Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Downregulation of Cx43 reduces endothelial cell migration. ( a ) Western blot analysis of Cx43, Cx40, and Cx37 revealed that Cx43 is expressed in both human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC), whereas Cx40 and Cx37 are not expressed in HMEC. Cx43 (Cx43), Cx40 (Cx), and Cx37 (Cx) expressing HeLa cells were used as the positive controls. CTL: Wildtype HeLa cell lysate. ( b ) Western blot analysis of Cx43 in HMEC transfected with 100 nM siRNA against Cx43 or non-silencing control siRNA (Ctrl) for 48 h. The Cx43 expression was analysed using a polyclonal Cx43 antibody. Immunostaining for GAPDH was used as the loading control. ( c ) Serum-induced cell migration of HMEC transfected with a Cx43-siRNA or a control siRNA (Ctrl) in response to 10% FCS was assessed in a wound assay. Downregulation of Cx43 significantly decreased cell migration, represented as accumulated distance (*** p < 0.001 Ctrl-siRNA vs. Cx43-siRNA, n = 6, 3 independent cell cultures). ( d ) Representative single cell traces of migrated HMEC.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Migration, Western Blot, Expressing, Transfection, Control, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Downregulation of Cx43 impairs vessel sprouting ex vivo. ( a ) Downregulation of Cx43 in the mouse aortae was achieved by transfection with Cx43 siRNA (500 nM) or control (Ctrl) siRNA, as assessed by Western blot 48 h later. ( b ) Representative images of aortic sprouts in matrigel. Scale bar represents 200 µm. ( c ) The vessel sprouting area (** p < 0.01 Ctrl-siRNA vs. Cx43-siRNA, n = 2 aortae with eight aortic segments each at 3 days; n = 3–4 aortae with 6–8 aortic segments each at 6 days) and ( d ) the number of sprout bifurcations (** p < 0.01 Ctrl-siRNA vs. Cx43-siRNA, n = 2 aortae with 8 aortic segments each at 3 days; *** p < 0.001 Ctrl-siRNA vs. Cx43-siRNA n = 3–4 aortae with 6–8 aortic segments each at 6 days) were significantly reduced upon downregulation of Cx43, as assessed 3 days and 6 days after transfection in a matrigel assay.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Ex Vivo, Transfection, Control, Western Blot, Matrigel Assay

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Interaction of Cx43 and SHP-2 induces SHP-2 phosphatase activity. ( a ) Upon immunoprecipitation (IP) of SHP-2 from HeLa cells expressing only Cx43, Cx43 was co-immunoprecipitated. As a control, cells transfected with an empty vector (control (CTL)) were used. L: Whole cell lysates. ( b ) SHP-2 was detected upon immunoblotting after immunoprecipitation of Cx43 from HeLa cells expressing only Cx43 in contrast to the control cells (CTL). L: Whole cell lysates. Images originates from the same blot, which was cropped; ( c ) SHP-2 was immunoprecipitated together with Cx43 in HMEC; ( d ) Co-localization of SHP-2 and Cx43 was detected by immunofluorescent staining of HMEC followed by confocal microscopic imaging. Scale bar represents 20 µm; ( e ) SHP-2 phosphatase activity was significantly increased in HeLa cells expressing Cx43 compared to HeLa cells transfected with an empty vector (* p < 0.05, n = 4 independent cell cultures), as assessed by detection of dephosphorylation of a phosphate analogue in SHP-2 immunoprecipitates.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activity Assay, Immunoprecipitation, Expressing, Control, Transfection, Plasmid Preparation, Western Blot, Staining, Imaging, De-Phosphorylation Assay

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: Inactivation of SHP-2 reduces endothelial cell migration. ( a ) Overexpression of a dominant negative substrate trapping mutant SHP-2 (SHP-2 CS) in HMEC significantly reduced serum (10%) induced endothelial cell migration, as assessed by the accumulated distance in µm in an in vitro wound assay (* p < 0.05, n = 3 independent cell cultures) compared to SHP-2 WT. Additional knock-down of Cx43 (Cx43 siRNA) did not further decrease migration (ns: not significant, n = 3 independent cell cultures). ( b ) Representative single cell traces of migrated HMEC.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Migration, Over Expression, Dominant Negative Mutation, Mutagenesis, In Vitro, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

doi: 10.3390/ijms23010294

Figure Lengend Snippet: The Cx43 and SHP-2 interaction is vital for endothelial migration. ( a ) Treatment of HMEC overexpressing a constitutively active mutant SHP-2 (SHP-2 EA) with Cx43 siRNA did not rescue the reduced migratory response caused by Cx43 knock-down (Cx43 siRNA) (ns: not significant, n = 3 independent cell cultures) compared to SHP-2 EA cells treated with control siRNA (Ctrl) (* p < 0.05, n = 3 independent cell cultures), as assessed by the accumulated distance in µm in an in vitro wound assay (* p < 0.05, n = 3 independent cell cultures). ( b ) Representative single cell traces of migrated HMEC.

Article Snippet: For immunoprecipitation, the supernatants were incubated over night at 4 °C with a mouse anti-SHP-2 antibody (Santa Cruz) or a rabbit anti-Cx43 antibody (Sigma Aldrich), and were magnetic beads coated with protein A (Cx43) or G (SHP-2) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Migration, Mutagenesis, Knockdown, Control, In Vitro

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Western blot showing expression of Cx43 in WT (lane 1) and in Cx43-null astrocytes transfected with full length Cx43 (lane 3), with Cx43 carboxyl terminus (lane 4) and with Cx43M257 (lane 5). Lane 2 corresponds to untransfected Cx43-null cells. Lanes 1–4 correspond to immunoblots performed with Cx43-18A antibody and lane 5 with Cx43-16A. (B) Time courses of calcein FRAP obtained from untreated (black squares) and 48 h carbenoxolone-treated (open triangles) WT astrocytes, and from untransfected (open circles) and transfected Cx43-null astrocytes with full length Cx43 (open squares) and Cx43M257 (black circles). Data were obtained from astrocytes cultured from a minimum of 3 litters of mice.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Western Blot, Expressing, Transfection, Cell Culture

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Dose-response curves obtained for untreated (black squares) and 48 h carbenoxolone-treated (open triangles) WT and untransfected Cx43-null spinal cord astrocytes exposed to the P2Y1R agonist 2-MeS-ATP. Note that blockade of gap junctional communication in WT astrocytes does not alter the half-maximal response (EC50 value) induced by the P2Y1R agonist. Mean values were obtained from four to seven independent experiments. (B) Dose-response curves obtained for WT (black squares), untransfected (open circles) and transfected Cx43-null astrocytes with full length Cx43 (open squares), Cx43M257 (black circles) and Cx43CT (black triangles). Note that both full length Cx43 and Cx43CT but not Cx43M257 shifted the EC50 values of 2-MeS-ATP obtained for Cx43-null astrocytes to those obtained in WT cells. Data were obtained from 5 to 8 litters.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Transfection

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: Bar histograms showing the mean values of P2Y1R expression levels in WT, untransfected Cx43-null (KO), and in Cx43-null transfected with Cx43 CT, Cx43 truncated at position 257 (M257) and with full length Cx43. An example of western blots for Cx43 and β-actin is shown above.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Expressing, Transfection, Western Blot

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Dose-response curves obtained for WT (black squares) and untransfected (open circles) and Cx43Δ260–280 transfected (black triangles) Cx43-null astrocytes exposed to 2-MeS-ATP, showing that deletion of the Cx43 SH3 domain does not rescue P2Y1 receptor function. Mean±SE values are from 100 to 200 cells obtained from three litters. (B) Dose-response curves obtained from WT (black squares), and Cx43-null astrocytes untreated (open circles) and treated (black triangles) with a membrane permeant peptide corresponding to amino acids 260–280 of Cx43CT. (C) Bar histograms of the mean±SE values of intracellular calcium mobilization induced by 100 nM 2-MeS-ATP recorded from WT, Cx43-null and Cx43-null transfected with Cx43CT and Cx43M257 in the absence and presence of 5 µM PP2. Note that only untransfected and Cx43M257 transfected Cx43-null astrocytes that were not exposed to PP2 did not respond to agonist with intracellular calcium levels similar to WT astrocytes. Mean ± SE values are from 4 litters (**P < 0.001; ANOVA followed by Newman-Keuls’ Multiple Comparison Test).

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Transfection

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Western blot showing decreased expression levels of Cx43 following exposure of spinal cord astrocytes to IL-1β (B) Dose-response curves obtained for 2-MeS-ATP performed on Fura-2 loaded WT and Cx43 KO astrocytes treated for 24 h with IL-1β (20 ng/mL). Note that exposure to the cytokine altered the agonist EC50 values in WT astrocytes. About 180 cells from three independent experiments were used in each condition.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Western Blot, Expressing

Journal: International journal of molecular medicine

Article Title: Secretome of EMSCs neutralizes LPS‑induced acute lung injury via aerosol administration.

doi: 10.3892/ijmm.2023.5307

Figure Lengend Snippet: Figure 1. Morphology and fluorescence identification of EMSCs. (A) EMSCs in P0 and P3. (B) Expression of MSC markers (CD44, Vimentin) and neural crest‑related biomarkers (Cx43, Sox9). EMSCs, ectodermal mesenchymal stem cells; Sox9, SRY‑related high‑mobility group box‑containing protein 9; Cx43, Connexin43.

Article Snippet: Briefly, samples in 24‐well plates were incubated with primary antibodies for CD44 (1:100, Boster, A00052), Cx43 (1:100, Boster, BA1727), Sox9 (1:100, Boster, PA1026‐1), Vimentin (1:100, Boster, PB9359) after being blocked with BSA.

Techniques: Fluorescence, Expressing

Journal: Viruses

Article Title: SARS-CoV-2 Structural Proteins Modulated Blood-Testis Barrier-Related Proteins through Autophagy in the Primary Sertoli Cells.

doi: 10.3390/v15061272

Figure Lengend Snippet: Figure 2. The effects of SARS-CoV-2 SPs on the expression of BTB-related proteins. (A) qPCR analysis of the mRNA expression of BTB-related genes. (A-a–A-f) Sertoli cells were transiently transfected with SARS-CoV-2 SP, then total RNAs were extracted for qPCR to analyze the mRNA expressions of ZO-1, claudin11, occludin, N-cadherin, β-catenin, and CX43. β-actin was the internal control. (B) Immunoblotting analysis of the expressions of BTB-related proteins. (B-a) Sertoli cells were transiently transfected with SARS-CoV-2 SPs, and then total cellular extracts were analyzed by immunoblotting of ZO-1, Claudin11, occludin, N-cadherin, β-Catenin, CX43, and GAPDH as the internal control. (B-b–B-g) The relative levels of the targeted proteins were shown by histograms representing density readings of the gel bands, and the ratios were calculated relative to the GAPDH control. The data represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, calculated using the Student’s t-test of SARS-CoV-2 SPs transfected cells vs. empty plasmid (GFP-V) transfected cells.

Article Snippet: Rabbit antibodies specific to CX43 (A00599), N-cadherin (BA0673), and β-catenin (BA0426) were purchased from Boster (Wuhan, China).

Techniques: Expressing, Transfection, Control, Western Blot, Plasmid Preparation

Journal: Viruses

Article Title: SARS-CoV-2 Structural Proteins Modulated Blood-Testis Barrier-Related Proteins through Autophagy in the Primary Sertoli Cells.

doi: 10.3390/v15061272

Figure Lengend Snippet: Figure 5. Inhibiting autophagy with 3-MA suppressed SARS-CoV-2 SPs’ effects on BTB-related proteins. (a) Sertoli cells were pretreated with 3-MA (5 mM) for 6 h, then transiently transfected with SARS-CoV-2 SPs. The expression of LC3, ZO-1, claudin11, occludin, N-cadherin, β-catenin, CX43, and GAPDH (internal control) was analyzed by immunoblotting with specific antibodies as described in Materials and Methods. (b–g) The relative levels of the targeted proteins were shown by histograms representing density readings of the gel bands, and the ratios were calculated relative to the GAPDH control. The data represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, calculated using the Student’s t-test of SARS-CoV-2 SPs transfected cells vs. empty plasmid transfected cells.

Article Snippet: Rabbit antibodies specific to CX43 (A00599), N-cadherin (BA0673), and β-catenin (BA0426) were purchased from Boster (Wuhan, China).

Techniques: Transfection, Expressing, Control, Western Blot, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Senolytic activity of small molecular polyphenols from olive restores chondrocyte redifferentiation and promotes a pro-regenerative environment in osteoarthritis

doi: 10.18632/aging.103801

Figure Lengend Snippet: Downregulation of Cx43 during chondrogenesis improves differentiation towards chondrocytes. ( A ) Treatment of OACs with oleuropein (Oleu) or olive extract (OE) for 2 h significantly downregulates Cx43 protein detected by western-blot and flow cytometry. Median fluorescence intensity (MFI) ratios of oleuropein and OE treatments with respect to their untreated controls of each experiment are represented (n=10 independent experiments, P =0.0003). ( B ) Differentiation capacity of hMSCs isolated from bone marrow grown in adipogenic (top, 21 days) or osteogenic (bottom, 21 days) medium supplemented with 10 μM oleuropein or 10 μM OE. hMSCs cultured in growth medium were used as a control. Top, adipogenic evaluation by oil red O for lipid staining and by PPARγ gene expression. Data represent the ratio of cells containing lipid deposits to the total number of cells (n=3 independent experiments, P <0.0001). Values were normalized to hMSCs differentiated in adipogenic medium without treatment (AM). On the right, PPARγ gene expression (n=4 independent experiments, P< 0.0001). Alizarin red staining was used to detect calcium deposits for osteogenic differentiation. Values were obtained by counting red pixels and normalized to those of hMSCs differentiated in osteogenic medium without treatment (OM) (n=4-6 independent experiments, P =0.0317). OSTCN gene expression was measured to confirm osteogenic differentiation (n=4 independent experiments, P =0.0055). ( C ) Differentiation capacity of hMSCs isolated from bone marrow grown in chondrogenic medium as micromasses for 30 days. Representative images for Col2A1. The quantification is shown on the right (n=5–6 micromasses from independent experiments, P =0.0423). Chondrogenesis was also evaluated by ACAN gene expression quantification (n=3–4 independent experiments, P <0.0001). ( D ) Cx43 protein levels in hMSCs, isolated from bone marrow and from inguinal fat, differentiated for 7 and 14 days in the presence of chondrogenic medium (CM) in comparison to untreated hMSCs cultured in normal growth medium (GM). ( E ) Cx43 RNA expression of hMSCs cultured for 14 days in the presence of chondrogenic medium (CM) alone or supplemented with 10 μM oleuropein. Data were normalized to HPRT-1 levels (n=5-6 independent experiments, P <0.0001). ( F ) Cx43 protein levels were analyzed by western blot in OACs differentiated for 7 days in the presence of chondrogenic medium (CM), supplemented with 10 μM oleuropein. The graph represents the quantification from 3 independent experiments ( P =0.0004). Data is expressed as mean±SD, one-way ANOVA; * P <0.05, ** P <0.01 and *** P <0.0001.

Article Snippet: Finally, cells were incubated with APC-conjugated anti-human Cx43 antibody (R&D Systems, FAB7737A) for 30 min at 4°C.

Techniques: Western Blot, Flow Cytometry, Fluorescence, Isolation, Cell Culture, Control, Staining, Gene Expression, Comparison, RNA Expression

Journal: Aging (Albany NY)

Article Title: Senolytic activity of small molecular polyphenols from olive restores chondrocyte redifferentiation and promotes a pro-regenerative environment in osteoarthritis

doi: 10.18632/aging.103801

Figure Lengend Snippet: Downregulation of Cx43 by oleuropein decreases GJIC and improves the phenotype of OACs. ( A ) Oleuropein (Oleu) treatment significantly decreases GJIC evaluated by an SL/DT assay when OACs were exposed with this molecule for 2 h (top, n=6 independent experiments; Student’s t test, P <0.0001). The results were confirmed by calcein transfer by flow cytometry (n=4 independent experiments; Student’s t test, P =0.0037). ( B ) Graph showing the effect of oleuropein on GJIC when healthy chondrocytes (N) were exposed to 10 μM oleuropein compared with OACs (n=5 independent experiments; one-way ANOVA, P =0.0004). ( C ) OACs cultured for 7 days with 10 μM Oleu showed reduced expression of the mesenchymal markers CD105 and CD166, analyzed by flow cytometry. Student’s t test, P =0.0039 (CD105) and P =0.0022 (CD166), n=6 independent experiments. CD166 levels were also analyzed by western blot (n=3 independent experiments, Student’s t test, P =0.0046). ( D ) Downregulation of Cx43 increased Col2A1, detected by immunofluorescence in OACs treated with 10 μM oleuropein. Graphs represent the corrected total cell fluorescence (CTCF) of Cx43 and Col2a1 (n=4 independent experiments). Student’s t test, P <0.0001 (Cx43), and P =0.0007 (Col2a1). ( E ) mRNA levels of IL-1ß, IL-6, COX-2 and MMP-3 of OACs cultured in normal medium (UT) exposed to 10 μM oleuropein for 2 h. n=4–7 independent experiments. Student’s t test: P = 0.033 (IL-1ß), P <0.0001 (IL-6), P =0.1013 (COX-2), P =0.0466 (MMP-3). ( F ) IL-6 detected by ELISA when OACs were treated with oleuropein for 72 h (n=4 independent experiments, Student’s t test, P =0.0345). IL-6 (n=3 independent experiments) and COX-2 (n=4 independent experiments) protein levels detected by western-blot in OACs treated with 10 μM oleuropein for 72 h. Student’s t test, P =0.0193 (IL-6), P =0.0141 (COX-2). Data is expressed as mean±SD; * P <0.05, ** P <0.01 and *** P <0.0001.

Article Snippet: Finally, cells were incubated with APC-conjugated anti-human Cx43 antibody (R&D Systems, FAB7737A) for 30 min at 4°C.

Techniques: Flow Cytometry, Cell Culture, Expressing, Western Blot, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Senolytic activity of small molecular polyphenols from olive restores chondrocyte redifferentiation and promotes a pro-regenerative environment in osteoarthritis

doi: 10.18632/aging.103801

Figure Lengend Snippet: Oleuropein treatment enhances chondrocyte redifferentiation. ( A ) Immunohistochemistry of Col2A1 (4-6 independent experiments; one-way ANOVA, P =0.0019) and toluidine blue staining of proteoglycan subunits (n=6 independent experiments; one-way ANOVA, P =0.059) indicate significant enrichment in ECM components in OACs micromasses grown in 3D culture for 30 days in chondrogenic medium (CM) when supplemented with 10 μM oleuropein (Oleu) or OE. ( B ) Cx43 protein levels detected by western blot (and normalized to Ponceau staining) are reduced when OACs micromasses are exposed to CM supplemented with 10 μM oleuropein or OE for 21 days (n=3 independent experiments; one-way ANOVA, P =0.0328). ( C ) Oil red staining showing reduced OACs dedifferentiation upon exposure to Oleu or OE in adipogenic medium (n=5 independent experiments; one-way ANOVA, P =0.0001). ( D ) Nuclear levels of Twist-1 were decreased in OACs cultured with 10 μM oleuropein for 2 h. Lamin A was used as a loading control (n=3 independent experiments; Student’s t test, P =0.001). ( E ) Cx43 protein levels in primary OACs after 1-h treatment with oleuropein or oligomycin. Western blot represents n=4 independent experiments. Quantification is shown on the right (one-way ANOVA, P =0.0036). On the right, immunofluorescence for Twist-1 (red) in primary OACs treated with 5 μg/ml oligomycin and 10 μM oleuropein for 1 h. The graph represents the percentage of cells with Twist-1 nuclear localization (n=4 independent experiments; one-way ANOVA, P =0.0067). ( F ) The mRNA expression of the EMT markers Twist-1, N-Cadherin and Vimentin in OACs treated with 10 μM oleuropein for 2 h. Data were normalized to HPRT-1 levels. n= 5 independent experiments; Student’s t test: P <0.0001 (Twist-1), P = 0.0011 (N-Cad), P =0.0209 (Vim). Data is expressed as mean±SD; * P <0.05, ** P <0.01 and *** P <0.0001.

Article Snippet: Finally, cells were incubated with APC-conjugated anti-human Cx43 antibody (R&D Systems, FAB7737A) for 30 min at 4°C.

Techniques: Immunohistochemistry, Staining, Western Blot, Cell Culture, Control, Immunofluorescence, Expressing

Journal: Aging (Albany NY)

Article Title: Senolytic activity of small molecular polyphenols from olive restores chondrocyte redifferentiation and promotes a pro-regenerative environment in osteoarthritis

doi: 10.18632/aging.103801

Figure Lengend Snippet: Oleuropein modulates the Cx43 promoter activity in chondrocytes. ( A ) Treatment with 10 μM oleuropein for 2 h decreases Cx43 protein levels in T/C-28a2 cells (n=4 independent experiments, Student’s t test, P =0.0012), but this effect was not observed in the same cell line overexpressing Cx43 (pIRES-Cx43)(n=3 independent experiments, Student’s t test, P =0.0624). ( B ) Luciferase reporter assay indicating that oleuropein inhibits Cx43 promoter activity. The graphs indicate the normalized luminescence activity in the T/C-28a2 transfected with a pGL3-basic plasmid containing 300 base pairs of Cx43 promoter ligated to the luciferase gene. Cells were cultured in DMEM with 10% FBS (UT) and with 5 μg/ml oligomycin or 10 μM oleuropein for 1 h as indicated (n=4 independent experiments; one-way ANOVA, P =0.0012). On the right, Cx43 gene expression under 5 μg/ml oligomycin and 10 μM oleuropein treatment in OACs treated for 1 h (n=4 independent experiments; one-way ANOVA, P =0.0002). Data were normalized to HPRT-1 levels. ( C ) Immunofluorescence assays of Cx43 in OACs treated with 10 μM oleuropein or 5 μg/ml oligomycin for 1 h. Data were normalized to the untreated condition (n=3 independent experiments; one-way ANOVA, P <0.0001). Data is expressed as mean±SD; * P <0.05, ** P <0.01 and *** P <0.0001.

Article Snippet: Finally, cells were incubated with APC-conjugated anti-human Cx43 antibody (R&D Systems, FAB7737A) for 30 min at 4°C.

Techniques: Activity Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Cell Culture, Gene Expression, Immunofluorescence

Journal: Aging (Albany NY)

Article Title: Senolytic activity of small molecular polyphenols from olive restores chondrocyte redifferentiation and promotes a pro-regenerative environment in osteoarthritis

doi: 10.18632/aging.103801

Figure Lengend Snippet: Cx43 downregulation by oleuropein decreased chondrocyte senescence. ( A ) SA-βGal activity detected by flow cytometry in OACs treated with 10 μM oleuropein (Oleu) for 7 and 14 days (n=3–7 independent experiments; one-way ANOVA, P <0.0001). ( B ) The graphs show the comparative analysis of SA-βGal activity measured by flow cytometry of OACs exposed for 24 h to 10 μM oleuropein or 5 μg/ml oligomycin as indicated (n=5 independent experiments; one-way ANOVA, P =0.0003). On the right, SA-βGal activity determined by X-Gal cleavage and cell staining (blue), evaluated by microscopy in OACs treated for 7 days with 10 μM oleuropein or 5 μg/ml oligomycin (n=3 independent experiments; one-way ANOVA, P <0.0001). ( C ) p16 mRNA expression of OACs treated with 10 μM oleuropein for 2 h. Data were normalized to HPRT-1 levels (n=5 independent experiments; Student’s t test, P =0.0002). ( D ) Western blot of p53 (n=3 independent experiments), p21 (n=3 independent experiments) and p16 (n=4 independent experiments) in OACs treated with 10 μM oleuropein for 2 h. α-tubulin was used as a loading control. Student’s t test, P =0.001 (p53), P =0.0278 (p21), P =0.0286 (p16). ( E ) Cell proliferation evaluated by immunofluorescence of Ki-67 in T/C-28a2 chondrocytes treated with 10 μM palbociclib and/or 10 μM oleuropein for 24 h. Images represent n= 3 independent experiments. One-way ANOVA, P =0.0434 (UT vs Palbo); P =0.0096 (Palbo vs Palbo+Oleu). ( F ) Downregulation of Cx43 by oleuropein attenuates IL-6 and COX-2 upregulation when OACs are exposed to oligomycin for 1 h (n=3–9 independent experiments; one-way ANOVA). ( G ) Western blot (n=3 independent experiments) shows the effect of 10 μM oleuropein and 10 ng/mL TNFα treatments (for 1 h) on Cx43 protein levels in OACs (one-way ANOVA, P =0.0018). On the right, NF-κB detected by immunofluorescence in OACs treated with 10 ng/mL TNFα for 1 h. This effect is partially abolished by 1-h 10 μM oleuropein treatment. The graph represents the cell percentage with nuclear NF-κB staining (n=7 independent experiments; one-way ANOVA, P =0.0055). ( H ) Nuclear levels of NF-kß in OACs cultured with 10 μM oleuropein for 2 h. Lamin A was used as a loading control (n=3 independent experiments; Student’s t test, P =0.0021). Data is expressed as mean±SD; * P <0.05, ** P <0.01 and *** P <0.0001.

Article Snippet: Finally, cells were incubated with APC-conjugated anti-human Cx43 antibody (R&D Systems, FAB7737A) for 30 min at 4°C.

Techniques: Activity Assay, Flow Cytometry, Staining, Microscopy, Expressing, Western Blot, Control, Immunofluorescence, Cell Culture

Journal: Aging (Albany NY)

Article Title: Senolytic activity of small molecular polyphenols from olive restores chondrocyte redifferentiation and promotes a pro-regenerative environment in osteoarthritis

doi: 10.18632/aging.103801

Figure Lengend Snippet: Oleuropein treatment decreased cellular senescence in synoviocytes and bone cells isolated from patients. ( A ) Cx43 protein levels analyzed by western blot in synoviocytes treated with 10 μM oleuropein for 2 h (n=7 independent experiments, P =0.0313). ( B ) Treatment of synoviocytes with 10 μM of oleuropein for 7 days detected by SA-βGal activity (n=4 independent experiments, P <0.0001). ( C ) p16 mRNA levels of synoviocytes treated with 10 μM oleuropein for 2 h. Data were normalized to HPRT-1 levels (n= 4 independent experiments, P <0.0001). On the right, mRNA levels of IL-1ß, IL-6 and COX-2 of synoviocytes cultured in normal medium (DMEM 10% FBS) and exposed to 10 μM oleuropein for 2 h. Data were normalized to HPRT-1 levels. N=4 independent experiments, P <0.0001 (IL-1ß), P =0.0024 (IL-6), P =0.0025 (COX-2). ( D ) Cx43 protein levels analyzed by western blot in bone cells treated with 10 μM oleuropein for 2 h (n=4 independent experiments, P =0.0319). ( E ) 10 μM of oleuropein treatment for 7 days reduces senescence levels in bone cells as detected by SA-βGal and flow cytometry (n=3 independent experiments, P =0.0149). ( F ) p16 mRNA expression of bone cells treated with 10 μM oleuropein for 2 h. Data were normalized to HPRT-1 levels (n= 4 independent experiments, P =0.002). On the right, mRNA levels of IL-1ß, IL-6 and COX-2 of bone cells cultured in normal medium (DMEM 10% FBS) exposed to 10 μM oleuropein for 2 h. Data were normalized to HPRT-1 levels. N=3-4 independent experiments. P =0.0463 (IL-1ß), P =0.0077 (IL-6), P =0.0002 (COX-2). Data is expressed as mean±SD, Student’s t test; * P <0.05, ** P <0.01 and *** P <0.0001.

Article Snippet: Finally, cells were incubated with APC-conjugated anti-human Cx43 antibody (R&D Systems, FAB7737A) for 30 min at 4°C.

Techniques: Isolation, Western Blot, Activity Assay, Cell Culture, Flow Cytometry, Expressing

Journal: Aging (Albany NY)

Article Title: Senolytic activity of small molecular polyphenols from olive restores chondrocyte redifferentiation and promotes a pro-regenerative environment in osteoarthritis

doi: 10.18632/aging.103801

Figure Lengend Snippet: Cx43 overactivity contributes to disease progression. Cx43 overexpression leads to accumulation of dedifferentiated and senescent cells involved in disease progression in OA patients. These phenotypic changes results in the synthesis of ECM remodeling factors involved in tissue degradation (MMPs) and proinflammatory factors, such as IL-1ß and IL-6, which facilitate the dedifferentiation and reprogramming of neighboring cells. These factors may further spread senescence and dedifferentiation to surrounding tissues contributing to joint degeneration. Downregulation of Cx43 by oleuropein treatment contributes to the elimination of senescent cells and redifferentiation of osteoarthritic chondrocytes into fully differentiated cells, able to support the ECM composition and restoring the regenerative capacity of the tissue. However, oleuropein may have other targets that may contribute to the drug effect. In addition, oleuropein treatment might improve the effectiveness of stem cell therapy, by promoting chondrogenic and osteogenic differentiation, and by inhibiting adipogenesis.

Article Snippet: Finally, cells were incubated with APC-conjugated anti-human Cx43 antibody (R&D Systems, FAB7737A) for 30 min at 4°C.

Techniques: Biomarker Discovery, Over Expression