Journal: Bioactive materials

Article Title: Double hits with bioactive nanozyme based on cobalt-doped nanoglass for acute and diabetic wound therapies through anti-inflammatory and pro-angiogenic functions.

doi: 10.1016/j.bioactmat.2023.08.014

Figure Lengend Snippet: Fig. 5. Effects of CoNZ on pro-angiogenesis in vivo and in vitro. (A–C) In vivo experiments with STZ-induced diabetic skin wounds: (A) Immunohistochemical staining of tissue samples for CD31 and α-SMA (neovascularization) at weeks 1 and 3, with semi-quantification of signal intensity by ImageJ; both signals were expressed at significantly higher levels in the CoNZ group. (B) Western blot analysis of tissue samples for CD31 at week 1, showing significant differences between groups (defect control < BGn & CoNZ); the dotted line (intensity level ‘1’) represents the normal group. (C) ELISA analysis of tissue samples for VEGF at day 3. For the in vivo studies, nanoparticles of 10 μL from a concentration of 100 mg/1 mL were treated to each wound. (D–G) In vitro experiments with high glucose/H2O2-challenged inflammatory cells. (D) Schematic representation of the in vitro design; HUVECs challenged with high glucose and H2O2 were analyzed for angiogenic events. (E) Intracellular ROS level at 12 h; ROS levels substantially increased with high glucose/H2O2 challenge, which were significantly reduced by CoNZ treatment; ROS scavenging ability was observed in the order, CoNZ > CoNZ-ions ~ BGn > high glucose/H2O2 control. Nanoparticle concentration used for the in vitro study was 20 μg/mL. (F) Cellular migration was analyzed over 24 h by assessing the ability to fill a scratched multicellular gap; images of four representative groups at 12 h are presented; cell migration was recorded in the order, CoNZ ~ CoNZ-ions » BGn > high glucose/H2O2 control. (G) Tubular formation of cells was examined and quantified in terms of tubule and node number at 6 h and 24 h on a Matrigel substrate; cell images of four representative groups at 24 h are presented. Statistical significance was calculated between groups using one-way ANOVA with *p < 0.05, **p < 0.01, and ***p < 0.001 denoting significance compared to the defect group (in vivo) or HG (+) & H2O2 (+) group (in vitro); and +p < 0.05, ++p < 0.01, and +++p < 0.001 indicating significance compared to the BGn group (in vivo). All data are presented as mean ± one standard deviation and the sample size was n = 5.

Article Snippet: Quantikine murine VEGF ELISA kit (MMV00, R&D Systems) was used to detect the VEGF quantity released from HUVECs after culturing for 3 days, following the manufacturer’s protocols.

Techniques: In Vivo, In Vitro, Immunohistochemical staining, Staining, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Migration, Standard Deviation

Journal: Bone research

Article Title: Metformin accelerates bone fracture healing by promoting type H vessel formation through inhibition of YAP1/TAZ expression.

doi: 10.1038/s41413-023-00279-4

Figure Lengend Snippet: Fig. 1 Metformin promotes angiogenesis under hypoxic conditions in vitro. Representative images (a) of the transwell migration assay with quantification of crystal violet-stained migrated HMECs (b) treated with metformin under different oxygen concentrations (21% and 1% O2). Met: metformin. Scale bar: 100 μm. n = 3 per group. Representative images (c) of tube formation of HMECs on Matrigel with quantification of the total branching points (d), total tube length (e), and total loop numbers (f). Scale bar: 200 μm. n = 3 per group. g CCK-8 analysis of the proliferation of HMECs. n = 4 per group. h qRT‒PCR analysis of the mRNA levels of the HIF-1α target genes Vegfa and Lrg1 in HMECs with or without metformin treatment under different oxygen concentrations. n = 3 per group. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) The concentrations of OCN and VEGFA were determined using the Mouse OCN (Elabscience, E-EL-M0864c, Wuhan, China) or VEGFA (Elabscience, E-EL-M1292c) ELISA kit according to the manufacturers’ instructions.

Techniques: In Vitro, Transwell Migration Assay, Staining, CCK-8 Assay

Journal: Bone research

Article Title: Metformin accelerates bone fracture healing by promoting type H vessel formation through inhibition of YAP1/TAZ expression.

doi: 10.1038/s41413-023-00279-4

Figure Lengend Snippet: Fig. 3 Metformin promotes type H vessel formation in osteoporotic fracture mice. a Representative CD31 and Emcn coimmunostaining images (left) with quantification of the type H vessel ratio in calluses (right) from the osteoporotic mice treated with PBS, Met, PTH, and ALN at 3, 6, and 9 weeks post-fracture. ca: callus. The dotted line represents the boundary of the callus. Met metformin, ALN alendronate, PTH parathyroid hormone. Scale bar: 100 μm. n = 5 per group. b Representative Ki67 and Emcn coimmunostaining images (left) with quantification of the number of Ki67-positive endothelial cells in calluses (right) from the osteoporotic mice treated with PBS, Met, PTH, and ALN at 3, 6, and 9 weeks post-fracture. Scale bar: 100 μm. n = 5 per group. ELISAs for the serum (c) and bone marrow (d) concentrations of VEGFA at 9 weeks post-osteoporotic fracture. n = 8 per group; Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) The concentrations of OCN and VEGFA were determined using the Mouse OCN (Elabscience, E-EL-M0864c, Wuhan, China) or VEGFA (Elabscience, E-EL-M1292c) ELISA kit according to the manufacturers’ instructions.

Techniques:

Journal: Bone research

Article Title: Metformin accelerates bone fracture healing by promoting type H vessel formation through inhibition of YAP1/TAZ expression.

doi: 10.1038/s41413-023-00279-4

Figure Lengend Snippet: Fig. 4 Metformin promotes the expression of HIF-1α by inhibiting the expression of YAP1/TAZ in HMECs under hypoxic conditions. a qRT‒PCR analysis of the inhibitory efficiency of siRNAs targeting HIF-1α. n = 3 per group. b qRT‒PCR analysis of the expression of HIF-1α and its target genes Vegfa and Lrg1 in the si-HIF-1α-transfected HMECs with or without metformin treatment under hypoxic conditions (1% O2). Met: metformin. n = 3 per group. Immunofluorescence staining images and quantification showing the protein levels of HIF-1α (c), YAP1 (d), and TAZ (e) in the HMECs treated with PBS (control) or metformin under hypoxic conditions. Scale bar: 20 μm. n = 9 per group. f qRT‒PCR analysis of the inhibitory efficiency of siRNAs targeting YAP1 or TAZ. n = 3 per group. g Immunofluorescence staining images and quantification showing the protein level of HIF-1α in the hypoxia-cultured HMECs from the si-Con, si-YAP1, si-TAZ, si-Y/T, and si-Y/T + Met groups. Y/T: YAP1 and TAZ. Scale bar: 20 μm. n = 3 per group. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) The concentrations of OCN and VEGFA were determined using the Mouse OCN (Elabscience, E-EL-M0864c, Wuhan, China) or VEGFA (Elabscience, E-EL-M1292c) ELISA kit according to the manufacturers’ instructions.

Techniques: Expressing, Transfection, Staining, Control, Cell Culture

Journal: Molecular and Cellular Biology

Article Title: Placental but Not Heart Defects Are Associated with Elevated Hypoxia-Inducible Factor α Levels in Mice Lacking Prolyl Hydroxylase Domain Protein 2

doi: 10.1128/mcb.00425-06

Figure Lengend Snippet: FIG. 6. Molecular analyses of Phd2/ embryos. (A) Anti-PHD2 Western blot of total cell lysates from wild-type E7.5 to E12.5 embryos (left) or E12.5 embryos from Phd2/ intercrosses (right). (B) Q-PCR for Phd1, Phd2, and Phd3 using wild-type E11.5 hearts. Data are shown as relative abundances to Hprt mRNA level (n 6). (C) RT-PCR for Phd1 and Phd3 using E11.5 hearts. Hprt was used as control. (D) Western blots of embryonic nuclear extracts. Developmental stages and antibodies used are indicated in the figure. (E) Northern blots of RNA prepared from whole embryos using cDNA probes specific for Vegfa, Pgk1, Hif-1, and Hif-2. (F) Western blots of E11.5 embryonic heart total extracts or nuclear extracts from the remaining tissues of embryo proper after removal of the heart. Antibodies are indicated in the figure. PC, positive controls, including nuclear extract of E11.5 wild-type embryo for HIF-1, total extracts of E11.5 wild-type placenta for HIF-2, and CoCl2-treated NIH 3T3 cell total lysate for HIF-3. (G) ELISA for VEGF-A in heart total lysates. Values are shown as means of protein concentration (n 4). Genotypes in all panels refer to the Phd2 locus.

Article Snippet: To quantify VEGF-A by ELISA, E11.5 hearts were sonicated in 100 l of 250 mM Tris-HCl (pH 7.5) and cleared by centrifugation, and supernatants were subjected to ELISA using a Quantikine Mouse VEGF Immunoassay kit (MMV00; R&D Systems).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Northern Blot, Enzyme-linked Immunosorbent Assay, Protein Concentration