α-actinin Search Results


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  • 99
    Millipore monoclonal anti alpha actinin sarcomeric antibody
    Distribution pattern and levels of histone H4 acetylation (green) and <t>α-actinin</t> (red) in ( Aa ) wt mESCs and ( Ab ) HDAC1 dn mESCs differentiated at dd20 and dd25. Panel ( Ba ) shows non-treated wt mESCs and HDCA1 dn mESCs at dd25, and panels ( Bb – d ) show wt mESCs and HDCA1 dn mESCs treated by HDACi at dd25: ( Bb ) TSA treatment, ( Bc ) SAHA treatment, and ( Bd ) VPA treatment. DAPI (blue) was used as a counterstain of the cell nuclei.
    Monoclonal Anti Alpha Actinin Sarcomeric Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3053 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti α actinin
    Laser confocal images of induced Muse double-stained for <t>α-actinin</t> (green) and connexin 43 (red)/diamidino-2-phenylindole (blue) in the adherent, Sus+Ad, and Sus+Ad+DN groups. Scale bar: 100 µm.
    Anti α Actinin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 844 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam α actinin
    Laser confocal images of induced Muse double-stained for <t>α-actinin</t> (green) and connexin 43 (red)/diamidino-2-phenylindole (blue) in the adherent, Sus+Ad, and Sus+Ad+DN groups. Scale bar: 100 µm.
    α Actinin, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 370 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sarcomeric α actinin
    Wnt2+ and Gli1+ cells define a cardiopulmonary progenitor (CPP) and generate mesoderm derivatives of the lung and cardiac inflow tract Wnt2 is expressed in the ventral mesoderm surrounding the anterior foregut and overlapping the posterior pole of the heart (a and b, arrows). Wnt2+ cells tagged at E8.5 can generate cells within the cardiac inflow tract as well as in the lung mesoderm (c and d). Wnt2+ cells tagged at E8.5 generate myocardium of the PV, smooth muscle of the PA, endothelium of the proximal PA, and Pdgfrβ+ pericyte-like cells in the lung by E17.5 (e–h). Gli1+ cells generate derivatives within the inflow tract and early lung mesoderm similar to Wnt2+ cells (i and j) and generate mesoderm lineages within the lung including airway and vascular smooth muscle (k and l), and endothelium of the proximal pulmonary vessels (n) as well as the myocardium of the atria (m). CPPs are located in a region of overlapping Wnt2, Gli1, and Isl1 expression between the developing heart and the anterior foregut (o). AP=anterior pole of the heart, PP=posterior pole of the heart, AFG=anterior foregut, AT=atria, OFT=outflow tract, PA=pulmonary artery, PV=pulmonary vein, LB=lung bud, ASM=airway smooth muscle, VSM=vascular smooth muscle, SMA=smooth muscle actin, SAA= sarcomeric <t>α-actinin,</t> VWF=von Willibrand factor. Scale bars b=100 μm, e–h=50 μm, k–n=50 μm.
    Sarcomeric α Actinin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 597 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology α actinin
    Induction of apolipoprotein (APOL) 1 expression in human embryonic kidney (HEK) cells induces the expression of transition markers. A: To determine APOL1 induction in kidney cells with undetectable APOL1 protein expression, HEK cells were transfected with empty vector (EV) or APOL1 plasmids. Protein blots were probed for APOL1 and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The same cellular lysates were probed for <t>α-actinin,</t> Wilms' tumor 1 (WT1), CD2AP, and podocalyxin (PDX) and reprobed for GAPDH. Gels from two different lysates are displayed. B: Cumulative densitometric data from the gels generated from the above-mentioned lysates are displayed. APOL1 induction in parietal epithelial cells (PECs) is associated with the expression of transition markers in HEK cells. C: To determine the effect of APOL1 induction on PEC markers in HEK cells, RNAs were extracted from the above-mentioned lysates. cDNAs were amplified for PAX2 and claudin 1. Cumulative data are shown. APOL1 induction in HEK cells down-regulates the transcription of PEC markers. D: To assess the effect of APOL1 induction on PEC transition markers in HEK cells, RNAs were extracted from the above-mentioned lysates. cDNAs were amplified for APOL1 , WT1 , PDX , α-actinin, and CD2AP . Cumulative data are shown in a bar diagram. The induction of APOL1 in HEK cells is associated with enhanced transcription of transition markers in HEK cells. n = 4 ( A ). ∗ P
    α Actinin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 404 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti alpha actinin antibody
    Induction of apolipoprotein (APOL) 1 expression in human embryonic kidney (HEK) cells induces the expression of transition markers. A: To determine APOL1 induction in kidney cells with undetectable APOL1 protein expression, HEK cells were transfected with empty vector (EV) or APOL1 plasmids. Protein blots were probed for APOL1 and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The same cellular lysates were probed for <t>α-actinin,</t> Wilms' tumor 1 (WT1), CD2AP, and podocalyxin (PDX) and reprobed for GAPDH. Gels from two different lysates are displayed. B: Cumulative densitometric data from the gels generated from the above-mentioned lysates are displayed. APOL1 induction in parietal epithelial cells (PECs) is associated with the expression of transition markers in HEK cells. C: To determine the effect of APOL1 induction on PEC markers in HEK cells, RNAs were extracted from the above-mentioned lysates. cDNAs were amplified for PAX2 and claudin 1. Cumulative data are shown. APOL1 induction in HEK cells down-regulates the transcription of PEC markers. D: To assess the effect of APOL1 induction on PEC transition markers in HEK cells, RNAs were extracted from the above-mentioned lysates. cDNAs were amplified for APOL1 , WT1 , PDX , α-actinin, and CD2AP . Cumulative data are shown in a bar diagram. The induction of APOL1 in HEK cells is associated with enhanced transcription of transition markers in HEK cells. n = 4 ( A ). ∗ P
    Monoclonal Anti Alpha Actinin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc α actinin
    Valproic acid causes marked morphological and ultrastructural changes, and promotes EndMT-like phenotypic switching in HUVECs. (A) Diluent-treated control HUVECs, cultured on a two-dimensional plate, formed a confluent monolayer with the typical EC ‘cobblestone’ morphology (left panel). VPA treatment resulted in marked morphological changes whereby HUVECs took on an enlarged spindle-shaped appearance with smooth surfaces (right panel). Both micrographs were taken at the same magnification (10X). (B) Immunofluorescent micrographs demonstrating cytoskeletal protein re-organization in HUVECs following VPA treatment. <t>α-Actinin</t> positivity is indicated in green and nuclei were stained with DAPI (blue); scale bar = 10 μm. (C) HUVECs were treated with diluent control or VPA. Total RNA and protein were extracted at 24 and 48 h, respectively. Differential (C) transcript (qPCR) ∗∗ p
    α Actinin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti sarcomeric alpha actinin antibody ea 53
    Valproic acid causes marked morphological and ultrastructural changes, and promotes EndMT-like phenotypic switching in HUVECs. (A) Diluent-treated control HUVECs, cultured on a two-dimensional plate, formed a confluent monolayer with the typical EC ‘cobblestone’ morphology (left panel). VPA treatment resulted in marked morphological changes whereby HUVECs took on an enlarged spindle-shaped appearance with smooth surfaces (right panel). Both micrographs were taken at the same magnification (10X). (B) Immunofluorescent micrographs demonstrating cytoskeletal protein re-organization in HUVECs following VPA treatment. <t>α-Actinin</t> positivity is indicated in green and nuclei were stained with DAPI (blue); scale bar = 10 μm. (C) HUVECs were treated with diluent control or VPA. Total RNA and protein were extracted at 24 and 48 h, respectively. Differential (C) transcript (qPCR) ∗∗ p
    Anti Sarcomeric Alpha Actinin Antibody Ea 53, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cytoskeleton Inc alpha actinin
    SIKE forms direct interactions with tubulin and <t>α‐actinin.</t> IVP reactions of 6xHis‐ SIKE ‐ FL ( SIKE or S) or FL ‐ SIKE ‐6xHis ( SIKE ‐26b or S‐26b) (8 μ m ) with monomeric tubulin (A, 4–11.2 μ m ), α‐actinin (B, C, 4–11.2 μ m ), or lysozyme (D, 4–11.2 μ m ) were precipitated with Ni‐ NTA resin and separated by SDS / PAGE (12% (A), 4–15% (B, C), or 15% (D) Tris/glycine) and stained with SimplyBlue SafeStain. Control reactions of individual proteins incubated with Ni‐ NTA resin were subjected to the same reaction conditions and separated by SDS / PAGE (15% (E), 4–15% (F)). UB , unbound sample; B, bound sample; 1 : 0.5 molar ratio, 1 : 1 molar ratio, 1 : 1.4 molar ratio. (A) Lanes: 1 – SIKE ‐ FL starting material; 2 – tubulin starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. B) Lanes: 1 – SIKE ‐ FL starting material; 2 – α‐actinin starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. (C) Gel 1 Lanes: 1 – SIKE ‐26b starting material; 2 – blank; 3 – α‐actinin starting material. Gel 2 Lanes: 1 – 1 : 0.5 UB ; 2 – 1 : 0.5 B; 3 – 1 : 1 UB ; 4 – 1 : 1 B; 5 – 1 : 1.4 UB ; 6 – 1 : 1.4 B. D) Lanes: 1 – SIKE ‐ FL starting material; 2 – lysozyme starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. (E) Lanes: 1 – SIKE ‐ FL starting material; 2 – UB ; 3 – B; 4 – tubulin starting material; 5 – UB ; 6 – B; 7 – lysozyme starting material; 8 – UB ; 9 – B. F) Gel 1 Lanes: 1 – α‐actinin starting material; 5 – UB ; 6 – B. Gel 2 Lanes: 1 – SIKE ‐26 starting material; 2 – UB ; 3 – B; control reactions show that only SIKE is bound to the Ni‐ NTA resin. The C‐terminal 6xHis‐tagged SIKE is not all bound to the Ni‐ NTA , but can be found in the unbound and bound fractions. SIKE does not interact nonspecifically with the negative control, lysozyme. In tubulin reactions, SIKE precipitates tubulin indicating a direct interaction. When SIKE is 6xHis‐tagged at the N terminus, α‐actinin pre‐incubation inhibits SIKE 's ability to bind to the resin, suggesting α‐actinin may complex to SIKE and mask SIKE 's 6xHis tag. With a C‐terminal 6xHis tag, SIKE precipitates α‐actinin indicating a direct interaction. Gels are representative of three independent experiments.
    Alpha Actinin, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 90/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore α actinin antibodies
    SIKE forms direct interactions with tubulin and <t>α‐actinin.</t> IVP reactions of 6xHis‐ SIKE ‐ FL ( SIKE or S) or FL ‐ SIKE ‐6xHis ( SIKE ‐26b or S‐26b) (8 μ m ) with monomeric tubulin (A, 4–11.2 μ m ), α‐actinin (B, C, 4–11.2 μ m ), or lysozyme (D, 4–11.2 μ m ) were precipitated with Ni‐ NTA resin and separated by SDS / PAGE (12% (A), 4–15% (B, C), or 15% (D) Tris/glycine) and stained with SimplyBlue SafeStain. Control reactions of individual proteins incubated with Ni‐ NTA resin were subjected to the same reaction conditions and separated by SDS / PAGE (15% (E), 4–15% (F)). UB , unbound sample; B, bound sample; 1 : 0.5 molar ratio, 1 : 1 molar ratio, 1 : 1.4 molar ratio. (A) Lanes: 1 – SIKE ‐ FL starting material; 2 – tubulin starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. B) Lanes: 1 – SIKE ‐ FL starting material; 2 – α‐actinin starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. (C) Gel 1 Lanes: 1 – SIKE ‐26b starting material; 2 – blank; 3 – α‐actinin starting material. Gel 2 Lanes: 1 – 1 : 0.5 UB ; 2 – 1 : 0.5 B; 3 – 1 : 1 UB ; 4 – 1 : 1 B; 5 – 1 : 1.4 UB ; 6 – 1 : 1.4 B. D) Lanes: 1 – SIKE ‐ FL starting material; 2 – lysozyme starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. (E) Lanes: 1 – SIKE ‐ FL starting material; 2 – UB ; 3 – B; 4 – tubulin starting material; 5 – UB ; 6 – B; 7 – lysozyme starting material; 8 – UB ; 9 – B. F) Gel 1 Lanes: 1 – α‐actinin starting material; 5 – UB ; 6 – B. Gel 2 Lanes: 1 – SIKE ‐26 starting material; 2 – UB ; 3 – B; control reactions show that only SIKE is bound to the Ni‐ NTA resin. The C‐terminal 6xHis‐tagged SIKE is not all bound to the Ni‐ NTA , but can be found in the unbound and bound fractions. SIKE does not interact nonspecifically with the negative control, lysozyme. In tubulin reactions, SIKE precipitates tubulin indicating a direct interaction. When SIKE is 6xHis‐tagged at the N terminus, α‐actinin pre‐incubation inhibits SIKE 's ability to bind to the resin, suggesting α‐actinin may complex to SIKE and mask SIKE 's 6xHis tag. With a C‐terminal 6xHis tag, SIKE precipitates α‐actinin indicating a direct interaction. Gels are representative of three independent experiments.
    α Actinin Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam sarcomeric α actinin
    Confocal images and Western blotting analysis of myotubes cultured under various stimulation regimes. Skeletal myotubes cultured under various stimulation regimes were immunostained for <t>α-actinin</t> (green; blue = nuclei) to show striation. (a–c) Application of 5 or 10% cyclic strain to cells cultured on aligned fibers resulted in overstretching and appeared to drive the myoblasts into proliferative instead of differentiative state; (d, i) Pre-stretching of the myoblasts before application of cyclic loading led to enhanced differentiation; (e, f, i) Electrical stimulation applied to the myotubes at earlier time points (Day 0 and 4 post-differentiation) showed detrimental effect in the development of striated myotubes; (g, i) When electrical stimulation was applied post-myotube assembly (day 7), it induced contraction and led to an increase in striation; (h, i) Cells cultured under synchronized stimulation showed insignificant increase in myotube diameter but significant increase in percent of striated myotubes; (j) Western blotting demonstrated an upregulation of contractile proteins mechanical, electrical or electromechanical stimuli was applied. There is a significant level of increase in fast myosin heavy chain when cells were cultured under synchronized stimulation. Scale bar: 20 μ m.
    Sarcomeric α Actinin, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam alpha actinin
    Confocal images and Western blotting analysis of myotubes cultured under various stimulation regimes. Skeletal myotubes cultured under various stimulation regimes were immunostained for <t>α-actinin</t> (green; blue = nuclei) to show striation. (a–c) Application of 5 or 10% cyclic strain to cells cultured on aligned fibers resulted in overstretching and appeared to drive the myoblasts into proliferative instead of differentiative state; (d, i) Pre-stretching of the myoblasts before application of cyclic loading led to enhanced differentiation; (e, f, i) Electrical stimulation applied to the myotubes at earlier time points (Day 0 and 4 post-differentiation) showed detrimental effect in the development of striated myotubes; (g, i) When electrical stimulation was applied post-myotube assembly (day 7), it induced contraction and led to an increase in striation; (h, i) Cells cultured under synchronized stimulation showed insignificant increase in myotube diameter but significant increase in percent of striated myotubes; (j) Western blotting demonstrated an upregulation of contractile proteins mechanical, electrical or electromechanical stimuli was applied. There is a significant level of increase in fast myosin heavy chain when cells were cultured under synchronized stimulation. Scale bar: 20 μ m.
    Alpha Actinin, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Distribution pattern and levels of histone H4 acetylation (green) and α-actinin (red) in ( Aa ) wt mESCs and ( Ab ) HDAC1 dn mESCs differentiated at dd20 and dd25. Panel ( Ba ) shows non-treated wt mESCs and HDCA1 dn mESCs at dd25, and panels ( Bb – d ) show wt mESCs and HDCA1 dn mESCs treated by HDACi at dd25: ( Bb ) TSA treatment, ( Bc ) SAHA treatment, and ( Bd ) VPA treatment. DAPI (blue) was used as a counterstain of the cell nuclei.

    Journal: International Journal of Molecular Sciences

    Article Title: Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells

    doi: 10.3390/ijms19082425

    Figure Lengend Snippet: Distribution pattern and levels of histone H4 acetylation (green) and α-actinin (red) in ( Aa ) wt mESCs and ( Ab ) HDAC1 dn mESCs differentiated at dd20 and dd25. Panel ( Ba ) shows non-treated wt mESCs and HDCA1 dn mESCs at dd25, and panels ( Bb – d ) show wt mESCs and HDCA1 dn mESCs treated by HDACi at dd25: ( Bb ) TSA treatment, ( Bc ) SAHA treatment, and ( Bd ) VPA treatment. DAPI (blue) was used as a counterstain of the cell nuclei.

    Article Snippet: The procedure was performed at room temperature (RT) for 1 h. After washing with PBS for 15 min, samples were incubated overnight at 4 °C with the monoclonal antibodies of interest: α-actinin (#A-7811, Sigma-Aldrich), H3K9ac (#06-942, Merc Millipore, MA, USA) and H4ac (#382160, Merc Millipore).

    Techniques:

    Histone acetylation and methylation in HDAC1 wt and HDAC1 dn mESCs induced into cardiomyocytes and treated with HDACi. The level of H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in ( A ) HDAC1 wt mESCs and ( B ) HDAC1 dn mESCs. In three biological replicates, Western blots were performed on one gel. For the data presented in panel A or B, the gel was separated by Photoshop to show samples that were compared in one relevant subset. Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized and quantified to the level of GAPDH ( C ). In wt and HDAC1 dn non-treated cells and in TSA-, SAHA-, or VPA-treated mESCs, panel ( Ca ) shows the levels of H4ac, ( Cb ) shows H4K20ac, and ( Cc ) shows the levels of α-actinin. The total protein levels were measured using a µQuant spectrophotometer for each sample, and an identical protein amount was loaded on the gels. In panel ( A , B ), the levels of histone markers are also shown for embryonic hearts (e15). Quantification of the protein levels in panel ( C ) was performed using ImageJ software (NIH, freeware). Statistical analyses were performed using Student’s t -test; asterisks (*) in panel ( Ca – c ) show statistically significant differences at p ≤ 0.05. Note that the y -axis-scale in panel ( Ca ) is different (red frames) for the wt and HDAC1 dn cells for technical purposes. In panel ( Ca ), the level of H4ac is significantly less in the wt mESCs when compared with the HDAC1 dn cells ( Cb ).

    Journal: International Journal of Molecular Sciences

    Article Title: Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells

    doi: 10.3390/ijms19082425

    Figure Lengend Snippet: Histone acetylation and methylation in HDAC1 wt and HDAC1 dn mESCs induced into cardiomyocytes and treated with HDACi. The level of H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in ( A ) HDAC1 wt mESCs and ( B ) HDAC1 dn mESCs. In three biological replicates, Western blots were performed on one gel. For the data presented in panel A or B, the gel was separated by Photoshop to show samples that were compared in one relevant subset. Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized and quantified to the level of GAPDH ( C ). In wt and HDAC1 dn non-treated cells and in TSA-, SAHA-, or VPA-treated mESCs, panel ( Ca ) shows the levels of H4ac, ( Cb ) shows H4K20ac, and ( Cc ) shows the levels of α-actinin. The total protein levels were measured using a µQuant spectrophotometer for each sample, and an identical protein amount was loaded on the gels. In panel ( A , B ), the levels of histone markers are also shown for embryonic hearts (e15). Quantification of the protein levels in panel ( C ) was performed using ImageJ software (NIH, freeware). Statistical analyses were performed using Student’s t -test; asterisks (*) in panel ( Ca – c ) show statistically significant differences at p ≤ 0.05. Note that the y -axis-scale in panel ( Ca ) is different (red frames) for the wt and HDAC1 dn cells for technical purposes. In panel ( Ca ), the level of H4ac is significantly less in the wt mESCs when compared with the HDAC1 dn cells ( Cb ).

    Article Snippet: The procedure was performed at room temperature (RT) for 1 h. After washing with PBS for 15 min, samples were incubated overnight at 4 °C with the monoclonal antibodies of interest: α-actinin (#A-7811, Sigma-Aldrich), H3K9ac (#06-942, Merc Millipore, MA, USA) and H4ac (#382160, Merc Millipore).

    Techniques: Methylation, Western Blot, Spectrophotometry, Software

    Histone post-translational modifications studied in mouse embryonic hearts (e15) treated with HDACi. ( A ) Western blots showed changes in H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in e15 embryonic hearts treated with HDACi (TSA, SAHA, and VPA). Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized to the level of GAPDH. An identical protein amount for each experimental event was loaded on the gel. ( B ) Data from panel ( A ) were normalized to the relevant reference protein GAPDH, and the density of Western blot fragments was statistically analyzed using Student’s t -test; asterisks show statistically significant differences at p ≤ 0.05. GAPDH was used for data normalization, and α-actinin was used as a marker of cardiomyocytes. ( C ) The distribution pattern of H3K9ac (red) in the e15 mouse embryonic hearts is shown. DAPI (blue) was used as a counterstain of the cell nuclei. Arrows show the accumulation of H3K9ac in ventricular portions.

    Journal: International Journal of Molecular Sciences

    Article Title: Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells

    doi: 10.3390/ijms19082425

    Figure Lengend Snippet: Histone post-translational modifications studied in mouse embryonic hearts (e15) treated with HDACi. ( A ) Western blots showed changes in H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in e15 embryonic hearts treated with HDACi (TSA, SAHA, and VPA). Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized to the level of GAPDH. An identical protein amount for each experimental event was loaded on the gel. ( B ) Data from panel ( A ) were normalized to the relevant reference protein GAPDH, and the density of Western blot fragments was statistically analyzed using Student’s t -test; asterisks show statistically significant differences at p ≤ 0.05. GAPDH was used for data normalization, and α-actinin was used as a marker of cardiomyocytes. ( C ) The distribution pattern of H3K9ac (red) in the e15 mouse embryonic hearts is shown. DAPI (blue) was used as a counterstain of the cell nuclei. Arrows show the accumulation of H3K9ac in ventricular portions.

    Article Snippet: The procedure was performed at room temperature (RT) for 1 h. After washing with PBS for 15 min, samples were incubated overnight at 4 °C with the monoclonal antibodies of interest: α-actinin (#A-7811, Sigma-Aldrich), H3K9ac (#06-942, Merc Millipore, MA, USA) and H4ac (#382160, Merc Millipore).

    Techniques: Western Blot, Marker

    Effects of CCRR on the gap junction distribution of Cx43 and CIP85. a Immunostaining of cardiac slices showing the co-localization (yellow) of CIP85 (red) and Cx43 (green) proteins at the intercalated discs in healthy hearts (upper panel), and the effects of CCRR on the gap junction distribution of these proteins (600× magnification, lower panel). Knockdown of endogenous CCRR by Lv-siCCRR drastically diminished the presence of Cx43 (stained in green) in the intercalated discs in healthy mouse hearts, resembling the changes caused by HF. In contrast, CCRR overexpression by Lv-CCRR normalized the HF-induced expression repression of Cx43. DAPI (blue) was used to stain nuclei and α-actinin (red) to identify the cell contour. Similar results were observed in another three separate experiments. b Immunostaining of cardiac slices showing the co-localization of CIP85 (stained in red) and Cx43 (green) proteins at the intercalated discs in healthy control subjects (600× magnification). Cx43 in the intercalated discs in HF patient significantly diminished compared with healthy control subjects. DAPI (blue) was used to stain nuclei and α-actinin (red) to identify the cell contour. Similar results were observed in another two separate experiments

    Journal: Nature Communications

    Article Title: Long non-coding RNA CCRR controls cardiac conduction via regulating intercellular coupling

    doi: 10.1038/s41467-018-06637-9

    Figure Lengend Snippet: Effects of CCRR on the gap junction distribution of Cx43 and CIP85. a Immunostaining of cardiac slices showing the co-localization (yellow) of CIP85 (red) and Cx43 (green) proteins at the intercalated discs in healthy hearts (upper panel), and the effects of CCRR on the gap junction distribution of these proteins (600× magnification, lower panel). Knockdown of endogenous CCRR by Lv-siCCRR drastically diminished the presence of Cx43 (stained in green) in the intercalated discs in healthy mouse hearts, resembling the changes caused by HF. In contrast, CCRR overexpression by Lv-CCRR normalized the HF-induced expression repression of Cx43. DAPI (blue) was used to stain nuclei and α-actinin (red) to identify the cell contour. Similar results were observed in another three separate experiments. b Immunostaining of cardiac slices showing the co-localization of CIP85 (stained in red) and Cx43 (green) proteins at the intercalated discs in healthy control subjects (600× magnification). Cx43 in the intercalated discs in HF patient significantly diminished compared with healthy control subjects. DAPI (blue) was used to stain nuclei and α-actinin (red) to identify the cell contour. Similar results were observed in another two separate experiments

    Article Snippet: After blocking, sections were incubated with the primary anti-α-actinin antibody (Cat#A7811, 1:200; Sigma, Saint Louis, USA), anti-Cx43 (Cat#ab11370,1:2000; Abcam, Cambridge, UK), anti-Cx43 (Cat#ab11369, 1:1000; Abcam, Cambridge, UK), or anti-CIP85 antibodies (Cat#sc-86867, 1:100; Santa Cruz Biotechnology, CA) at 4 °C overnight.

    Techniques: Immunostaining, Staining, Over Expression, Expressing

    Laser confocal images of induced Muse double-stained for α-actinin (green) and connexin 43 (red)/diamidino-2-phenylindole (blue) in the adherent, Sus+Ad, and Sus+Ad+DN groups. Scale bar: 100 µm.

    Journal: Cell Transplantation

    Article Title: Cardiotrophic Growth Factor–Driven Induction of Human Muse Cells Into Cardiomyocyte-Like Phenotype

    doi: 10.1177/0963689717721514

    Figure Lengend Snippet: Laser confocal images of induced Muse double-stained for α-actinin (green) and connexin 43 (red)/diamidino-2-phenylindole (blue) in the adherent, Sus+Ad, and Sus+Ad+DN groups. Scale bar: 100 µm.

    Article Snippet: Primary antibodies used were mouse anti α-actinin (Sigma-Aldrich; 1:700), mouse anti desmin (1:2,000; BD Pharmingen, San Diego, CA, USA), rabbit anti HCN4 (Abcam; 1:1,400), and mouse anti β-actin (Abcam; 1:7,000).

    Techniques: Staining

    (A) Western blotting data showing α-actinin and desmin protein expression level of the adherent, Sus+Ad, and Sus+Ad+DN groups. Sus+Ad+DN showed the highest protein expression level among all groups; (B) graphical representation of densitometry Western blotting quantitative data for β-actin, α-actinin, and desmin; (C) RT-PCR analysis of induced Muse stem cells for early and late cardiac markers; (D E) Addition of OAC2, CHIR99021, and PI3Kγ inhibitor to either the Sus+Ad or the Sus+Ad+DN groups led to increased number of cardiomyocyte-like cells showing striation-like pattern. Scale bar: 100 µm. HCN-4, hyperpolarization activated cyclic nucleotide gated-4; ANP, atrial natriuretic peptide; MLC1v, myosin light chain 1v; MLC1a, myosin light chain 1a;Myo-D, myogenic differentiation antigen; OAC-2, Oct-4 activator; CHIR99021, GSK-3 inhibitor; PI3Kγ, phosphatidyl inositol-3 kinase γ.

    Journal: Cell Transplantation

    Article Title: Cardiotrophic Growth Factor–Driven Induction of Human Muse Cells Into Cardiomyocyte-Like Phenotype

    doi: 10.1177/0963689717721514

    Figure Lengend Snippet: (A) Western blotting data showing α-actinin and desmin protein expression level of the adherent, Sus+Ad, and Sus+Ad+DN groups. Sus+Ad+DN showed the highest protein expression level among all groups; (B) graphical representation of densitometry Western blotting quantitative data for β-actin, α-actinin, and desmin; (C) RT-PCR analysis of induced Muse stem cells for early and late cardiac markers; (D E) Addition of OAC2, CHIR99021, and PI3Kγ inhibitor to either the Sus+Ad or the Sus+Ad+DN groups led to increased number of cardiomyocyte-like cells showing striation-like pattern. Scale bar: 100 µm. HCN-4, hyperpolarization activated cyclic nucleotide gated-4; ANP, atrial natriuretic peptide; MLC1v, myosin light chain 1v; MLC1a, myosin light chain 1a;Myo-D, myogenic differentiation antigen; OAC-2, Oct-4 activator; CHIR99021, GSK-3 inhibitor; PI3Kγ, phosphatidyl inositol-3 kinase γ.

    Article Snippet: Primary antibodies used were mouse anti α-actinin (Sigma-Aldrich; 1:700), mouse anti desmin (1:2,000; BD Pharmingen, San Diego, CA, USA), rabbit anti HCN4 (Abcam; 1:1,400), and mouse anti β-actin (Abcam; 1:7,000).

    Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Aqueous Normal-phase Chromatography

    Q-PCR analysis of cardiac markers. (A) α-actinin and GATA-4 expression in adherent, Sus+Ad, and Sus+Ad+DN groups at the final time point, together with samples 1 and 2; (B) MLC2a and (C) MLC2v expression in adherent, Sus+Ad, and Sus+Ad+DN groups at the final time point, together with sample 2, Sus+Ad+DN without cardiotrophin-1(Sus+Ad+DN(-CT)), and Sus+Ad+DN without hepatocyte growth factor (Sus+Ad+DN(-HGF)); (D) Q-PCR analysis of α-actinin expression in Muse cells induced with either cardiotrophin-1(CT-1), cardiotrophin-1 with Posphatidyl inositol-3 kinase inhibitor (CT-1/PI3Ki), cardiotrophin-1 with mitogen activated protein kinase 1,2 inhibitor (CT-1/MEKi), and glycogen synthetase-3 kinase inhibitor (GSK3i); * P

    Journal: Cell Transplantation

    Article Title: Cardiotrophic Growth Factor–Driven Induction of Human Muse Cells Into Cardiomyocyte-Like Phenotype

    doi: 10.1177/0963689717721514

    Figure Lengend Snippet: Q-PCR analysis of cardiac markers. (A) α-actinin and GATA-4 expression in adherent, Sus+Ad, and Sus+Ad+DN groups at the final time point, together with samples 1 and 2; (B) MLC2a and (C) MLC2v expression in adherent, Sus+Ad, and Sus+Ad+DN groups at the final time point, together with sample 2, Sus+Ad+DN without cardiotrophin-1(Sus+Ad+DN(-CT)), and Sus+Ad+DN without hepatocyte growth factor (Sus+Ad+DN(-HGF)); (D) Q-PCR analysis of α-actinin expression in Muse cells induced with either cardiotrophin-1(CT-1), cardiotrophin-1 with Posphatidyl inositol-3 kinase inhibitor (CT-1/PI3Ki), cardiotrophin-1 with mitogen activated protein kinase 1,2 inhibitor (CT-1/MEKi), and glycogen synthetase-3 kinase inhibitor (GSK3i); * P

    Article Snippet: Primary antibodies used were mouse anti α-actinin (Sigma-Aldrich; 1:700), mouse anti desmin (1:2,000; BD Pharmingen, San Diego, CA, USA), rabbit anti HCN4 (Abcam; 1:1,400), and mouse anti β-actin (Abcam; 1:7,000).

    Techniques: Polymerase Chain Reaction, Expressing

    Wnt2+ and Gli1+ cells define a cardiopulmonary progenitor (CPP) and generate mesoderm derivatives of the lung and cardiac inflow tract Wnt2 is expressed in the ventral mesoderm surrounding the anterior foregut and overlapping the posterior pole of the heart (a and b, arrows). Wnt2+ cells tagged at E8.5 can generate cells within the cardiac inflow tract as well as in the lung mesoderm (c and d). Wnt2+ cells tagged at E8.5 generate myocardium of the PV, smooth muscle of the PA, endothelium of the proximal PA, and Pdgfrβ+ pericyte-like cells in the lung by E17.5 (e–h). Gli1+ cells generate derivatives within the inflow tract and early lung mesoderm similar to Wnt2+ cells (i and j) and generate mesoderm lineages within the lung including airway and vascular smooth muscle (k and l), and endothelium of the proximal pulmonary vessels (n) as well as the myocardium of the atria (m). CPPs are located in a region of overlapping Wnt2, Gli1, and Isl1 expression between the developing heart and the anterior foregut (o). AP=anterior pole of the heart, PP=posterior pole of the heart, AFG=anterior foregut, AT=atria, OFT=outflow tract, PA=pulmonary artery, PV=pulmonary vein, LB=lung bud, ASM=airway smooth muscle, VSM=vascular smooth muscle, SMA=smooth muscle actin, SAA= sarcomeric α-actinin, VWF=von Willibrand factor. Scale bars b=100 μm, e–h=50 μm, k–n=50 μm.

    Journal: Nature

    Article Title: Coordination of heart and lung co-development by a multipotent cardiopulmonary progenitor

    doi: 10.1038/nature12358

    Figure Lengend Snippet: Wnt2+ and Gli1+ cells define a cardiopulmonary progenitor (CPP) and generate mesoderm derivatives of the lung and cardiac inflow tract Wnt2 is expressed in the ventral mesoderm surrounding the anterior foregut and overlapping the posterior pole of the heart (a and b, arrows). Wnt2+ cells tagged at E8.5 can generate cells within the cardiac inflow tract as well as in the lung mesoderm (c and d). Wnt2+ cells tagged at E8.5 generate myocardium of the PV, smooth muscle of the PA, endothelium of the proximal PA, and Pdgfrβ+ pericyte-like cells in the lung by E17.5 (e–h). Gli1+ cells generate derivatives within the inflow tract and early lung mesoderm similar to Wnt2+ cells (i and j) and generate mesoderm lineages within the lung including airway and vascular smooth muscle (k and l), and endothelium of the proximal pulmonary vessels (n) as well as the myocardium of the atria (m). CPPs are located in a region of overlapping Wnt2, Gli1, and Isl1 expression between the developing heart and the anterior foregut (o). AP=anterior pole of the heart, PP=posterior pole of the heart, AFG=anterior foregut, AT=atria, OFT=outflow tract, PA=pulmonary artery, PV=pulmonary vein, LB=lung bud, ASM=airway smooth muscle, VSM=vascular smooth muscle, SMA=smooth muscle actin, SAA= sarcomeric α-actinin, VWF=von Willibrand factor. Scale bars b=100 μm, e–h=50 μm, k–n=50 μm.

    Article Snippet: Antibodies used are anti-smooth muscle actin (mouse anti-SMA 1:200 Abcam), CD31 (rat anti-CD31 1:500 BD Pharmingen), Von Willibrand Factor (rabbit anti-VWF 1:200 Sigma), MF20 (mouse anti-MF20 1:20 Abcam), Isl1 (mouse anti-Isl1 1:10 HybridomaBank), Nkx2.5 (goat anti-Nkx2.5 1:10 Santa Cruz), Sarcomeric α-actinin (mouse anti-SAA 1:100 Sigma), NG2 (rabbit anti-NG2 1:100 Millipore), and GFP (goat anti-GFP 1:100 Abcam).

    Techniques: Conditioned Place Preference, Expressing

    Clonal analysis reveals that CPPs generate related lineages within the cardiopulmonary system Single clones of Wnt2+ CPPs contribute to both the developing cardiac inflow tract as well as the mesoderm of the lung (a–c). Clonal analysis with the Gli1 creERT2 :R26R confetti line shows that a single CPP clone (YFP+) can generate myocardium (SAA+) within the cardiac inflow tract (d, arrow) as well as lung mesodermal lineages such as smooth muscle (SM22α+) around the airway and PA (e, arrows). Clonal analysis of Gli1 creERT2 :R26R confetti lungs show that vascular and airway smooth muscle and endothelium of the proximal vessels share a common Gli1+ progenitor at E8.5 (f and g). A cell lineage tree showing the relationship of CPPs to differentiated lineages within the cardiopulmonary system (h). AT=atria, OFT=outflow tract, LB=lung bud, SV=sinus venosus, ASM=airway smooth muscle, VSM=vascular smooth muscle, SAA= sarcomeric α-actinin.

    Journal: Nature

    Article Title: Coordination of heart and lung co-development by a multipotent cardiopulmonary progenitor

    doi: 10.1038/nature12358

    Figure Lengend Snippet: Clonal analysis reveals that CPPs generate related lineages within the cardiopulmonary system Single clones of Wnt2+ CPPs contribute to both the developing cardiac inflow tract as well as the mesoderm of the lung (a–c). Clonal analysis with the Gli1 creERT2 :R26R confetti line shows that a single CPP clone (YFP+) can generate myocardium (SAA+) within the cardiac inflow tract (d, arrow) as well as lung mesodermal lineages such as smooth muscle (SM22α+) around the airway and PA (e, arrows). Clonal analysis of Gli1 creERT2 :R26R confetti lungs show that vascular and airway smooth muscle and endothelium of the proximal vessels share a common Gli1+ progenitor at E8.5 (f and g). A cell lineage tree showing the relationship of CPPs to differentiated lineages within the cardiopulmonary system (h). AT=atria, OFT=outflow tract, LB=lung bud, SV=sinus venosus, ASM=airway smooth muscle, VSM=vascular smooth muscle, SAA= sarcomeric α-actinin.

    Article Snippet: Antibodies used are anti-smooth muscle actin (mouse anti-SMA 1:200 Abcam), CD31 (rat anti-CD31 1:500 BD Pharmingen), Von Willibrand Factor (rabbit anti-VWF 1:200 Sigma), MF20 (mouse anti-MF20 1:20 Abcam), Isl1 (mouse anti-Isl1 1:10 HybridomaBank), Nkx2.5 (goat anti-Nkx2.5 1:10 Santa Cruz), Sarcomeric α-actinin (mouse anti-SAA 1:100 Sigma), NG2 (rabbit anti-NG2 1:100 Millipore), and GFP (goat anti-GFP 1:100 Abcam).

    Techniques: Clone Assay, Conditioned Place Preference

    Induction of apolipoprotein (APOL) 1 expression in human embryonic kidney (HEK) cells induces the expression of transition markers. A: To determine APOL1 induction in kidney cells with undetectable APOL1 protein expression, HEK cells were transfected with empty vector (EV) or APOL1 plasmids. Protein blots were probed for APOL1 and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The same cellular lysates were probed for α-actinin, Wilms' tumor 1 (WT1), CD2AP, and podocalyxin (PDX) and reprobed for GAPDH. Gels from two different lysates are displayed. B: Cumulative densitometric data from the gels generated from the above-mentioned lysates are displayed. APOL1 induction in parietal epithelial cells (PECs) is associated with the expression of transition markers in HEK cells. C: To determine the effect of APOL1 induction on PEC markers in HEK cells, RNAs were extracted from the above-mentioned lysates. cDNAs were amplified for PAX2 and claudin 1. Cumulative data are shown. APOL1 induction in HEK cells down-regulates the transcription of PEC markers. D: To assess the effect of APOL1 induction on PEC transition markers in HEK cells, RNAs were extracted from the above-mentioned lysates. cDNAs were amplified for APOL1 , WT1 , PDX , α-actinin, and CD2AP . Cumulative data are shown in a bar diagram. The induction of APOL1 in HEK cells is associated with enhanced transcription of transition markers in HEK cells. n = 4 ( A ). ∗ P

    Journal: The American Journal of Pathology

    Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

    doi: 10.1016/j.ajpath.2018.07.025

    Figure Lengend Snippet: Induction of apolipoprotein (APOL) 1 expression in human embryonic kidney (HEK) cells induces the expression of transition markers. A: To determine APOL1 induction in kidney cells with undetectable APOL1 protein expression, HEK cells were transfected with empty vector (EV) or APOL1 plasmids. Protein blots were probed for APOL1 and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The same cellular lysates were probed for α-actinin, Wilms' tumor 1 (WT1), CD2AP, and podocalyxin (PDX) and reprobed for GAPDH. Gels from two different lysates are displayed. B: Cumulative densitometric data from the gels generated from the above-mentioned lysates are displayed. APOL1 induction in parietal epithelial cells (PECs) is associated with the expression of transition markers in HEK cells. C: To determine the effect of APOL1 induction on PEC markers in HEK cells, RNAs were extracted from the above-mentioned lysates. cDNAs were amplified for PAX2 and claudin 1. Cumulative data are shown. APOL1 induction in HEK cells down-regulates the transcription of PEC markers. D: To assess the effect of APOL1 induction on PEC transition markers in HEK cells, RNAs were extracted from the above-mentioned lysates. cDNAs were amplified for APOL1 , WT1 , PDX , α-actinin, and CD2AP . Cumulative data are shown in a bar diagram. The induction of APOL1 in HEK cells is associated with enhanced transcription of transition markers in HEK cells. n = 4 ( A ). ∗ P

    Article Snippet: Separated proteins were transferred to polyvinylidene difluoride (EMD Millipore, Toronto, ON, Canada) membranes and processed further for immunostaining with primary antibodies against APOL1 (1G12D11; 1.57 μg/mL; mouse monoclonal, catalog number 661241-LG; ProteinTech), WT1 (C-19; 0.2 μg/mL; catalog number SC-192; rabbit polyclonal, Santa Cruz Biotechnology), synaptopodin (P-19; 0.2 μg/mL; catalog number SC21537; rabbit polyclonal, Santa Cruz), CD2AP (B-4; 0.2 μg/mL; catalog number SC25272; mouse monoclonal, Santa Cruz), podocalyxin (0.2 μg/mL; catalog number PAI-46170; rabbit polyclonal, Life Techologies), α-actinin (H-2; 0.2 μg/mL; catalog number SC17829; mouse monoclonal, Santa Cruz), PAX2 (0.29 μg/mL; catalog number ab23799; rabbit polyclonal, Abcam), and claudin 1 (0.35 μg/mL; catalog number ab15078; rabbit polyclonal, Abcam).

    Techniques: Expressing, Transfection, Plasmid Preparation, Wilms Tumor Assay, Generated, Amplification

    HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n = 4 ( A and B ); n = 3 ( E and F ). ∗ P

    Journal: The American Journal of Pathology

    Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

    doi: 10.1016/j.ajpath.2018.07.025

    Figure Lengend Snippet: HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n = 4 ( A and B ); n = 3 ( E and F ). ∗ P

    Article Snippet: Separated proteins were transferred to polyvinylidene difluoride (EMD Millipore, Toronto, ON, Canada) membranes and processed further for immunostaining with primary antibodies against APOL1 (1G12D11; 1.57 μg/mL; mouse monoclonal, catalog number 661241-LG; ProteinTech), WT1 (C-19; 0.2 μg/mL; catalog number SC-192; rabbit polyclonal, Santa Cruz Biotechnology), synaptopodin (P-19; 0.2 μg/mL; catalog number SC21537; rabbit polyclonal, Santa Cruz), CD2AP (B-4; 0.2 μg/mL; catalog number SC25272; mouse monoclonal, Santa Cruz), podocalyxin (0.2 μg/mL; catalog number PAI-46170; rabbit polyclonal, Life Techologies), α-actinin (H-2; 0.2 μg/mL; catalog number SC17829; mouse monoclonal, Santa Cruz), PAX2 (0.29 μg/mL; catalog number ab23799; rabbit polyclonal, Abcam), and claudin 1 (0.35 μg/mL; catalog number ab15078; rabbit polyclonal, Abcam).

    Techniques: Expressing, Incubation, Wilms Tumor Assay, Transduction, Plasmid Preparation, Amplification, Labeling, Fluorescence

    Apolipoprotein (APO) L1 is critical for the functionality of APOL1-miR193a axis. A: To evaluate the effect of APOL1 silencing on parietal epithelial cell (PEC) markers, PECs (undifferentiated) were transfected with scrambled (SCR) or APOL1 siRNAs. Protein blots control cells and transfected cells were probed for PAX2, claudin 1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from three different lysate preparations are displayed. B: Cumulative densitometric data from the above-mentioned lysates are shown in a bar diagram. APOL1 silencing in PECs with undetectable APOL1 protein enhances the expression of PEC markers. C: To assess whether APOL1 is critical for the expression of PEC transition markers, PECs were transfected with SCR or APOL1 siRNA. Control (C) and transfected cells were incubated in media that contained vehicle, vitamin D receptor (VDR) agonist (100 nmol/L), interferon (IFN)-γ (10 ng/mL), or miR193a inhibitor (miR, 50 nmol/L) for 48 hours. In parallel sets of experiments, C and transfected cells were transduced with HIV (NL4-3; 10 3 green fluorescent protein–expressing units/mL). Protein blots were probed for APOL1 and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Representative protein gel blots are displayed. D: Cumulative densitometric data of protein blots from C are shown in bar graphs. E: Protein gel blots from the cellular lysates used in 8C were probed for α-actinin, podocalyxin (PDX), and GAPDH. Representative blots are displayed. F: Cumulative densitometric data of protein blots from E are shown in a bar diagram. n = 4. ∗ P

    Journal: The American Journal of Pathology

    Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

    doi: 10.1016/j.ajpath.2018.07.025

    Figure Lengend Snippet: Apolipoprotein (APO) L1 is critical for the functionality of APOL1-miR193a axis. A: To evaluate the effect of APOL1 silencing on parietal epithelial cell (PEC) markers, PECs (undifferentiated) were transfected with scrambled (SCR) or APOL1 siRNAs. Protein blots control cells and transfected cells were probed for PAX2, claudin 1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from three different lysate preparations are displayed. B: Cumulative densitometric data from the above-mentioned lysates are shown in a bar diagram. APOL1 silencing in PECs with undetectable APOL1 protein enhances the expression of PEC markers. C: To assess whether APOL1 is critical for the expression of PEC transition markers, PECs were transfected with SCR or APOL1 siRNA. Control (C) and transfected cells were incubated in media that contained vehicle, vitamin D receptor (VDR) agonist (100 nmol/L), interferon (IFN)-γ (10 ng/mL), or miR193a inhibitor (miR, 50 nmol/L) for 48 hours. In parallel sets of experiments, C and transfected cells were transduced with HIV (NL4-3; 10 3 green fluorescent protein–expressing units/mL). Protein blots were probed for APOL1 and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Representative protein gel blots are displayed. D: Cumulative densitometric data of protein blots from C are shown in bar graphs. E: Protein gel blots from the cellular lysates used in 8C were probed for α-actinin, podocalyxin (PDX), and GAPDH. Representative blots are displayed. F: Cumulative densitometric data of protein blots from E are shown in a bar diagram. n = 4. ∗ P

    Article Snippet: Separated proteins were transferred to polyvinylidene difluoride (EMD Millipore, Toronto, ON, Canada) membranes and processed further for immunostaining with primary antibodies against APOL1 (1G12D11; 1.57 μg/mL; mouse monoclonal, catalog number 661241-LG; ProteinTech), WT1 (C-19; 0.2 μg/mL; catalog number SC-192; rabbit polyclonal, Santa Cruz Biotechnology), synaptopodin (P-19; 0.2 μg/mL; catalog number SC21537; rabbit polyclonal, Santa Cruz), CD2AP (B-4; 0.2 μg/mL; catalog number SC25272; mouse monoclonal, Santa Cruz), podocalyxin (0.2 μg/mL; catalog number PAI-46170; rabbit polyclonal, Life Techologies), α-actinin (H-2; 0.2 μg/mL; catalog number SC17829; mouse monoclonal, Santa Cruz), PAX2 (0.29 μg/mL; catalog number ab23799; rabbit polyclonal, Abcam), and claudin 1 (0.35 μg/mL; catalog number ab15078; rabbit polyclonal, Abcam).

    Techniques: Transfection, Expressing, Incubation, Transduction, Wilms Tumor Assay

    The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n = 4 ( A and C–F ). ∗ P

    Journal: The American Journal of Pathology

    Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

    doi: 10.1016/j.ajpath.2018.07.025

    Figure Lengend Snippet: The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n = 4 ( A and C–F ). ∗ P

    Article Snippet: Separated proteins were transferred to polyvinylidene difluoride (EMD Millipore, Toronto, ON, Canada) membranes and processed further for immunostaining with primary antibodies against APOL1 (1G12D11; 1.57 μg/mL; mouse monoclonal, catalog number 661241-LG; ProteinTech), WT1 (C-19; 0.2 μg/mL; catalog number SC-192; rabbit polyclonal, Santa Cruz Biotechnology), synaptopodin (P-19; 0.2 μg/mL; catalog number SC21537; rabbit polyclonal, Santa Cruz), CD2AP (B-4; 0.2 μg/mL; catalog number SC25272; mouse monoclonal, Santa Cruz), podocalyxin (0.2 μg/mL; catalog number PAI-46170; rabbit polyclonal, Life Techologies), α-actinin (H-2; 0.2 μg/mL; catalog number SC17829; mouse monoclonal, Santa Cruz), PAX2 (0.29 μg/mL; catalog number ab23799; rabbit polyclonal, Abcam), and claudin 1 (0.35 μg/mL; catalog number ab15078; rabbit polyclonal, Abcam).

    Techniques: Expressing, Incubation, Wilms Tumor Assay, Generated, Amplification, Labeling, Microscopy, Fluorescence

    HIV, interferon (IFN)-γ, and a vitamin D receptor (VDR) agonist induce apolipoprotein (APO) L1 and transition markers in human embryonic kidney (HEK) cells. A: To examine the effect of APOL1 stimulators on the induction of APOL1 and associated downstream signaling in kidney cells with undetectable APOL1 expression, HEK cells were transduced with HIV (NL4-3, 10 3 green fluorescent protein–expressing units/mL) or treated with IFN-γ (10 ng/mL) or a VDR agonist (EB1089, 100 nmol/L) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels from two different lysates are shown. B: Cumulative densitometric data from the protein blots of lysates from A are shown in a bar diagram. APOL1 inducers enhance the expression of parietal epithelial cell (PEC) transition markers in HEK cells. C: To visualize the downstream effects of APOL1 inducers in kidney cells with undetectable APOL1 expression, HEK cells grown on coverslips were treated with IFN-γ (10 ng/mL) or VDR agonist (100 nmol/L) for 48 hours. Subsequently, cells were labeled for APOL1, PDX, α-actinin, and WT1 and examined under a confocal microscope. Representative fluoromicrographs are displayed. VDR agonist and IFN-γ induce the expression APOL1, PDX, α-actinin, and WT1 (indicated by green fluorescence) in HEK cells. D: Quantification of fluorescence data (25 to 40 cells in each group) from C is shown in bar graphs. B and D: APOL1 stimulators induce the expression of transition markers in HEK cells. n = 4 ( A ); n = 3 ( B ). ∗ P

    Journal: The American Journal of Pathology

    Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

    doi: 10.1016/j.ajpath.2018.07.025

    Figure Lengend Snippet: HIV, interferon (IFN)-γ, and a vitamin D receptor (VDR) agonist induce apolipoprotein (APO) L1 and transition markers in human embryonic kidney (HEK) cells. A: To examine the effect of APOL1 stimulators on the induction of APOL1 and associated downstream signaling in kidney cells with undetectable APOL1 expression, HEK cells were transduced with HIV (NL4-3, 10 3 green fluorescent protein–expressing units/mL) or treated with IFN-γ (10 ng/mL) or a VDR agonist (EB1089, 100 nmol/L) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels from two different lysates are shown. B: Cumulative densitometric data from the protein blots of lysates from A are shown in a bar diagram. APOL1 inducers enhance the expression of parietal epithelial cell (PEC) transition markers in HEK cells. C: To visualize the downstream effects of APOL1 inducers in kidney cells with undetectable APOL1 expression, HEK cells grown on coverslips were treated with IFN-γ (10 ng/mL) or VDR agonist (100 nmol/L) for 48 hours. Subsequently, cells were labeled for APOL1, PDX, α-actinin, and WT1 and examined under a confocal microscope. Representative fluoromicrographs are displayed. VDR agonist and IFN-γ induce the expression APOL1, PDX, α-actinin, and WT1 (indicated by green fluorescence) in HEK cells. D: Quantification of fluorescence data (25 to 40 cells in each group) from C is shown in bar graphs. B and D: APOL1 stimulators induce the expression of transition markers in HEK cells. n = 4 ( A ); n = 3 ( B ). ∗ P

    Article Snippet: Separated proteins were transferred to polyvinylidene difluoride (EMD Millipore, Toronto, ON, Canada) membranes and processed further for immunostaining with primary antibodies against APOL1 (1G12D11; 1.57 μg/mL; mouse monoclonal, catalog number 661241-LG; ProteinTech), WT1 (C-19; 0.2 μg/mL; catalog number SC-192; rabbit polyclonal, Santa Cruz Biotechnology), synaptopodin (P-19; 0.2 μg/mL; catalog number SC21537; rabbit polyclonal, Santa Cruz), CD2AP (B-4; 0.2 μg/mL; catalog number SC25272; mouse monoclonal, Santa Cruz), podocalyxin (0.2 μg/mL; catalog number PAI-46170; rabbit polyclonal, Life Techologies), α-actinin (H-2; 0.2 μg/mL; catalog number SC17829; mouse monoclonal, Santa Cruz), PAX2 (0.29 μg/mL; catalog number ab23799; rabbit polyclonal, Abcam), and claudin 1 (0.35 μg/mL; catalog number ab15078; rabbit polyclonal, Abcam).

    Techniques: Expressing, Transduction, Wilms Tumor Assay, Labeling, Microscopy, Fluorescence

    Effects of affixin knockdown by siRNA on cell morphology. (A) IMR-90 (left) and HT1080 (both panels) human fibroblasts were transfected with the indicated siRNA duplex. The specificity of each siRNA was assessed by immunoblot analysis 48 h after transfection. Note that #1 and #3 siRNAs specifically suppressed affixin expression without significant effects on the expression of α-actinin (both panels), ILK, vinculin, or actin (right). (B) The percentage of cells with multiple blebs or round morphology was estimated 48 h after transfection. The values provided represent mean values (±SD) of three independent experiments. (C) DIC images of cells transfected with control RNA (a and b) or affixin #3 siRNA (c and d). Note that #3 siRNA-transfected cells were surrounded by various sized spherical out-pouchings called blebs. Although relatively well-spread cells with multiple blebs were observed (c), they moved slowly and gradually reduced in size, and finally resulted in a round morphology (d). Bar, 10 μm. An animated time-lapse version of this process is shown in Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200308141/DC1 .

    Journal: The Journal of Cell Biology

    Article Title: Affixin interacts with ?-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction

    doi: 10.1083/jcb.200308141

    Figure Lengend Snippet: Effects of affixin knockdown by siRNA on cell morphology. (A) IMR-90 (left) and HT1080 (both panels) human fibroblasts were transfected with the indicated siRNA duplex. The specificity of each siRNA was assessed by immunoblot analysis 48 h after transfection. Note that #1 and #3 siRNAs specifically suppressed affixin expression without significant effects on the expression of α-actinin (both panels), ILK, vinculin, or actin (right). (B) The percentage of cells with multiple blebs or round morphology was estimated 48 h after transfection. The values provided represent mean values (±SD) of three independent experiments. (C) DIC images of cells transfected with control RNA (a and b) or affixin #3 siRNA (c and d). Note that #3 siRNA-transfected cells were surrounded by various sized spherical out-pouchings called blebs. Although relatively well-spread cells with multiple blebs were observed (c), they moved slowly and gradually reduced in size, and finally resulted in a round morphology (d). Bar, 10 μm. An animated time-lapse version of this process is shown in Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200308141/DC1 .

    Article Snippet: Reagents Anti-ILK and anti-α-actinin mAbs were obtained from Upstate Biotechnology; anti-paxillin and anti-Mena mAbs were from Transduction Laboratories; anti-vinculin, anti-actin, and anti-flag mAbs were from Sigma-Aldrich; anti-flag pAb was from Zymed Laboratories; and anti-α-actinin and anti-T7 pAbs were from Santa Cruz Biotechnology, Inc. Anti-affixin antibodies were generated as described previously ( ).

    Techniques: Transfection, Expressing

    Knockdown of affixin expression impaired FA and SF formation. HT1080 cells were transfected with control RNA or affixin-targeted siRNA #3 duplex as indicated. 48 h after transfection, the cells were fixed with 0.5% PFA, permeabilized with 0.2% Triton X-100, and double stained with affixin antibody (A, D, G, J, M, and P) and FITC-phalloidin (B and E), or anti-α-actinin antibody (H and K) or anti-vinculin antibody (N and Q). Merged views of each staining are also presented in C, F, I, L, O, and R. Asterisks indicate cells that were considered to evade affixin siRNA transfection. Nuclear stainings in the affixin stain (A, D, G, J, M, and P) are nonspecific signals raised by the secondary antibody under this fixation method. Note that the expression of affixin was markedly reduced in #3 siRNA-transfected cells. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Affixin interacts with ?-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction

    doi: 10.1083/jcb.200308141

    Figure Lengend Snippet: Knockdown of affixin expression impaired FA and SF formation. HT1080 cells were transfected with control RNA or affixin-targeted siRNA #3 duplex as indicated. 48 h after transfection, the cells were fixed with 0.5% PFA, permeabilized with 0.2% Triton X-100, and double stained with affixin antibody (A, D, G, J, M, and P) and FITC-phalloidin (B and E), or anti-α-actinin antibody (H and K) or anti-vinculin antibody (N and Q). Merged views of each staining are also presented in C, F, I, L, O, and R. Asterisks indicate cells that were considered to evade affixin siRNA transfection. Nuclear stainings in the affixin stain (A, D, G, J, M, and P) are nonspecific signals raised by the secondary antibody under this fixation method. Note that the expression of affixin was markedly reduced in #3 siRNA-transfected cells. Bar, 10 μm.

    Article Snippet: Reagents Anti-ILK and anti-α-actinin mAbs were obtained from Upstate Biotechnology; anti-paxillin and anti-Mena mAbs were from Transduction Laboratories; anti-vinculin, anti-actin, and anti-flag mAbs were from Sigma-Aldrich; anti-flag pAb was from Zymed Laboratories; and anti-α-actinin and anti-T7 pAbs were from Santa Cruz Biotechnology, Inc. Anti-affixin antibodies were generated as described previously ( ).

    Techniques: Expressing, Transfection, Staining

    α -Actinin interacts with affixin in an integrin stimulation–dependent manner. (A) CHO-K1 cells were lysed in a lysis buffer 3 h after replating on FN-coated dishes or nonadhesive plastic dishes. The anti-affixin antibody and control IgG immunoprecipitates were subjected to SDS-PAGE followed by Western blotting with anti-α-actinin, -affixin, -paxillin, -actin, and -vinculin antibodies. Note that lysate from FN-coated dishes (but not nonadhesive plastic dishes) exhibited coimmunoprecipitation of α-actinin with endogenous affixin. On the other hand, the negative interaction between affixin and actin (or paxillin and vinculin) demonstrates that the association is specific and is not the result of contamination of large cytoskeletal pellets. (B) HT1080 cells were trypsinized, washed three times in PBS, and replated on FN-coated dishes. After the cells were collected by lysis buffer at times indicated, the lysates were subjected to immunoprecipitation assay by anti-affixin antibody. Note that the coimmunoprecipitation of endogenous affixin and α-actinin increases gradually after replating with a peak at 60 min.

    Journal: The Journal of Cell Biology

    Article Title: Affixin interacts with ?-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction

    doi: 10.1083/jcb.200308141

    Figure Lengend Snippet: α -Actinin interacts with affixin in an integrin stimulation–dependent manner. (A) CHO-K1 cells were lysed in a lysis buffer 3 h after replating on FN-coated dishes or nonadhesive plastic dishes. The anti-affixin antibody and control IgG immunoprecipitates were subjected to SDS-PAGE followed by Western blotting with anti-α-actinin, -affixin, -paxillin, -actin, and -vinculin antibodies. Note that lysate from FN-coated dishes (but not nonadhesive plastic dishes) exhibited coimmunoprecipitation of α-actinin with endogenous affixin. On the other hand, the negative interaction between affixin and actin (or paxillin and vinculin) demonstrates that the association is specific and is not the result of contamination of large cytoskeletal pellets. (B) HT1080 cells were trypsinized, washed three times in PBS, and replated on FN-coated dishes. After the cells were collected by lysis buffer at times indicated, the lysates were subjected to immunoprecipitation assay by anti-affixin antibody. Note that the coimmunoprecipitation of endogenous affixin and α-actinin increases gradually after replating with a peak at 60 min.

    Article Snippet: Reagents Anti-ILK and anti-α-actinin mAbs were obtained from Upstate Biotechnology; anti-paxillin and anti-Mena mAbs were from Transduction Laboratories; anti-vinculin, anti-actin, and anti-flag mAbs were from Sigma-Aldrich; anti-flag pAb was from Zymed Laboratories; and anti-α-actinin and anti-T7 pAbs were from Santa Cruz Biotechnology, Inc. Anti-affixin antibodies were generated as described previously ( ).

    Techniques: Lysis, SDS Page, Western Blot, Immunoprecipitation

    Affixin 249–272 –expressing cells showed blockade of cell spreading and abnormal distribution of F-actin, α -actinin, and Mena. (A) CHO-K1 cells transfected with GFP-affixin 249–272 or GFP vector were cultured on FN-coated plates and video microscopy data were collected from 24 to 48 h after transfection at 37°C in a humidified atmosphere of 5% CO 2 . Fluorescence microscopic data obtained 38 h after transfection to only visualize GFP-expressing cells under low magnification (a and b; Videos 1 and 2) are shown, and DIC images obtained at 24 h (c and d) or 30 h (e and f) demonstrate cell morphology under enhanced magnification. In c–f, arrows indicate GFP-expressing cells that were confirmed by fluorescence microscopy (not depicted) and arrowheads indicate GFP-negative cells that divided during time-lapse observation (see Videos 3 and 4). Note that cells transfected with GFP-affixin 249–272 were arrested at the early stage of cell spreading with peripheral blebs. Bars: 20 μm (A, in a and b), 10 μm (C). An animated time-lapse version of this figure is available at http://www.jcb.org/cgi/content/full/jcb.200308141/DC1 . (B) The percentage of round cells among GFP-positive cells were estimated 24 h after transfection. The values provided represent mean values (±SD) of three independent experiments. (C) GFP-affixin 249–272 was overexpressed in CHO-K1 cells, and 24 h later the cells were fixed with 100% cold methanol (a, b, e, and f) or 2% PFA (c and d) and stained with the anti-α-actinin antibody (a) or rhodamine phalloidin (c). In e and f, GFP-expressing cells were doubly stained with anti-affixin (e, Cy3) and Mena (f, Cy5). Note that in GFP-affixin 249-272 –overexpressing cells, α-actinin demonstrated punctate staining at cell periphery, whereas F-actin formed a weak peripheral staining with a high concentration in a limited number of blebs. Colocalization of affixin and Mena was not observed.

    Journal: The Journal of Cell Biology

    Article Title: Affixin interacts with ?-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction

    doi: 10.1083/jcb.200308141

    Figure Lengend Snippet: Affixin 249–272 –expressing cells showed blockade of cell spreading and abnormal distribution of F-actin, α -actinin, and Mena. (A) CHO-K1 cells transfected with GFP-affixin 249–272 or GFP vector were cultured on FN-coated plates and video microscopy data were collected from 24 to 48 h after transfection at 37°C in a humidified atmosphere of 5% CO 2 . Fluorescence microscopic data obtained 38 h after transfection to only visualize GFP-expressing cells under low magnification (a and b; Videos 1 and 2) are shown, and DIC images obtained at 24 h (c and d) or 30 h (e and f) demonstrate cell morphology under enhanced magnification. In c–f, arrows indicate GFP-expressing cells that were confirmed by fluorescence microscopy (not depicted) and arrowheads indicate GFP-negative cells that divided during time-lapse observation (see Videos 3 and 4). Note that cells transfected with GFP-affixin 249–272 were arrested at the early stage of cell spreading with peripheral blebs. Bars: 20 μm (A, in a and b), 10 μm (C). An animated time-lapse version of this figure is available at http://www.jcb.org/cgi/content/full/jcb.200308141/DC1 . (B) The percentage of round cells among GFP-positive cells were estimated 24 h after transfection. The values provided represent mean values (±SD) of three independent experiments. (C) GFP-affixin 249–272 was overexpressed in CHO-K1 cells, and 24 h later the cells were fixed with 100% cold methanol (a, b, e, and f) or 2% PFA (c and d) and stained with the anti-α-actinin antibody (a) or rhodamine phalloidin (c). In e and f, GFP-expressing cells were doubly stained with anti-affixin (e, Cy3) and Mena (f, Cy5). Note that in GFP-affixin 249-272 –overexpressing cells, α-actinin demonstrated punctate staining at cell periphery, whereas F-actin formed a weak peripheral staining with a high concentration in a limited number of blebs. Colocalization of affixin and Mena was not observed.

    Article Snippet: Reagents Anti-ILK and anti-α-actinin mAbs were obtained from Upstate Biotechnology; anti-paxillin and anti-Mena mAbs were from Transduction Laboratories; anti-vinculin, anti-actin, and anti-flag mAbs were from Sigma-Aldrich; anti-flag pAb was from Zymed Laboratories; and anti-α-actinin and anti-T7 pAbs were from Santa Cruz Biotechnology, Inc. Anti-affixin antibodies were generated as described previously ( ).

    Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture, Microscopy, Fluorescence, Staining, Concentration Assay

    α -Actinin colocalizes with affixin at leading edge, FA, and along SFs. Double-immunofluorescence staining with anti-T7 and anti-flag mAbs of CHO-K1 cells transfected with T7-tagged affixin and flag-tagged α1-actinin (A–C) or with anti-affixin and anti-α-actinin antibodies of CHO-K1 cells (D–I). Note that both affixin and α-actinin are colocalized at FAs (A–F, arrowheads), at the tip of the leading edge (A–C, G–I; arrows). In A–C, fixation was performed using 2% PFA in PBS, whereas in D–I, 0.5% PFA in PBS was used. Bars, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Affixin interacts with ?-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction

    doi: 10.1083/jcb.200308141

    Figure Lengend Snippet: α -Actinin colocalizes with affixin at leading edge, FA, and along SFs. Double-immunofluorescence staining with anti-T7 and anti-flag mAbs of CHO-K1 cells transfected with T7-tagged affixin and flag-tagged α1-actinin (A–C) or with anti-affixin and anti-α-actinin antibodies of CHO-K1 cells (D–I). Note that both affixin and α-actinin are colocalized at FAs (A–F, arrowheads), at the tip of the leading edge (A–C, G–I; arrows). In A–C, fixation was performed using 2% PFA in PBS, whereas in D–I, 0.5% PFA in PBS was used. Bars, 10 μm.

    Article Snippet: Reagents Anti-ILK and anti-α-actinin mAbs were obtained from Upstate Biotechnology; anti-paxillin and anti-Mena mAbs were from Transduction Laboratories; anti-vinculin, anti-actin, and anti-flag mAbs were from Sigma-Aldrich; anti-flag pAb was from Zymed Laboratories; and anti-α-actinin and anti-T7 pAbs were from Santa Cruz Biotechnology, Inc. Anti-affixin antibodies were generated as described previously ( ).

    Techniques: Double Immunofluorescence Staining, Transfection

    Affixin requires kinase activity of ILK to directly bind to α -actinin in vitro. (A) GST full-length affixin (GST-affixin wt) or COOH-terminal CH domain (affixin 213–364 ) of affixin fusion protein (GST-CH2) was incubated with 20 μg purified chicken α2-actinin. Coprecipitated proteins were collected using glutathione-Sepharose, resolved by SDS-PAGE, and transferred to a PVDF membrane. The membrane was probed with the anti-α-actinin antibody (top) and stained with Coomassie brilliant blue (CBB) to confirm the amount of GST fusion proteins. (B and C) These affixin fusion proteins were preincubated with protein G–Sepharose with flag antibody immunoprecipitates from lysates of Cos-7 cells overexpressing flag-tagged ILK (B) or its kinase-dead mutant (C) before incubation with α-actinin. These GST fusion proteins were similarly processed as described in A. The membrane was probed with the anti-α-actinin antibody (top) and anti-flag pAb (bottom). (D) The GST-affixin specifically interacts with α-actinin after preincubation with ILK. GST-affixin wt, GST-CH2, or GST alone was separated by SDS-PAGE after preincubation with in vitro–translated ILK in the presence or absence of 10 μM ATP in phosphorylation buffer, and was transferred to membrane. Blot overlay assay was done using 10 μg/ml purified α-actinin after blocking.

    Journal: The Journal of Cell Biology

    Article Title: Affixin interacts with ?-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction

    doi: 10.1083/jcb.200308141

    Figure Lengend Snippet: Affixin requires kinase activity of ILK to directly bind to α -actinin in vitro. (A) GST full-length affixin (GST-affixin wt) or COOH-terminal CH domain (affixin 213–364 ) of affixin fusion protein (GST-CH2) was incubated with 20 μg purified chicken α2-actinin. Coprecipitated proteins were collected using glutathione-Sepharose, resolved by SDS-PAGE, and transferred to a PVDF membrane. The membrane was probed with the anti-α-actinin antibody (top) and stained with Coomassie brilliant blue (CBB) to confirm the amount of GST fusion proteins. (B and C) These affixin fusion proteins were preincubated with protein G–Sepharose with flag antibody immunoprecipitates from lysates of Cos-7 cells overexpressing flag-tagged ILK (B) or its kinase-dead mutant (C) before incubation with α-actinin. These GST fusion proteins were similarly processed as described in A. The membrane was probed with the anti-α-actinin antibody (top) and anti-flag pAb (bottom). (D) The GST-affixin specifically interacts with α-actinin after preincubation with ILK. GST-affixin wt, GST-CH2, or GST alone was separated by SDS-PAGE after preincubation with in vitro–translated ILK in the presence or absence of 10 μM ATP in phosphorylation buffer, and was transferred to membrane. Blot overlay assay was done using 10 μg/ml purified α-actinin after blocking.

    Article Snippet: Reagents Anti-ILK and anti-α-actinin mAbs were obtained from Upstate Biotechnology; anti-paxillin and anti-Mena mAbs were from Transduction Laboratories; anti-vinculin, anti-actin, and anti-flag mAbs were from Sigma-Aldrich; anti-flag pAb was from Zymed Laboratories; and anti-α-actinin and anti-T7 pAbs were from Santa Cruz Biotechnology, Inc. Anti-affixin antibodies were generated as described previously ( ).

    Techniques: Activity Assay, In Vitro, Incubation, Purification, SDS Page, Staining, Mutagenesis, Overlay Assay, Blocking Assay

    α -Actinin binds to affixin through its ABD. (A) Interaction between affixin and α-actinin in the two-hybrid system. cDNA fragments encoding chicken α1-actinin deletion mutants were subcloned into pGAD424 vectors. These vectors were cotransformed with pAS2-1 affixin 53–272 into yeast Y187(a), and the interaction was investigated by β-galactosidase filter assay. (B) The ABD of α-actinin was coimmunoprecipitated with affixin. The expression vector encoding the flag-tagged α-actinin or its ABD or ABD-truncated α-actinin mutant (SR) was cotransfected into Cos-7 cells with pSRD-T7-affixin. Immunoprecipitation assay was performed using the anti-flag antibody, and immunocomplexes were subjected to immunoblot analysis with anti-flag monoclonal and anti-T7 antibodies.

    Journal: The Journal of Cell Biology

    Article Title: Affixin interacts with ?-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction

    doi: 10.1083/jcb.200308141

    Figure Lengend Snippet: α -Actinin binds to affixin through its ABD. (A) Interaction between affixin and α-actinin in the two-hybrid system. cDNA fragments encoding chicken α1-actinin deletion mutants were subcloned into pGAD424 vectors. These vectors were cotransformed with pAS2-1 affixin 53–272 into yeast Y187(a), and the interaction was investigated by β-galactosidase filter assay. (B) The ABD of α-actinin was coimmunoprecipitated with affixin. The expression vector encoding the flag-tagged α-actinin or its ABD or ABD-truncated α-actinin mutant (SR) was cotransfected into Cos-7 cells with pSRD-T7-affixin. Immunoprecipitation assay was performed using the anti-flag antibody, and immunocomplexes were subjected to immunoblot analysis with anti-flag monoclonal and anti-T7 antibodies.

    Article Snippet: Reagents Anti-ILK and anti-α-actinin mAbs were obtained from Upstate Biotechnology; anti-paxillin and anti-Mena mAbs were from Transduction Laboratories; anti-vinculin, anti-actin, and anti-flag mAbs were from Sigma-Aldrich; anti-flag pAb was from Zymed Laboratories; and anti-α-actinin and anti-T7 pAbs were from Santa Cruz Biotechnology, Inc. Anti-affixin antibodies were generated as described previously ( ).

    Techniques: Expressing, Plasmid Preparation, Mutagenesis, Immunoprecipitation

    Mapping of α-actinin–binding region on affixin molecule by yeast two-hybrid assay. (A) Isolated positive clones by yeast two-hybrid screening. (B) Specific binding site of α-actinin on affixin molecule. cDNA fragments encoding human l -affixin deletion mutants and full-length α1-actinin were subcloned into pAS2-1 and pGAD10 vectors, respectively. These vectors, obtained clone 94, and pGAD424 full-length ILK were cotransformed into yeast Y187(a) in the indicated combinations, and 5 d later the interactions were examined by β-galactosidase filter assay.

    Journal: The Journal of Cell Biology

    Article Title: Affixin interacts with ?-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction

    doi: 10.1083/jcb.200308141

    Figure Lengend Snippet: Mapping of α-actinin–binding region on affixin molecule by yeast two-hybrid assay. (A) Isolated positive clones by yeast two-hybrid screening. (B) Specific binding site of α-actinin on affixin molecule. cDNA fragments encoding human l -affixin deletion mutants and full-length α1-actinin were subcloned into pAS2-1 and pGAD10 vectors, respectively. These vectors, obtained clone 94, and pGAD424 full-length ILK were cotransformed into yeast Y187(a) in the indicated combinations, and 5 d later the interactions were examined by β-galactosidase filter assay.

    Article Snippet: Reagents Anti-ILK and anti-α-actinin mAbs were obtained from Upstate Biotechnology; anti-paxillin and anti-Mena mAbs were from Transduction Laboratories; anti-vinculin, anti-actin, and anti-flag mAbs were from Sigma-Aldrich; anti-flag pAb was from Zymed Laboratories; and anti-α-actinin and anti-T7 pAbs were from Santa Cruz Biotechnology, Inc. Anti-affixin antibodies were generated as described previously ( ).

    Techniques: Binding Assay, Y2H Assay, Isolation, Clone Assay, Two Hybrid Screening

    Schematic illustration of the role of affixin. The integrin–ECM interaction acutely activates ILK, which is considered to induce phosphorylation of the COOH-terminal CH domain of affixin. It triggers the interaction between affixin and α-actinin, which promotes the recruitment of α-actinin into these nascent FAs. Zyxin and its binding partner, Mena, are recruited into FAs by their interaction with α-actinin. On the other hand, the NH 2 -terminal CH domain of affixin is considered to transmit integrin–ILK signals to activate Cdc42/Rac1 through interaction with αPIX, which results in the recruitment of PAK to FAs. Affixin-mediated formation of the signaling complex at nascent FAs should cooperatively promote actin polymerization and lead to cell spreading.

    Journal: The Journal of Cell Biology

    Article Title: Affixin interacts with ?-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction

    doi: 10.1083/jcb.200308141

    Figure Lengend Snippet: Schematic illustration of the role of affixin. The integrin–ECM interaction acutely activates ILK, which is considered to induce phosphorylation of the COOH-terminal CH domain of affixin. It triggers the interaction between affixin and α-actinin, which promotes the recruitment of α-actinin into these nascent FAs. Zyxin and its binding partner, Mena, are recruited into FAs by their interaction with α-actinin. On the other hand, the NH 2 -terminal CH domain of affixin is considered to transmit integrin–ILK signals to activate Cdc42/Rac1 through interaction with αPIX, which results in the recruitment of PAK to FAs. Affixin-mediated formation of the signaling complex at nascent FAs should cooperatively promote actin polymerization and lead to cell spreading.

    Article Snippet: Reagents Anti-ILK and anti-α-actinin mAbs were obtained from Upstate Biotechnology; anti-paxillin and anti-Mena mAbs were from Transduction Laboratories; anti-vinculin, anti-actin, and anti-flag mAbs were from Sigma-Aldrich; anti-flag pAb was from Zymed Laboratories; and anti-α-actinin and anti-T7 pAbs were from Santa Cruz Biotechnology, Inc. Anti-affixin antibodies were generated as described previously ( ).

    Techniques: Binding Assay

    Affixin 249–272 specifically inhibits α -actinin–affixin interaction. (A) The minimum α-actinin–binding site of affixin was coimmunoprecipitated with α-actinin. The expression vector encoding the flag-tagged α-actinin was cotransfected into CHO-K1 cells with pEGFP-affixin 249–272 . Immunoprecipitation assay was performed using the anti-flag antibody, and immunocomplexes were subjected to immunoblot analysis with monoclonal anti-flag and anti-GFP antibodies. (B) Endogenous affixin was immunoprecipitated from lysates of CHO-K1 cells harvested from fully spreading cells transfected with GFP-affixin 249–272 or GFP vector alone and cultured for 24 h. Affixin immunoprecipitates were analyzed by Western blotting with anti-α-actinin, affixin, GFP, ILK, paxillin, vinculin, and actin antibodies as indicated. Note that the coimmunoprecipitation of α-actinin, but not ILK, was abolished in GFP-affixin 249–272 –expressing cells.

    Journal: The Journal of Cell Biology

    Article Title: Affixin interacts with ?-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction

    doi: 10.1083/jcb.200308141

    Figure Lengend Snippet: Affixin 249–272 specifically inhibits α -actinin–affixin interaction. (A) The minimum α-actinin–binding site of affixin was coimmunoprecipitated with α-actinin. The expression vector encoding the flag-tagged α-actinin was cotransfected into CHO-K1 cells with pEGFP-affixin 249–272 . Immunoprecipitation assay was performed using the anti-flag antibody, and immunocomplexes were subjected to immunoblot analysis with monoclonal anti-flag and anti-GFP antibodies. (B) Endogenous affixin was immunoprecipitated from lysates of CHO-K1 cells harvested from fully spreading cells transfected with GFP-affixin 249–272 or GFP vector alone and cultured for 24 h. Affixin immunoprecipitates were analyzed by Western blotting with anti-α-actinin, affixin, GFP, ILK, paxillin, vinculin, and actin antibodies as indicated. Note that the coimmunoprecipitation of α-actinin, but not ILK, was abolished in GFP-affixin 249–272 –expressing cells.

    Article Snippet: Reagents Anti-ILK and anti-α-actinin mAbs were obtained from Upstate Biotechnology; anti-paxillin and anti-Mena mAbs were from Transduction Laboratories; anti-vinculin, anti-actin, and anti-flag mAbs were from Sigma-Aldrich; anti-flag pAb was from Zymed Laboratories; and anti-α-actinin and anti-T7 pAbs were from Santa Cruz Biotechnology, Inc. Anti-affixin antibodies were generated as described previously ( ).

    Techniques: Binding Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection, Cell Culture, Western Blot

    Valproic acid causes marked morphological and ultrastructural changes, and promotes EndMT-like phenotypic switching in HUVECs. (A) Diluent-treated control HUVECs, cultured on a two-dimensional plate, formed a confluent monolayer with the typical EC ‘cobblestone’ morphology (left panel). VPA treatment resulted in marked morphological changes whereby HUVECs took on an enlarged spindle-shaped appearance with smooth surfaces (right panel). Both micrographs were taken at the same magnification (10X). (B) Immunofluorescent micrographs demonstrating cytoskeletal protein re-organization in HUVECs following VPA treatment. α-Actinin positivity is indicated in green and nuclei were stained with DAPI (blue); scale bar = 10 μm. (C) HUVECs were treated with diluent control or VPA. Total RNA and protein were extracted at 24 and 48 h, respectively. Differential (C) transcript (qPCR) ∗∗ p

    Journal: Frontiers in Pharmacology

    Article Title: Valproic Acid Induces Endothelial-to-Mesenchymal Transition-Like Phenotypic Switching

    doi: 10.3389/fphar.2018.00737

    Figure Lengend Snippet: Valproic acid causes marked morphological and ultrastructural changes, and promotes EndMT-like phenotypic switching in HUVECs. (A) Diluent-treated control HUVECs, cultured on a two-dimensional plate, formed a confluent monolayer with the typical EC ‘cobblestone’ morphology (left panel). VPA treatment resulted in marked morphological changes whereby HUVECs took on an enlarged spindle-shaped appearance with smooth surfaces (right panel). Both micrographs were taken at the same magnification (10X). (B) Immunofluorescent micrographs demonstrating cytoskeletal protein re-organization in HUVECs following VPA treatment. α-Actinin positivity is indicated in green and nuclei were stained with DAPI (blue); scale bar = 10 μm. (C) HUVECs were treated with diluent control or VPA. Total RNA and protein were extracted at 24 and 48 h, respectively. Differential (C) transcript (qPCR) ∗∗ p

    Article Snippet: For immunoblotting analysis, the following primary antibodies were utilized at a 1:1000 dilution: CD31 (Cell Signaling #3528), VE-Cadherin (Santa Cruz Biotechnology #6458), Tie2 (Santa Cruz Biotechnology #324), N -Cadherin (abcam #ab76057), FSP1 (Abnova #H00006275-M01), αSMA (abcam #ab5694), α-actinin (Cell Signaling #3134), TGFβ1 (abcam #ab9758), SMAD2 (Cell Signaling #3122), pSMAD2 (Cell Signaling #3101), SMAD3 (abcam #ab28379 and Cell Signaling #9513), pSMAD3 (abcam #ab51451), SMAD5 (Cell Signaling #12534), pSMAD5 (abcam #ab92698), CTGF (abcam #ab6992), eNOS (Cell Signaling #9572), peNOS (Cell Signaling #9570), AKT (Cell Signaling #9272), pAKT (Cell Signaling #9271), β-catenin (Cell Signaling #8480), phospho-β-catenin (Cell Signaling #9561), and GAPDH (Millipore #MAB374).

    Techniques: Cell Culture, Staining, Real-time Polymerase Chain Reaction

    SIKE forms direct interactions with tubulin and α‐actinin. IVP reactions of 6xHis‐ SIKE ‐ FL ( SIKE or S) or FL ‐ SIKE ‐6xHis ( SIKE ‐26b or S‐26b) (8 μ m ) with monomeric tubulin (A, 4–11.2 μ m ), α‐actinin (B, C, 4–11.2 μ m ), or lysozyme (D, 4–11.2 μ m ) were precipitated with Ni‐ NTA resin and separated by SDS / PAGE (12% (A), 4–15% (B, C), or 15% (D) Tris/glycine) and stained with SimplyBlue SafeStain. Control reactions of individual proteins incubated with Ni‐ NTA resin were subjected to the same reaction conditions and separated by SDS / PAGE (15% (E), 4–15% (F)). UB , unbound sample; B, bound sample; 1 : 0.5 molar ratio, 1 : 1 molar ratio, 1 : 1.4 molar ratio. (A) Lanes: 1 – SIKE ‐ FL starting material; 2 – tubulin starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. B) Lanes: 1 – SIKE ‐ FL starting material; 2 – α‐actinin starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. (C) Gel 1 Lanes: 1 – SIKE ‐26b starting material; 2 – blank; 3 – α‐actinin starting material. Gel 2 Lanes: 1 – 1 : 0.5 UB ; 2 – 1 : 0.5 B; 3 – 1 : 1 UB ; 4 – 1 : 1 B; 5 – 1 : 1.4 UB ; 6 – 1 : 1.4 B. D) Lanes: 1 – SIKE ‐ FL starting material; 2 – lysozyme starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. (E) Lanes: 1 – SIKE ‐ FL starting material; 2 – UB ; 3 – B; 4 – tubulin starting material; 5 – UB ; 6 – B; 7 – lysozyme starting material; 8 – UB ; 9 – B. F) Gel 1 Lanes: 1 – α‐actinin starting material; 5 – UB ; 6 – B. Gel 2 Lanes: 1 – SIKE ‐26 starting material; 2 – UB ; 3 – B; control reactions show that only SIKE is bound to the Ni‐ NTA resin. The C‐terminal 6xHis‐tagged SIKE is not all bound to the Ni‐ NTA , but can be found in the unbound and bound fractions. SIKE does not interact nonspecifically with the negative control, lysozyme. In tubulin reactions, SIKE precipitates tubulin indicating a direct interaction. When SIKE is 6xHis‐tagged at the N terminus, α‐actinin pre‐incubation inhibits SIKE 's ability to bind to the resin, suggesting α‐actinin may complex to SIKE and mask SIKE 's 6xHis tag. With a C‐terminal 6xHis tag, SIKE precipitates α‐actinin indicating a direct interaction. Gels are representative of three independent experiments.

    Journal: FEBS Open Bio

    Article Title: Suppressor of IKKepsilon forms direct interactions with cytoskeletal proteins, tubulin and α‐actinin, linking innate immunity to the cytoskeleton

    doi: 10.1002/2211-5463.12454

    Figure Lengend Snippet: SIKE forms direct interactions with tubulin and α‐actinin. IVP reactions of 6xHis‐ SIKE ‐ FL ( SIKE or S) or FL ‐ SIKE ‐6xHis ( SIKE ‐26b or S‐26b) (8 μ m ) with monomeric tubulin (A, 4–11.2 μ m ), α‐actinin (B, C, 4–11.2 μ m ), or lysozyme (D, 4–11.2 μ m ) were precipitated with Ni‐ NTA resin and separated by SDS / PAGE (12% (A), 4–15% (B, C), or 15% (D) Tris/glycine) and stained with SimplyBlue SafeStain. Control reactions of individual proteins incubated with Ni‐ NTA resin were subjected to the same reaction conditions and separated by SDS / PAGE (15% (E), 4–15% (F)). UB , unbound sample; B, bound sample; 1 : 0.5 molar ratio, 1 : 1 molar ratio, 1 : 1.4 molar ratio. (A) Lanes: 1 – SIKE ‐ FL starting material; 2 – tubulin starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. B) Lanes: 1 – SIKE ‐ FL starting material; 2 – α‐actinin starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. (C) Gel 1 Lanes: 1 – SIKE ‐26b starting material; 2 – blank; 3 – α‐actinin starting material. Gel 2 Lanes: 1 – 1 : 0.5 UB ; 2 – 1 : 0.5 B; 3 – 1 : 1 UB ; 4 – 1 : 1 B; 5 – 1 : 1.4 UB ; 6 – 1 : 1.4 B. D) Lanes: 1 – SIKE ‐ FL starting material; 2 – lysozyme starting material; 3 – 1 : 0.5 UB ; 4 – 1 : 0.5 B; 5 – 1 : 1 UB ; 6 – 1 : 1 B; 7 – 1 : 1.4 UB ; 8 – 1 : 1.4 B. (E) Lanes: 1 – SIKE ‐ FL starting material; 2 – UB ; 3 – B; 4 – tubulin starting material; 5 – UB ; 6 – B; 7 – lysozyme starting material; 8 – UB ; 9 – B. F) Gel 1 Lanes: 1 – α‐actinin starting material; 5 – UB ; 6 – B. Gel 2 Lanes: 1 – SIKE ‐26 starting material; 2 – UB ; 3 – B; control reactions show that only SIKE is bound to the Ni‐ NTA resin. The C‐terminal 6xHis‐tagged SIKE is not all bound to the Ni‐ NTA , but can be found in the unbound and bound fractions. SIKE does not interact nonspecifically with the negative control, lysozyme. In tubulin reactions, SIKE precipitates tubulin indicating a direct interaction. When SIKE is 6xHis‐tagged at the N terminus, α‐actinin pre‐incubation inhibits SIKE 's ability to bind to the resin, suggesting α‐actinin may complex to SIKE and mask SIKE 's 6xHis tag. With a C‐terminal 6xHis tag, SIKE precipitates α‐actinin indicating a direct interaction. Gels are representative of three independent experiments.

    Article Snippet: In vitro precipitation SIKE (8 μm final concentration) and cytoskeletal proteins, tubulin and α‐actinin (Cytoskeleton, Inc., Denver, CO, USA), or control protein, lysozyme, were incubated at molar ratios of 1 : 0.5, 1 : 1, or 1 : 1.4 in 50 mm NaH2 PO4 , pH 8, and 0.3 m NaCl (EQ buffer) at 4 °C.

    Techniques: SDS Page, Staining, Incubation, Negative Control

    In epithelial cells, SIKE colocalizes with actin cytoskeletal markers. DOV 13 cells were fixed, permeabilized, and stained with mouse anti‐cytoskeletal marker antibody as indicated followed by anti‐mouse Alexa Fluor 555 (red), rabbit anti‐ SIKE antibody followed by anti‐rabbit Alexa Fluor 488 (green) and Hoechst 33342 (blue). F‐actin was stained with Alexa Fluor 555 Phalloidin. Cytoskeletal markers: actin, α‐actinin, α‐tubulin, ezrin, myosin light chain 2 ( MLC 2), focal adhesion kinase ( FAK ), β‐catenin. Images are maximum intensity of z ‐stack projections. Scale bar, 10 μm. Images are representative of two independent experiments, five images/experiment. Images are maximum intensity z ‐stack projections. Intersection of fluorescence for SIKE to target was significant (≥ 0.5) for actin, α‐actinin, and ezrin.

    Journal: FEBS Open Bio

    Article Title: Suppressor of IKKepsilon forms direct interactions with cytoskeletal proteins, tubulin and α‐actinin, linking innate immunity to the cytoskeleton

    doi: 10.1002/2211-5463.12454

    Figure Lengend Snippet: In epithelial cells, SIKE colocalizes with actin cytoskeletal markers. DOV 13 cells were fixed, permeabilized, and stained with mouse anti‐cytoskeletal marker antibody as indicated followed by anti‐mouse Alexa Fluor 555 (red), rabbit anti‐ SIKE antibody followed by anti‐rabbit Alexa Fluor 488 (green) and Hoechst 33342 (blue). F‐actin was stained with Alexa Fluor 555 Phalloidin. Cytoskeletal markers: actin, α‐actinin, α‐tubulin, ezrin, myosin light chain 2 ( MLC 2), focal adhesion kinase ( FAK ), β‐catenin. Images are maximum intensity of z ‐stack projections. Scale bar, 10 μm. Images are representative of two independent experiments, five images/experiment. Images are maximum intensity z ‐stack projections. Intersection of fluorescence for SIKE to target was significant (≥ 0.5) for actin, α‐actinin, and ezrin.

    Article Snippet: In vitro precipitation SIKE (8 μm final concentration) and cytoskeletal proteins, tubulin and α‐actinin (Cytoskeleton, Inc., Denver, CO, USA), or control protein, lysozyme, were incubated at molar ratios of 1 : 0.5, 1 : 1, or 1 : 1.4 in 50 mm NaH2 PO4 , pH 8, and 0.3 m NaCl (EQ buffer) at 4 °C.

    Techniques: Staining, Marker, Fluorescence

    Quantitation of colocalization between SIKE and cytoskeletal markers. volocity software (PerkinElmer) was used to define SIKE or cytoskeletal marker volumes on thresholded images. Fraction of intersecting volumes was determined for SIKE to marker compared to total SIKE volume and marker to SIKE compared to total marker volume (A, C). These graphs represent co‐occurrence. Significant co‐occurrence was noted when values were > 0.5 with a 95% confidence interval. Thresholded PCC was determined (B, D) to assess codistribution of SIKE with cytoskeletal markers. Error bars represent standard error, n = 5. (A) In epithelial cells, co‐occurrence of actin, α‐actinin, and ezrin was observed for SIKE fluorescence. In myeloid cells, co‐occurrence of α‐actinin was observed for SIKE fluorescence. No significant (≥ 0.5, as noted in Ref. 27 ) codistribution of cytoskeletal marker and SIKE fluorescence was observed for either epithelial (B) or myeloid (D) cells.

    Journal: FEBS Open Bio

    Article Title: Suppressor of IKKepsilon forms direct interactions with cytoskeletal proteins, tubulin and α‐actinin, linking innate immunity to the cytoskeleton

    doi: 10.1002/2211-5463.12454

    Figure Lengend Snippet: Quantitation of colocalization between SIKE and cytoskeletal markers. volocity software (PerkinElmer) was used to define SIKE or cytoskeletal marker volumes on thresholded images. Fraction of intersecting volumes was determined for SIKE to marker compared to total SIKE volume and marker to SIKE compared to total marker volume (A, C). These graphs represent co‐occurrence. Significant co‐occurrence was noted when values were > 0.5 with a 95% confidence interval. Thresholded PCC was determined (B, D) to assess codistribution of SIKE with cytoskeletal markers. Error bars represent standard error, n = 5. (A) In epithelial cells, co‐occurrence of actin, α‐actinin, and ezrin was observed for SIKE fluorescence. In myeloid cells, co‐occurrence of α‐actinin was observed for SIKE fluorescence. No significant (≥ 0.5, as noted in Ref. 27 ) codistribution of cytoskeletal marker and SIKE fluorescence was observed for either epithelial (B) or myeloid (D) cells.

    Article Snippet: In vitro precipitation SIKE (8 μm final concentration) and cytoskeletal proteins, tubulin and α‐actinin (Cytoskeleton, Inc., Denver, CO, USA), or control protein, lysozyme, were incubated at molar ratios of 1 : 0.5, 1 : 1, or 1 : 1.4 in 50 mm NaH2 PO4 , pH 8, and 0.3 m NaCl (EQ buffer) at 4 °C.

    Techniques: Quantitation Assay, Software, Marker, Periodic Counter-current Chromatography, Fluorescence

    α‐Actinin, but not actin, immunoprecipitates SIKE . Five hundred microgram of protein from six independent transfections of HEK 293 cells with pm EGFP ‐β‐actin (A, B) or pEGFP ‐α‐actinin (C, D) and pCMV 5a‐ SIKE ‐ FLAG was exposed to protein G Dynabeads charged with anti‐ GFP antibody that had been cross‐linked to beads with bis(sulfosuccinimidyl)suberate as per manufacturers’ protocol. Proteins were eluted from beads into 1× Nu PAGE SDS / PAGE sample buffer and were separated by Tris/glycine SDS / PAGE , transferred to nitrocellulose, and immunoblotted for the cytoskeleton protein and FLAG . Three lysates were from cells that had been stimulated with 50 μg· mL −1 polyriboinosinic : polyribocytidylic acid (ds RNA ) 12 h prior to harvesting to mimic a viral infection and induce SIKE phosphorylation. Actin served as a loading control. In each panel, (top) IP / IB of cytoskeletal protein with IB of lysate for cytoskeletal protein control and loading control; (bottom) IP of cytoskeletal protein IB of SIKE with IB of lysate for SIKE protein control and loading control. (A) basal state immunoprecipitation of actin, (B) ds RNA ‐stimulated immunoprecipitation of actin, (C) basal state immunoprecipitation of α‐actinin, (D) ds RNA ‐stimulated immunoprecipitation of α‐actinin. Molecular weights of proteins: SIKE ‐ FLAG (27 kDa), EGFP ‐actin (67 kDa), and EGFP ‐α‐actinin (132 kDa). *Nonspecific band observed with anti‐ FLAG antibody. Blots are representative of at least 5–6 independent experiments (2–3 shown).

    Journal: FEBS Open Bio

    Article Title: Suppressor of IKKepsilon forms direct interactions with cytoskeletal proteins, tubulin and α‐actinin, linking innate immunity to the cytoskeleton

    doi: 10.1002/2211-5463.12454

    Figure Lengend Snippet: α‐Actinin, but not actin, immunoprecipitates SIKE . Five hundred microgram of protein from six independent transfections of HEK 293 cells with pm EGFP ‐β‐actin (A, B) or pEGFP ‐α‐actinin (C, D) and pCMV 5a‐ SIKE ‐ FLAG was exposed to protein G Dynabeads charged with anti‐ GFP antibody that had been cross‐linked to beads with bis(sulfosuccinimidyl)suberate as per manufacturers’ protocol. Proteins were eluted from beads into 1× Nu PAGE SDS / PAGE sample buffer and were separated by Tris/glycine SDS / PAGE , transferred to nitrocellulose, and immunoblotted for the cytoskeleton protein and FLAG . Three lysates were from cells that had been stimulated with 50 μg· mL −1 polyriboinosinic : polyribocytidylic acid (ds RNA ) 12 h prior to harvesting to mimic a viral infection and induce SIKE phosphorylation. Actin served as a loading control. In each panel, (top) IP / IB of cytoskeletal protein with IB of lysate for cytoskeletal protein control and loading control; (bottom) IP of cytoskeletal protein IB of SIKE with IB of lysate for SIKE protein control and loading control. (A) basal state immunoprecipitation of actin, (B) ds RNA ‐stimulated immunoprecipitation of actin, (C) basal state immunoprecipitation of α‐actinin, (D) ds RNA ‐stimulated immunoprecipitation of α‐actinin. Molecular weights of proteins: SIKE ‐ FLAG (27 kDa), EGFP ‐actin (67 kDa), and EGFP ‐α‐actinin (132 kDa). *Nonspecific band observed with anti‐ FLAG antibody. Blots are representative of at least 5–6 independent experiments (2–3 shown).

    Article Snippet: In vitro precipitation SIKE (8 μm final concentration) and cytoskeletal proteins, tubulin and α‐actinin (Cytoskeleton, Inc., Denver, CO, USA), or control protein, lysozyme, were incubated at molar ratios of 1 : 0.5, 1 : 1, or 1 : 1.4 in 50 mm NaH2 PO4 , pH 8, and 0.3 m NaCl (EQ buffer) at 4 °C.

    Techniques: Transfection, Polyacrylamide Gel Electrophoresis, SDS Page, Infection, Immunoprecipitation

    In myeloid cells, SIKE colocalizes with actin and microtubule markers. RAW 264.7 cells were fixed, permeabilized, and stained with mouse anti‐cytoskeletal marker antibody as indicated followed by anti‐mouse Alexa Fluor 555 (red), rabbit anti‐ SIKE antibody followed by anti‐rabbit Alexa Fluor 488 (green) and Hoechst 33342 (blue). F‐actin was stained with Alexa Fluor 555 Phalloidin. Cytoskeletal markers: actin, α‐actinin, α‐tubulin, ezrin, MLC 2, focal adhesion kinase ( FAK ), β‐catenin. Images are maximum intensity of z ‐stack projections. Scale bar, 10 μm. Images are representative of two independent experiments, five images/experiment. Images are maximum intensity z ‐stack projections. Intersection of fluorescence for SIKE to target was significant (≥ 0.5) for α‐actinin.

    Journal: FEBS Open Bio

    Article Title: Suppressor of IKKepsilon forms direct interactions with cytoskeletal proteins, tubulin and α‐actinin, linking innate immunity to the cytoskeleton

    doi: 10.1002/2211-5463.12454

    Figure Lengend Snippet: In myeloid cells, SIKE colocalizes with actin and microtubule markers. RAW 264.7 cells were fixed, permeabilized, and stained with mouse anti‐cytoskeletal marker antibody as indicated followed by anti‐mouse Alexa Fluor 555 (red), rabbit anti‐ SIKE antibody followed by anti‐rabbit Alexa Fluor 488 (green) and Hoechst 33342 (blue). F‐actin was stained with Alexa Fluor 555 Phalloidin. Cytoskeletal markers: actin, α‐actinin, α‐tubulin, ezrin, MLC 2, focal adhesion kinase ( FAK ), β‐catenin. Images are maximum intensity of z ‐stack projections. Scale bar, 10 μm. Images are representative of two independent experiments, five images/experiment. Images are maximum intensity z ‐stack projections. Intersection of fluorescence for SIKE to target was significant (≥ 0.5) for α‐actinin.

    Article Snippet: In vitro precipitation SIKE (8 μm final concentration) and cytoskeletal proteins, tubulin and α‐actinin (Cytoskeleton, Inc., Denver, CO, USA), or control protein, lysozyme, were incubated at molar ratios of 1 : 0.5, 1 : 1, or 1 : 1.4 in 50 mm NaH2 PO4 , pH 8, and 0.3 m NaCl (EQ buffer) at 4 °C.

    Techniques: Staining, Marker, Fluorescence

    Confocal images and Western blotting analysis of myotubes cultured under various stimulation regimes. Skeletal myotubes cultured under various stimulation regimes were immunostained for α-actinin (green; blue = nuclei) to show striation. (a–c) Application of 5 or 10% cyclic strain to cells cultured on aligned fibers resulted in overstretching and appeared to drive the myoblasts into proliferative instead of differentiative state; (d, i) Pre-stretching of the myoblasts before application of cyclic loading led to enhanced differentiation; (e, f, i) Electrical stimulation applied to the myotubes at earlier time points (Day 0 and 4 post-differentiation) showed detrimental effect in the development of striated myotubes; (g, i) When electrical stimulation was applied post-myotube assembly (day 7), it induced contraction and led to an increase in striation; (h, i) Cells cultured under synchronized stimulation showed insignificant increase in myotube diameter but significant increase in percent of striated myotubes; (j) Western blotting demonstrated an upregulation of contractile proteins mechanical, electrical or electromechanical stimuli was applied. There is a significant level of increase in fast myosin heavy chain when cells were cultured under synchronized stimulation. Scale bar: 20 μ m.

    Journal: Cellular and molecular bioengineering

    Article Title: Effect of Electromechanical Stimulation on the Maturation of Myotubes on Aligned Electrospun Fibers

    doi: 10.1007/s12195-008-0021-y

    Figure Lengend Snippet: Confocal images and Western blotting analysis of myotubes cultured under various stimulation regimes. Skeletal myotubes cultured under various stimulation regimes were immunostained for α-actinin (green; blue = nuclei) to show striation. (a–c) Application of 5 or 10% cyclic strain to cells cultured on aligned fibers resulted in overstretching and appeared to drive the myoblasts into proliferative instead of differentiative state; (d, i) Pre-stretching of the myoblasts before application of cyclic loading led to enhanced differentiation; (e, f, i) Electrical stimulation applied to the myotubes at earlier time points (Day 0 and 4 post-differentiation) showed detrimental effect in the development of striated myotubes; (g, i) When electrical stimulation was applied post-myotube assembly (day 7), it induced contraction and led to an increase in striation; (h, i) Cells cultured under synchronized stimulation showed insignificant increase in myotube diameter but significant increase in percent of striated myotubes; (j) Western blotting demonstrated an upregulation of contractile proteins mechanical, electrical or electromechanical stimuli was applied. There is a significant level of increase in fast myosin heavy chain when cells were cultured under synchronized stimulation. Scale bar: 20 μ m.

    Article Snippet: The cells were immunostained for sarcomeric α-actinin (1:100 dilution, EA-53, Abcam) and counterstained with DAPI and Alexa488 phalloidin (Invitrogen).

    Techniques: Western Blot, Cell Culture

    Differentiation of myotubes on various topographical surfaces. Skeletal myoblasts cultured on various topographical surfaces were immunostained for α-actinin (green; blue = nuclei) to show striation. Myotubes were randomly oriented and less striated when cultured on (a) film and (b) random fiber surfaces; (c) When cultured on aligned PU fibers (600 nm with stiffness of 0.5 MPa), myotubes were highly aligned with 70% of myotubes showing striation; (d, e) The degree of striation decreased when myoblasts were cultured on stiffer aligned PU fibers (1 MPa and 22 MPa); (f, g) Degree of striation in the myotubes was comparable among the groups with fiber diameters of 600 nm to 10 μ m; (h) Average myotube diameter did not significantly differ among various topographical surfaces, while (i) percentage of striated myotubes was dependent on topographical features and matrix stiffness. Scale bar: 20 μ m.

    Journal: Cellular and molecular bioengineering

    Article Title: Effect of Electromechanical Stimulation on the Maturation of Myotubes on Aligned Electrospun Fibers

    doi: 10.1007/s12195-008-0021-y

    Figure Lengend Snippet: Differentiation of myotubes on various topographical surfaces. Skeletal myoblasts cultured on various topographical surfaces were immunostained for α-actinin (green; blue = nuclei) to show striation. Myotubes were randomly oriented and less striated when cultured on (a) film and (b) random fiber surfaces; (c) When cultured on aligned PU fibers (600 nm with stiffness of 0.5 MPa), myotubes were highly aligned with 70% of myotubes showing striation; (d, e) The degree of striation decreased when myoblasts were cultured on stiffer aligned PU fibers (1 MPa and 22 MPa); (f, g) Degree of striation in the myotubes was comparable among the groups with fiber diameters of 600 nm to 10 μ m; (h) Average myotube diameter did not significantly differ among various topographical surfaces, while (i) percentage of striated myotubes was dependent on topographical features and matrix stiffness. Scale bar: 20 μ m.

    Article Snippet: The cells were immunostained for sarcomeric α-actinin (1:100 dilution, EA-53, Abcam) and counterstained with DAPI and Alexa488 phalloidin (Invitrogen).

    Techniques: Cell Culture

    Western blots of contractile proteins. (a) All contractile proteins were upregulated in myotubes cultured on aligned fibers compared to random fibers and film; (b) The upregulation of myosin, myogenin, and α-actinin was statistically higher in aligned fibers compared to random fibers and film samples. Western blotting analysis of myotubes cultured on different fiber sizes did not reveal significant difference among the groups. Values were normalized to GAPDH level.

    Journal: Cellular and molecular bioengineering

    Article Title: Effect of Electromechanical Stimulation on the Maturation of Myotubes on Aligned Electrospun Fibers

    doi: 10.1007/s12195-008-0021-y

    Figure Lengend Snippet: Western blots of contractile proteins. (a) All contractile proteins were upregulated in myotubes cultured on aligned fibers compared to random fibers and film; (b) The upregulation of myosin, myogenin, and α-actinin was statistically higher in aligned fibers compared to random fibers and film samples. Western blotting analysis of myotubes cultured on different fiber sizes did not reveal significant difference among the groups. Values were normalized to GAPDH level.

    Article Snippet: The cells were immunostained for sarcomeric α-actinin (1:100 dilution, EA-53, Abcam) and counterstained with DAPI and Alexa488 phalloidin (Invitrogen).

    Techniques: Western Blot, Cell Culture