Journal: Regenerative Biomaterials

Article Title: Stiff matrix drives microglial cell migration through Piezo1/Ca 2+ /AKT/cofilin signaling axis-regulated F-actin reassembly

doi: 10.1093/rb/rbaf124

Figure Lengend Snippet: The Piezo1 channel is involved in stiff substrate-induced BV2 cell migration. ( A , B ) Cell migration over an 8-h period cultured on substrates of 500 Pa, 1 kPa and 20 kPa. ( A ) Migration trajectories of individual cells; ( B ) Quantitative analysis of total migration distance (left) and average velocity (right) ( n = 20 cells). ( C ) Representative Western blots showing (top) and quantification (bottom) of the Piezo1 protein expression in cells on 500 Pa, 1 kPa and 20 kPa substrates ( n = 7 repeats). ( D ) Representative immunofluorescence images showing the Piezo1 protein expression (left) and quantification of fluorescence intensity (right) for cells on 500 Pa, 1 kPa and 20 kPa substrates (scale bar: 10 µm; n = 30 cells). ( E , F ) Cell migration on soft substrates (500 Pa), which were pretreated with DMSO or 5 μM Yoda1 for 1 h and subsequently tracked for 8 h. ( E ) Migration trajectories of individual cells; ( F ) Quantitative analysis of total migration distance (left) and average velocity (right) ( n = 20 cells). ( G , H ) Cell migration on stiff substrates (20 kPa) that were pretreated with DMSO or 1 μM GsMTx4 for 1 h and subsequently tracked for 8 h. ( G ) Migration trajectories of individual cells; ( H ) Quantitative analysis of total migration distance (left), and average velocity (right) ( n = 20 cells). ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies and dilutions were as follows: Piezo1 (1:1000, Proteintech, 28511-1-AP, USA), AKT (1:400, Cell signaling Technology, 4691, USA) and cofilin (1:200, Santa Cruz, sc376476, USA).

Techniques: Migration, Cell Culture, Western Blot, Expressing, Immunofluorescence, Fluorescence

Journal: Regenerative Biomaterials

Article Title: Stiff matrix drives microglial cell migration through Piezo1/Ca 2+ /AKT/cofilin signaling axis-regulated F-actin reassembly

doi: 10.1093/rb/rbaf124

Figure Lengend Snippet: Piezo1-Mediated Ca 2+ influx promotes migration and F-actin assembly in BV2 cells on stiff substrates. ( A , B ) Fluo-4 fluorescence intensity in cells on 500 Pa or 20 kPa substrates in the absence and then presence of Ca 2+ and 1 μM GsMTx4 over a 5-min period. 2 mM CaCl 2 was added at 60 s, and GsMTx4 at 180 s as indicated ( n = 3 cells). ( A ) Temporal changes in fluo-4 fluorescence intensity for individual cells; ( B ) Quantitative analysis of fluo-4 fluorescence intensity ratios after and before the addition of CaCl 2 (left), and before and after treatment with GsMTx4 (right) ( n = 20 cells). ( C , D ) Migration of cells seeded on 20 kPa substrates that were pretreated with DMSO or 10 μM BAPTA-AM for 1 h and subsequently tracked for 8 h. ( C ) Migration trajectories of individual cells; ( D ) Quantitative analysis of total migration distance (left) and average velocity (right) ( n = 20 cells). ( E , F ) Representative immunofluorescence images showing F-actin and quantification of fluorescence intensity in cells seeded on 500 Pa and 20 kPa substrates. Cells were pretreated with 1 μM GsMTx4 ( E ) or 10 μM BAPTA-AM ( F ) for 1 h and subsequently cultured for an additional 12 h (scale bar: 5 µm; n = 30 cells). ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies and dilutions were as follows: Piezo1 (1:1000, Proteintech, 28511-1-AP, USA), AKT (1:400, Cell signaling Technology, 4691, USA) and cofilin (1:200, Santa Cruz, sc376476, USA).

Techniques: Migration, Fluorescence, Immunofluorescence, Cell Culture

Journal: Regenerative Biomaterials

Article Title: Stiff matrix drives microglial cell migration through Piezo1/Ca 2+ /AKT/cofilin signaling axis-regulated F-actin reassembly

doi: 10.1093/rb/rbaf124

Figure Lengend Snippet: The Piezo1 channel mediates stiff substrate-induced migration of primary microglial cells. ( A , B ) Migration analysis of cells over an 8-h period seeded on 500 Pa and 20 kPa substrates. ( A ) Migration trajectories of individual cells; ( B ) Quantitative analysis of total migration distance (left) and average velocity (right) ( n = 20 cells). ( C ) Quantitative analysis of the Piezo1 mRNA expression in cells seeded on 500 Pa and 20 kPa substrates ( n = 4 repeats). ( D ) Representative immunofluorescence images showing Piezo1 expression (left) and quantification of fluorescence intensity (right) in cells on 500 Pa and 20 kPa substrates (scale bar: 10 µm, n = 30 cells). ( E , F ) Migration analysis of cells seeded on soft substrates (500 Pa) pretreated with DMSO or 5 μM Yoda1 for 1 h and subsequently tracked for 8 h. ( E ) Migration trajectories of individual cells; ( F ) Quantitative analysis of total migration distance (left) and average velocity (right) ( n = 20 cells). ( G , H ) Migration analysis of cells on stiff substrates (20 kPa) pretreated with DMSO or 1 μM GsMTx4 for 1 h and subsequently tracked for 8 h ( n = 20 cells). ( G ) Migration trajectories of individual cells; ( H ) Quantitative analysis of total migration distance (left) and average velocity (right) ( n = 20 cells). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies and dilutions were as follows: Piezo1 (1:1000, Proteintech, 28511-1-AP, USA), AKT (1:400, Cell signaling Technology, 4691, USA) and cofilin (1:200, Santa Cruz, sc376476, USA).

Techniques: Migration, Expressing, Immunofluorescence, Fluorescence

Journal: Regenerative Biomaterials

Article Title: Stiff matrix drives microglial cell migration through Piezo1/Ca 2+ /AKT/cofilin signaling axis-regulated F-actin reassembly

doi: 10.1093/rb/rbaf124

Figure Lengend Snippet: Piezo1-mediated Ca 2+ influx promotes migration and F-actin reassembly in primary microglial cells on stiff substrates. ( A , B ) Fluo-4 fluorescence intensity in BV2 cells on 500 Pa or 20 kPa substrates in the absence and presence of Ca 2+ and 1 μM GsMTx4 over a 5-min period. 2 mM CaCl 2 was added at 60 s, and GsMTx4 at 180 s as indicated ( n = 3 cells). ( A ) Temporal changes in fluo-4 fluorescence intensity for individual cells; ( B ) Quantitative analysis of fluorescence intensity ratios after and before the addition of CaCl 2 (left), and before and after treatment with GsMTx4 (right) ( n = 15 cells). ( C , D ) Migration of primary microglial cells on 20 kPa substrates pretreated with DMSO or 10 μM BAPTA-AM for 1 h and subsequently tracked for 8 h. ( C ) Migration trajectories of individual cells; ( D ) Quantitative analysis of total migration distance (left) and average velocity (right) ( n = 20 cells). ( E , F ) Representative immunofluorescence images showing F-actin and quantification of fluorescence intensity in primary microglial cells seeded on 500 Pa and 20 kPa substrates. Cells were pretreated with 1 μM GsMTx4 ( E ) or 10 μM BAPTA-AM ( F ) for 1 h and subsequently cultured for an additional 12 h (scale bar: 5 µm; n = 30 cells). ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies and dilutions were as follows: Piezo1 (1:1000, Proteintech, 28511-1-AP, USA), AKT (1:400, Cell signaling Technology, 4691, USA) and cofilin (1:200, Santa Cruz, sc376476, USA).

Techniques: Migration, Fluorescence, Immunofluorescence, Cell Culture

Journal: Regenerative Biomaterials

Article Title: Stiff matrix drives microglial cell migration through Piezo1/Ca 2+ /AKT/cofilin signaling axis-regulated F-actin reassembly

doi: 10.1093/rb/rbaf124

Figure Lengend Snippet: RNA-Seq analysis of the signaling pathways driving substrate stiffness-induced regulation of migration of primary microglial cells. ( A ) Statistical analysis of DEGs in primary microglial cells cultured on 500 Pa (soft) or 20 kPa (stiff) substrates, including a volcano map (left) and cluster heatmaps (right). ( B ) KEGG pathway enrichment analysis of DEGs associated with environmental information processing influenced by substrate stiffness in primary microglial cells. Significant signaling pathways are highlighted by rectangles. ( C ) KEGG enrichment analysis of DEGs involved in cell migration affected by substrate stiffness in primary microglial cells. ( D ) Statistical analysis of DEGs in primary microglial cells following transfection with negative control siRNA (siNC) or Piezo1-specific siRNA (siPiezo1) and culture on stiff substrates, including a volcano plot (left) and cluster heatmaps (right). ( E ) KEGG pathway enrichment analysis of DEGs associated with environmental information processing following Piezo1 knockdown on stiff substrates. ( F ) KEGG pathway enrichment analysis of DEGs involved in cell migration processes affected by silencing Piezo1 expression in primary microglial cells cultured on stiff substrates.

Article Snippet: The primary antibodies and dilutions were as follows: Piezo1 (1:1000, Proteintech, 28511-1-AP, USA), AKT (1:400, Cell signaling Technology, 4691, USA) and cofilin (1:200, Santa Cruz, sc376476, USA).

Techniques: RNA Sequencing, Protein-Protein interactions, Migration, Cell Culture, Transfection, Negative Control, Knockdown, Expressing

Journal: Regenerative Biomaterials

Article Title: Stiff matrix drives microglial cell migration through Piezo1/Ca 2+ /AKT/cofilin signaling axis-regulated F-actin reassembly

doi: 10.1093/rb/rbaf124

Figure Lengend Snippet: Piezo1-mediated and Ca 2+ -dependent AKT activity is critical for substrate stiffness regulation of cell migration, cofilin phosphorylation and actin dynamics in BV2 cells. ( A , B ) Migration analysis of BV2 cells seeded on 20 kPa substrates and pretreated with DMSO or 20 μM LY for 1 h and subsequently tracked for 8 h. ( A ) Migration trajectories of individual cells; ( B ) Quantitative analysis of total migration distance (left) and average velocity (right) ( n = 20 cells). ( C ) Representative Western blots showing (top) and quantification of p-AKT protein expression (bottom) in cells on 500 Pa and 20 kPa substrates ( n = 4 repeats). Cells were pretreated with DMSO or 1 μM GsMTx4 for 1 h and subsequently cultured for a further 12 h. ( D , E ) Representative Western blots showing phosphorylation of cofilin (p-cofilin) and total cofilin (t-cofilin) expression (top) and quantification of p-cofilin to t-cofilin ratio (bottom) in cells seeded on 500 Pa and 20 kPa substrates ( n = 3 repeats). Cells were pretreated with 1 μM GsMTx4 ( D ) or 20 μM LY ( E ) for 1 h and subsequently cultured for a further 12 h. ( F , G ) Immunofluorescent staining of F-actin and G-actin in cells on 500 Pa and 20 kPa substrates. Cells were pretreated with DMSO or 1 μM GsMTx4 for 1 h and subsequently cultured for a further 12 h. ( F ) Representative images showing expression of F-actin and G-actin; ( G ) Quantification of F-actin protein expression (left) and G-actin/F-actin ratio (right) ( n = 30 cells). ( H , I ) Immunofluorescent staining of F-actin and G-actin in cells seeded on 500 Pa and 20 kPa substrates. Cells were pretreated with DMSO or 10 μM BAPTA-AM for 1 h and subsequently cultured for a further 12 h. ( H ) Representative images showing F-actin and G-actin. ( I ) Quantification of F-actin protein expression (left) and G-actin/F-actin ratio (right) ( n = 30 cells). ( J , K ) Immunofluorescent staining of F-actin and G-actin in cells on 500 Pa and 20 kPa substrates. Cells were pretreated with DMSO or 20 μM LY for 1 h and subsequently cultured for a further 12 h. ( J ) Representative images showing expression of F-actin and G-actin; ( K ) Quantification of F-actin protein expression (left) and G-actin/F-actin ratio (right) ( n = 30 cells). Scale bar: 10 µm. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies and dilutions were as follows: Piezo1 (1:1000, Proteintech, 28511-1-AP, USA), AKT (1:400, Cell signaling Technology, 4691, USA) and cofilin (1:200, Santa Cruz, sc376476, USA).

Techniques: Activity Assay, Migration, Phospho-proteomics, Western Blot, Expressing, Cell Culture, Staining

Journal: Regenerative Biomaterials

Article Title: Stiff matrix drives microglial cell migration through Piezo1/Ca 2+ /AKT/cofilin signaling axis-regulated F-actin reassembly

doi: 10.1093/rb/rbaf124

Figure Lengend Snippet: Activation of Piezo1-mediated and Ca 2+ -dependent AKT signaling pathway enhances migration of primary microglial cells on stiff substrates. ( A , B ) Migration analysis of cells seeded on 20 kPa substrates pretreated with DMSO or 20 μM LY for 1 h and subsequently tracked for 8 h ( n = 20 cells). ( A ) Migration trajectories of individual cells; ( B ) Quantitative analysis of total migration distance (left) and average velocity (right). ( C , D ) Representative immunofluorescence images showing (left) and quantification (right) of AKT expression in cells seeded on 500 Pa and 20 kPa substrates. Cells were pretreated with 1 μM GsMTx4 ( C ) or 10 μM BAPTA-AM ( D ) for 1 h and subsequently cultured for a further 12 h (scale bar: 10 µm, n = 30 cells). ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies and dilutions were as follows: Piezo1 (1:1000, Proteintech, 28511-1-AP, USA), AKT (1:400, Cell signaling Technology, 4691, USA) and cofilin (1:200, Santa Cruz, sc376476, USA).

Techniques: Activation Assay, Migration, Immunofluorescence, Expressing, Cell Culture

Journal: Regenerative Biomaterials

Article Title: Stiff matrix drives microglial cell migration through Piezo1/Ca 2+ /AKT/cofilin signaling axis-regulated F-actin reassembly

doi: 10.1093/rb/rbaf124

Figure Lengend Snippet: Piezo1/Ca 2+ /AKT Signaling-mediated regulation of cofilin activation and actin dynamics in primary microglial cells. ( A – C ) Representative immunofluorescence images showing (left) and quantification (right) of cofilin expression in cells seeded on 500 Pa and 20 kPa substrates. Cells were pretreated with 1 μM GsMTx4 ( A ) or 10 μM BAPTA-AM ( B ) or 20 μM LY ( C ) for 1 h and subsequently cultured for a further 12 h. Scale bar: 10 µm. ( D , E ) Representative immunofluorescence images showing expression of G-actin and F-actin ( D ) and quantification of F-actin protein expression (left of E ) and G-actin/F-actin ratio (right of E ) in cells on 500 Pa and 20 kPa substrates. Cells were pretreated with DMSO or 1 μM GsMTx4 for 1 h and subsequently cultured for a further 12 h. Scale bar: 5 µm. ( F , G ) Representative immunofluorescence images showing expression of G-actin and F-actin ( F ) and quantification of F-actin protein expression (left of G) and G-actin/F-actin ratio (right of G) in cells on 500 Pa and 20 kPa substrates. Cells were pretreated with DMSO or 10 μM BAPTA-AM for 1 h and subsequently cultured for a further 12 h. Scale bar: 5 µm ( H , I ). Representative immunofluorescence images showing expression of G-actin and F-actin ( H ) and quantification of F-actin protein expression (left of I ) and G-actin/F-actin ratio (right of I ) in cells on 500 Pa and 20 kPa substrates. Cells were pretreated with DMSO or 20 μM LY for 1 h and subsequently cultured for a further 12 h. Scale bar: 5 µm. n = 30 cells; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies and dilutions were as follows: Piezo1 (1:1000, Proteintech, 28511-1-AP, USA), AKT (1:400, Cell signaling Technology, 4691, USA) and cofilin (1:200, Santa Cruz, sc376476, USA).

Techniques: Activation Assay, Immunofluorescence, Expressing, Cell Culture