical stimuli compared to controls, measured by the Von Frey Test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. K . Fibulin-2 KO mice exhibit hypersensitivity to heat stimuli compared to controls, measured by the Hot-Plate test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. L . Fibulin-2 KO mice exhibit hypersensitivity to cold stimuli compared to controls, measured by the Cold-Plate test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. M . Representative immunofluorescence images of the hindpaw of control and Fibulin-2 KO mice immunostained for PGP9.5 (white) and DAPI (blue). Three sections from n=3 mouse per group were used. N . Quantification of intraepidermal nerve fiber density (IENFD). n=3 mice per genotype. T-test, ns- non-significant

Article Snippet: Fibulin-2 KO mice (B6.129S1-Fbln2tm1Chu/J) were obtained from The Jackson Laboratory.

Techniques: Control, Membrane, Comparison, Construct, Western Blot, Expressing, Hot Plate Test, Immunofluorescence

Journal: bioRxiv

Article Title: Satellite Glial Cells Control Sensory Neuron Excitability via the Release of Fibulin-2

doi: 10.64898/2026.02.13.705760

Figure Lengend Snippet: A. Sample traces of action potentials (APs) recorded from Control and rFibulin-2-treated DRG neurons. APs were evoked by ramp current injection (0.15 pA/ms) via recording pipettes. Traces within shaded areas were used to calculate input resistance at hyperpolarization (blue, summarized in O ) and depolarization (red, summarized in P ) states. B-F . rFibulin-2 treatment decreased excitability of DRG neurons, as shown by reduced number of APs ( B ), and increases in the initial inter-AP interval ( C ), AP rheobase ( D ), normalized rheobase ( E ), and rheobase charge transfer ( F ). Number of cells tested from 3 independent experiments: control n = 10; rFibulin-2: n = 12. G-K . rFibulin-2 did not affect multiple other AP parameters, including AP threshold ( G ), maximal rise rate ( H ), amplitude ( I ), duration ( J ) and fast afterhyperpolarization (fAHP) ( K ). Number of cells tested from 3 independent experiments: control n = 10; rFibulin-2: n = 12. L-N . The recorded cells have comparable size ( L ), membrane capacitance ( M ), and resting membrane potential (RMP) ( N ). Number of cells tested from 3 independent experiments: control n = 10–15; rFibulin-2: n = 12–13. O-P . rFibulin-2 decreased input resistance of DRG neurons at depolarization state ( P ). However, it did not affect the input resistance at hyperpolarization state ( O ). Number of cells tested from 3 independent experiments: control n = 8–9; rFibulin-2: n = 12. T-test; * P < 0.05; ** P < 0.01; ns, not significant.

Article Snippet: For rFibulin-2 treatment, the cells were exposed to 2 μg/ml rFibulin-2 protein (R&D System Catalog # 9559-FB-050) or MQ water as a control at the time of seeding, and they were incubated for an additional 24 hours.

Techniques: Control, Injection, Membrane

Journal: bioRxiv

Article Title: Satellite Glial Cells Control Sensory Neuron Excitability via the Release of Fibulin-2

doi: 10.64898/2026.02.13.705760

Figure Lengend Snippet: A . Voltage protocols for measurement of different types of K + currents: total ( I Total ), K-type ( I K ) and A-type ( I A ) K + currents. B . Sample traces of voltage-dependent K + currents I total (left), I K (middle) and I A (right) evoked by the protocols in ( A ) from Control (upper panel) and rFibulin-2 treated DRG cells (lower panel). C . rFibulin-2 increases voltage-dependent K + currents I Total (left), I K (middle) and I A (right) in DRG cells. Insert bar graphs are K + currents at membrane potential of -10 mV (around voltage threshold level), indicating that rFibulin-2 decreases excitability mainly mediated by enhancement of I A conductance, which reduces input resistance. Number of cells tested from 3 independent experiments: control n = 10; rFibulin-2: n = 8. D . Phrixotoxin-1 (PaTx1) was used to isolate Kv4 current evoked by voltage ramp (-100 to +20 mV, 100 mV/s). Sample traces of ramp-evoked K + currents before (a) and during (b) application of PaTx1, and the PaTx1-sensitive current (c, c = a - b). Currents were normalized to membrane capacitance for better comparison. E . I-V curves were constructed from the ramp-evoked Kv4 current (mean current value over 0.1 mV intervals from averages of five trials for each cell to approximate quasi-steady-state current). Note PaTx1 significantly increases the Kv4 current when the membrane potentials are depolarized to positive values greater than -25 mV. Number of cells tested from 3 independent experiments: control n = 6; rFibulin-2: n = 6; T-test; * P < 0.05; ** P < 0.01. F . Representative western blot of control and Fibulin-2 KO DRG lysate analyzed for Fibulin-2 and Kv4.2. GAPDH is used as a loading control. G . Quantification of Kv4.2 expression in control and Fibulin-2 KO mice. n=3 WT and n=3 Fibulin-2 KO mice. T-test; ** P < 0.01. H . Representative western blot of control and Fibulin-2 KO DRG lysate analyzed for Fibulin-2 and Kv4.3. GAPDH is used as a loading control. I . Quantification of Kv4.3 expression in control and Fibulin-2 KO mice. n=3 WT and n=3 Fibulin-2 KO mice. T-test; *** P < 0.001 J . Fibulin-2 KO mice show hypersensitivity to mechanical stimuli compared to controls, measured by the Von Frey Test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. K . Fibulin-2 KO mice exhibit hypersensitivity to heat stimuli compared to controls, measured by the Hot-Plate test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. L . Fibulin-2 KO mice exhibit hypersensitivity to cold stimuli compared to controls, measured by the Cold-Plate test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. M . Representative immunofluorescence images of the hindpaw of control and Fibulin-2 KO mice immunostained for PGP9.5 (white) and DAPI (blue). Three sections from n=3 mouse per group were used. N . Quantification of intraepidermal nerve fiber density (IENFD). n=3 mice per genotype. T-test, ns- non-significant

Article Snippet: For rFibulin-2 treatment, the cells were exposed to 2 μg/ml rFibulin-2 protein (R&D System Catalog # 9559-FB-050) or MQ water as a control at the time of seeding, and they were incubated for an additional 24 hours.

Techniques: Control, Membrane, Comparison, Construct, Western Blot, Expressing, Hot Plate Test, Immunofluorescence