mouse monoclonal igm connexin 43 cx43  (Santa Cruz Biotechnology)

Journal: Biomaterials advances

Article Title: Titanium nanotopography enhances mechano-response of osteocyte three-dimensional network toward osteoblast activation.

doi: 10.1016/j.bioadv.2024.213939

Figure Lengend Snippet: Fig. 2. Effects of titanium nanosurfaces on focal adhesion formation and cell shapes in osteocytes (A) Representative confocal laser microscopic images of F-actin, nuclei, and paxillin, (B) circularity and solidity as cytomorphometrical parameters, (C) numbers of connexin 43 per osteocyte, (D) expression of a focal adhesion gene marker of paxillin (Pxn) relative to glyceraldehyde 3-phosphate dehydrogenase (Gapdh) analyzed using reverse transcription-polymerase chain reaction (RT-PCR), (E) representative scanning electron microscopy (SEM) images, and (F) shining spot density within the peripheral region of the cell body in MLO-Y4 osteocyte cell line monolayer culture on polystyrene culture plates (Poly) or titanium discs with machined (MA), and nano-roughened (NR) surfaces at days 1, 3 and/or 7. Dashed lines in E indicate the margin of cells. Red and yellow rectangles in E indicate the regions corresponding to the high magnification images surrounded by the same colors. Note that the cells on the NR titanium surface form multiple projections with highly expressed paxillin at day 1 (white arrowheads in A), exhibiting shining spots corresponding to the vertices of surface nanospikes digging into the peripheral parts of the cell body consistently throughout days 1 to 7 (yellow arrowheads in E), and elongate cellular projections markedly at day 7 (yellow arrows in E). Data are presented as dot plots with mean value (N = 9–11 in [B], N = 20–23 in [C and D], N = 15–22 in [F]) and means ± standard deviation (SD) (N = 3 in [D]). Different letters or an asterisk indicate statistically significant differences (P < 0.05, Tukey's honestly significant difference [HSD] test, Bonferroni's multiple comparison test or Steel-Dwass test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The cells cultured in the monolayer and 3D cultures were fixed with 4 % paraformaldehyde in phosphate buffer solution (FUJIFILM Wako Pure Chemical Corporation) for 15 min. After washing with PBS, the cells were blocked for non-specific protein binding using blocking buffer containing 3.0 % bovine serum albumin (BSA) (FUJIFILM Wako Pure Chemical Corporation), 0.1 % Triton X-100 (FUJIFILM Wako Pure Chemical Corporation), and 0.01 % Tween 20 (Sigma-Aldrich) for 1 h. Next, the cells were incubated overnight at 4 ◦C in a 1/200 dilution of anti-rabbit paxillin monoclonal antibody (ab32084; Abcam), 1/250 dilution of mouse anti-Cx43 monoclonal antibody (sc-271,837, Santa Cruz Biotechnology, Dallas, TX, USA) or 1/200 anti-total YAP (sc101,199, Santa Cruz Biotechnology).

Techniques: Expressing, Marker, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Electron Microscopy, Standard Deviation, Comparison

Journal: Biomaterials advances

Article Title: Titanium nanotopography enhances mechano-response of osteocyte three-dimensional network toward osteoblast activation.

doi: 10.1016/j.bioadv.2024.213939

Figure Lengend Snippet: Fig. 3. Effects of titanium nanosurfaces on osteocyte network formation in association with mechanotransduction (A) Representative confocal laser microscopic images of F-actin, nuclei, and connexin 43, (B) numbers of connexin 43 per osteocyte, (C) gene expressions of a dendrite marker of podoplanin (Pdpn), a gap junction marker of connexin 43 (Gja1) and a prostaglandin marker of prostaglandin-endoperoxide synthase 2 (Ptgs2) relative to glyceraldehyde 3-phosphate dehydrogenase (Gapdh) analyzed by reverse transcription-polymerase chain reaction (RT-PCR), and (D) expressions of phosphorylated yes-associated protein (p-YAP], active YAP, total YAP and GAPDH analyzed using western blotting in MLO-Y4 osteocyte cell line monolayer culture co-incubated with or without an inhibitor of Rho-associated coiled-coil forming kinase (ROCK) on polystyrene culture plates (Poly) or titanium discs with machined (MA), and nano-roughened (NR) surfaces for 7 days. (E) Representative confocal laser microscopy images of F-actin, nuclei, and connexin 43 in MLO-Y4 cells cultured in three-dimensional collagen gels on Poly or the MA or NR titanium surfaces for 7 days. Note the cells on the NR titanium surface develop multiple projections with highly expressed connexin 43 both in a monolayer (double arrows in A) and 3D culture (double arrows in B). Data are presented as means ± standard deviation (SD) (N = 4 in B and N = 3 in C). Different letters indicate statistically significant differences (P < 0.05, Tukey's honestly significant difference [HSD] test).

Article Snippet: The cells cultured in the monolayer and 3D cultures were fixed with 4 % paraformaldehyde in phosphate buffer solution (FUJIFILM Wako Pure Chemical Corporation) for 15 min. After washing with PBS, the cells were blocked for non-specific protein binding using blocking buffer containing 3.0 % bovine serum albumin (BSA) (FUJIFILM Wako Pure Chemical Corporation), 0.1 % Triton X-100 (FUJIFILM Wako Pure Chemical Corporation), and 0.01 % Tween 20 (Sigma-Aldrich) for 1 h. Next, the cells were incubated overnight at 4 ◦C in a 1/200 dilution of anti-rabbit paxillin monoclonal antibody (ab32084; Abcam), 1/250 dilution of mouse anti-Cx43 monoclonal antibody (sc-271,837, Santa Cruz Biotechnology, Dallas, TX, USA) or 1/200 anti-total YAP (sc101,199, Santa Cruz Biotechnology).

Techniques: Marker, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation, Microscopy, Cell Culture, Standard Deviation

Journal: Biomaterials advances

Article Title: Titanium nanotopography enhances mechano-response of osteocyte three-dimensional network toward osteoblast activation.

doi: 10.1016/j.bioadv.2024.213939

Figure Lengend Snippet: Fig. 4. Effects of culture supernatant on osteocyte 3D network formation induced by titanium nanosurfaces. (A) Prostaglandin E2 (PGE2) concentration in culture supernatants from MLO-Y4 osteocyte cell line monolayer culture on polystyrene culture plates or titanium discs with machined (MA), and nano-roughened (NR) surfaces for 7 days. (B) Representative confocal laser microscopic images of F-actin, nuclei, and connexin 43, (C) numbers of connexin 43 per osteocyte, and (D) gene expressions of a dendrite marker of podoplanin (Pdpn), a gap junction marker of connexin 43 (Gja1) and a prostaglandin marker of prostaglandin-endoperoxide synthase 2 (Ptgs2) relative to that of glyceraldehyde 3-phosphate dehydrogenase (Gapdh), analyzed using reverse transcription-polymerase chain reaction (RT-PCR) in MLO-Y4 cell monolayer cultures with the culture supernatants on polystyrene culture plates for 7 days. The cells cultured in the supernatants from osteocyte monolayer culture on the NR titanium surface develop multiple projections and show a high expression of connexin 43 even on polystyrene culture plates (double arrows in B). Data represented as mean ± SD (N = 3 in A and D and N = 4 in C). Different letters indicate statistically significant differences (P < 0.05, Tukey's honestly significant difference [HSD] test).

Article Snippet: The cells cultured in the monolayer and 3D cultures were fixed with 4 % paraformaldehyde in phosphate buffer solution (FUJIFILM Wako Pure Chemical Corporation) for 15 min. After washing with PBS, the cells were blocked for non-specific protein binding using blocking buffer containing 3.0 % bovine serum albumin (BSA) (FUJIFILM Wako Pure Chemical Corporation), 0.1 % Triton X-100 (FUJIFILM Wako Pure Chemical Corporation), and 0.01 % Tween 20 (Sigma-Aldrich) for 1 h. Next, the cells were incubated overnight at 4 ◦C in a 1/200 dilution of anti-rabbit paxillin monoclonal antibody (ab32084; Abcam), 1/250 dilution of mouse anti-Cx43 monoclonal antibody (sc-271,837, Santa Cruz Biotechnology, Dallas, TX, USA) or 1/200 anti-total YAP (sc101,199, Santa Cruz Biotechnology).

Techniques: Concentration Assay, Marker, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Expressing

Journal: Biomaterials advances

Article Title: Titanium nanotopography enhances mechano-response of osteocyte three-dimensional network toward osteoblast activation.

doi: 10.1016/j.bioadv.2024.213939

Figure Lengend Snippet: Fig. 7. Effects of titanium nanosurfaces on osteocyte 3D network formation under cyclic compressive loading (A) Representative confocal laser microscopy images of F-actin, nuclei, and connexin 43, (B) the number of connexin 43 per osteocyte, and (C) western blotting images for expression of connexin 43 (Cx43), cyclooxygenase-2 (Cox-2), and β-actin in MLO-Y4 osteocyte cell lines cultured in 3D collagen gels on polystyrene culture plates, titanium discs with machined (MA), or nano-roughened (NR) surfaces for 7 days, followed by an additional 49 h culturing with 5 times repeated cyclic compressive loading. Data represented as mean ± SD (N = 3). Different letters indicate statistically significant differences (P < 0.05, Tukey's honestly significant difference [HSD] test).

Article Snippet: The cells cultured in the monolayer and 3D cultures were fixed with 4 % paraformaldehyde in phosphate buffer solution (FUJIFILM Wako Pure Chemical Corporation) for 15 min. After washing with PBS, the cells were blocked for non-specific protein binding using blocking buffer containing 3.0 % bovine serum albumin (BSA) (FUJIFILM Wako Pure Chemical Corporation), 0.1 % Triton X-100 (FUJIFILM Wako Pure Chemical Corporation), and 0.01 % Tween 20 (Sigma-Aldrich) for 1 h. Next, the cells were incubated overnight at 4 ◦C in a 1/200 dilution of anti-rabbit paxillin monoclonal antibody (ab32084; Abcam), 1/250 dilution of mouse anti-Cx43 monoclonal antibody (sc-271,837, Santa Cruz Biotechnology, Dallas, TX, USA) or 1/200 anti-total YAP (sc101,199, Santa Cruz Biotechnology).

Techniques: Microscopy, Western Blot, Expressing, Cell Culture

Journal: Biomaterials advances

Article Title: Titanium nanotopography enhances mechano-response of osteocyte three-dimensional network toward osteoblast activation.

doi: 10.1016/j.bioadv.2024.213939

Figure Lengend Snippet: Fig. 8. Role of PGE2 in synergistic activation of osteocyte three-dimensional network formation by cyclic compressive loading and titanium nano-surfaces (A) Scheme showing the established pathway for prostaglandin E2 (PGE2) synthesis and connexin 43 gene (Gja) expressions of osteocytes mediated by PGE2 under loading condition. Action point in arachidonic acid cascade for a PGE2 synthesis inhibitor used in this study is shown. (B) Expression of Gja1 and prostaglandin- endoperoxide synthase 2 (Ptgs2) relative to that of glyceraldehyde 3-phosphate dehydrogenase (Gapdh), analyzed using reverse transcription polymerase chain reaction (RT-PCR) in MLO-Y4 cells cultured in 3D collagen gels on the machined (MA) or nano-roughened (NR) titanium surfaces for 7 days, followed by an additional 49 h culturing with or without 5 times repeated cyclic compressive loading and co-culturing with the PGE2 synthesis inhibitor. Data are presented as mean ± standard deviation (SD) (N = 3). Different letters indicate statistically significant differences (P < 0.05, Tukey's honestly significant difference [HSD] test).

Article Snippet: The cells cultured in the monolayer and 3D cultures were fixed with 4 % paraformaldehyde in phosphate buffer solution (FUJIFILM Wako Pure Chemical Corporation) for 15 min. After washing with PBS, the cells were blocked for non-specific protein binding using blocking buffer containing 3.0 % bovine serum albumin (BSA) (FUJIFILM Wako Pure Chemical Corporation), 0.1 % Triton X-100 (FUJIFILM Wako Pure Chemical Corporation), and 0.01 % Tween 20 (Sigma-Aldrich) for 1 h. Next, the cells were incubated overnight at 4 ◦C in a 1/200 dilution of anti-rabbit paxillin monoclonal antibody (ab32084; Abcam), 1/250 dilution of mouse anti-Cx43 monoclonal antibody (sc-271,837, Santa Cruz Biotechnology, Dallas, TX, USA) or 1/200 anti-total YAP (sc101,199, Santa Cruz Biotechnology).

Techniques: Activation Assay, Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Standard Deviation

Journal: Biomaterials advances

Article Title: Titanium nanotopography enhances mechano-response of osteocyte three-dimensional network toward osteoblast activation.

doi: 10.1016/j.bioadv.2024.213939

Figure Lengend Snippet: Fig. 9. Synergetic effects of titanium nanotopography and cyclic compressive loading on osteocyte three-dimensional network development and bone turnover direction Possible cellular mechanisms underlying the synergistic effects of titanium nanotopography and cyclic compressive loading on 3D network development in osteo cytes. Osteocytes attached to normal titanium surfaces were not sufficiently activated to achieve intercellular connections until physiological cyclic compressive loading was applied (left panel). In contrast (a right panel), anisotropically-patterned dense nanospikes on nano-roughened titanium surfaces provide static me chanical stress on adherent osteocytes to promote maturation, dendrite formation, and connexin 43 (Cx43) expressions. Cells actively release prostaglandin E2 (PGE2) through Cx43 hemichannels. Secreted PGE2 induces the secretory and adjacent cells to mediate the autonomous development of 3D intercellular connections via autocrine and paracrine pathways. Well-developed osteocyte 3D networks exhibit high sensitivity to physiological cyclic compressive loading, for augmenting the mechano-responsive development of intercellular 3D architecture and regulation capability toward osteoblast activation as synergistic effects of static and dynamic mechanical stress.

Article Snippet: The cells cultured in the monolayer and 3D cultures were fixed with 4 % paraformaldehyde in phosphate buffer solution (FUJIFILM Wako Pure Chemical Corporation) for 15 min. After washing with PBS, the cells were blocked for non-specific protein binding using blocking buffer containing 3.0 % bovine serum albumin (BSA) (FUJIFILM Wako Pure Chemical Corporation), 0.1 % Triton X-100 (FUJIFILM Wako Pure Chemical Corporation), and 0.01 % Tween 20 (Sigma-Aldrich) for 1 h. Next, the cells were incubated overnight at 4 ◦C in a 1/200 dilution of anti-rabbit paxillin monoclonal antibody (ab32084; Abcam), 1/250 dilution of mouse anti-Cx43 monoclonal antibody (sc-271,837, Santa Cruz Biotechnology, Dallas, TX, USA) or 1/200 anti-total YAP (sc101,199, Santa Cruz Biotechnology).

Techniques: Activation Assay

Journal: JCI insight

Article Title: Connexin43 in mesenchymal lineage cells regulates body adiposity and energy metabolism in mice.

doi: 10.1172/jci.insight.170016

Figure Lengend Snippet: Figure 2. Cx43 is upregulated by HFD in BAT, and downregulated during adipogenic differentiation. (A) Expression of Gja1 (Cx43), Gja4 (Cx40), and Gjc1 (Cx45) mRNA by qPCR in inguinal WAT (iWAT) and BAT in 5-month-old male mice fed either a regular chow diet (RCD) or HFD for 12 weeks. Data are pre- sented as box-and-whisker plots representing the interquartile range (box bounds) with median (inside bar); whiskers represent maximum and minimum values. Groups were compared using 2-tailed Mann-Whitney U test. (B) Western blot of whole-cell lysates of confluent, undifferentiated cultures of Ing- WAT preadipocytic cells, cells isolated from the stromal vascular fraction (SVF) of WAT, and the osteogenic cell line, MC3T3-E1, as control. (C) Western blot of whole-cell lysates, Oil Red O stain, and (D) RT-qPCR analysis of mRNA of IngWAT cells before and during adipogenic differentiation (median and range, n = 6; P < 0.001 vs. time 0 at both time points). (E) Immunohistochemical staining (brown color) for Cx43 in WAT and BAT isolated from WT mice kept on either RCD or HFD and in cKOTW2 mice. Scale bar: 500 μm. Each image is representative of 3 mice per condition.

Article Snippet: Sections were then blocked using serum blocking solution (Invitrogen Histostain-SP Kit) and incubated overnight with primary antibody against mouse Cx43 (unconjugated F-7, mouse, Santa Cruz Biotechnology) diluted 1:200 in PBS/0.1% Triton X-100 (Sigma-Aldrich) 1 4 R E S E A R C H A R T I C L E JCI Insight 2024;9(6):e170016 https://doi.org/10.1172/jci.insight.170016 at 4°C.

Techniques: Expressing, Whisker Assay, MANN-WHITNEY, Western Blot, Isolation, Control, Staining, Quantitative RT-PCR, Immunohistochemical staining

Journal: JCI insight

Article Title: Connexin43 in mesenchymal lineage cells regulates body adiposity and energy metabolism in mice.

doi: 10.1172/jci.insight.170016

Figure Lengend Snippet: Figure 9. Schematic representation of the effect of Gja1 ablation on the metabolic response to an HFD. Left column: In normal mice, high dietary calorie intake changes energy metabolism, resulting in excess energy storage in fat depots and other organs, leading to obesity, hyperinsulinemia, high serum lipids, and glucose intolerance. In WAT depots (bottom row), fat accumulation occurs primarily by adipocyte hypertrophy; in BAT (top row), it leads to whitening as cells become engulfed by lipid droplets. Right column: Genetic ablation of Gja1 in the mesenchymal lineage (cKOTw2) mitigates these effects of high calorie intake, resulting in reduced BAT whitening and higher BAT activity (increased lipolysis, fatty acid oxidation, and oxidative phosphorylation), smaller WAT depots, and increased glucose uptake and utilization. At the organism level (middle row), Cx43-deficient mice are more active and more cold tolerant, burn more energy, and utilize more glucose than control littermates under high calorie intake. We propose that the increased energy consumption for physical activity and thermogenesis reduces fat accumulation, WAT hypertrophy, and BAT whitening, resulting in less severe obesity, par- tially preserved glucose tolerance, and better circulating lipid profile than in normal mice.

Article Snippet: Sections were then blocked using serum blocking solution (Invitrogen Histostain-SP Kit) and incubated overnight with primary antibody against mouse Cx43 (unconjugated F-7, mouse, Santa Cruz Biotechnology) diluted 1:200 in PBS/0.1% Triton X-100 (Sigma-Aldrich) 1 4 R E S E A R C H A R T I C L E JCI Insight 2024;9(6):e170016 https://doi.org/10.1172/jci.insight.170016 at 4°C.

Techniques: Activity Assay, Phospho-proteomics, Control