recombinant human fibulin 5  (R&D Systems)

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: Scientific Reports

Article Title: Effect of Protease Inhibitors in Healing of the Vaginal Wall

doi: 10.1038/s41598-019-48527-0

Figure Lengend Snippet: Effect of vaginal wall injury on FBLN5. Nulliparous young rats were either not injured or injured as described in Materials and Methods. Injured rats were treated with hydrogel (HG) alone or HG + FBLN5 (10 µg/ml) (n = 5 in each group). Data represent mean ± SEM. After 48 h, vaginal muscularis was harvested and analyzed for levels of endogenous or recombinant FBLN5. ( A ) Upper blot was incubated with anti-FBLN5 whereas the lower blot was incubated with anti-His tag antibody to recognize recombinant FBLN5. ( B ) Quantitative analysis of endogenous FBLN5 in non-injured and injured vagina treated with HG or HG + FBLN5. * P < 0.05 compared with control ( C ) Immunoblot analysis of urea extracts of muscularis from control uninjured rats before and 12, 24, 48 h and 7D after cut injury. The 26 kDa bands represent nonspecific binding to IgG. ( D ) Quantitative analysis of FBLN5 levels in control uninjured (Ctl) or injured animals as a function of time after cut (open symbols) or wedge injury (solid squares). Data represent mean ± SEM. Uninjured control rats (n = 6) are presented with 20 cut and 12 wedge injuries divided equally among the four time points At 7 days after injury, horizontal line represents mean, individual values are presented. Data represent mean ± SEM, * p < 0.05 compared with control; ** p < 0.05 compared with wedge.

Article Snippet: Recombinant human FBLN5 (R&D Systems #3095-FB, Minneapolis, MN, USA) was incubated with MMP-7 ± actinonin.

Techniques: Recombinant, Incubation, Control, Western Blot, Binding Assay

Journal: Scientific Reports

Article Title: Effect of Protease Inhibitors in Healing of the Vaginal Wall

doi: 10.1038/s41598-019-48527-0

Figure Lengend Snippet: Effect of systemic or local estrogen on vaginal FBLN5. ( A ) Representative immunoblot of baseline levels of FBLN5 in uninjured control rats treated with systemic estradiol injections (Systemic E2 inj., 50 µg/kg/d), or ovariectomized with either (i) vaginal conjugated equine estrogen (CEE) cream discontinued prior to vaginal wall injury, (ii) placebo cream, or (iii) continuous vaginal conjugated equine estrogen (CEE) cream continued following vaginal wall injury. ( B ) Effect of systemic E2 on FBLN5 levels after injury. Data represent mean ± SEM of 5 animals at each time point in both groups. ( C ) Effect of local CEE cream on FBLN5 levels after injury. Ovariectomized animals treated with placebo (OVX), 2 weeks of preoperative CEE cream (OVX + Preop CEE) or preoperative CEE cream + continuous cream after injury (OVX + Continuous CEE). Results represent mean ± SEM of 5 animals at each time point in each group. *P < 0.05, ANOVA.

Article Snippet: Recombinant human FBLN5 (R&D Systems #3095-FB, Minneapolis, MN, USA) was incubated with MMP-7 ± actinonin.

Techniques: Western Blot, Control, Cream

Journal: Scientific Reports

Article Title: Effect of Protease Inhibitors in Healing of the Vaginal Wall

doi: 10.1038/s41598-019-48527-0

Figure Lengend Snippet: Effect of two protease inhibitors (sivelestat and actinonin) on FBLN5 content in the rat vaginal wall 24 hours after injury. ( A ) Animals were injected with PBS, sivelestat, or actinonin, at the time of injury. Data represent mean ± SEM levels of FBLN-5 of 4 animals per group. *One-way analysis of variance (P < 0.05). ( B ) Silver-stained gel of rFBLN5 (200 ng), MMP-7 (1:36), actinonin (acti), FBLN5 + MMP-7 ± acti, or FBLN5 + MMP-7 incubated at 37 °C for 4 min. 17 kDa protein represents MMP-7 (arrow).

Article Snippet: Recombinant human FBLN5 (R&D Systems #3095-FB, Minneapolis, MN, USA) was incubated with MMP-7 ± actinonin.

Techniques: Injection, Staining, Incubation

Journal: Scientific Reports

Article Title: Effect of Protease Inhibitors in Healing of the Vaginal Wall

doi: 10.1038/s41598-019-48527-0

Figure Lengend Snippet: Effect of actinonin on FBLN5 content in the injured vaginal wall as a function of time. ( A ) Representative immunoblot and coomasie stained protein gel. 30 µg urea-extracted protein per lane. IgG represents nonspecific interacting protein without primary antibody. ( B ) Data represent mean ± SEM of 8 animals in each group. *Two-way analysis of variance (P < 0.05).

Article Snippet: Recombinant human FBLN5 (R&D Systems #3095-FB, Minneapolis, MN, USA) was incubated with MMP-7 ± actinonin.

Techniques: Western Blot, Staining