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5 protocols using integrin β1 sirna

1

Integrin and Fibronectin Knockdown Assay

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Cells were plated and left to grow overnight in 5% FBS/RPMI. 5%FBS/RPMI media was replaced with Opti-MEM (Invitrogen). Fibronectin pool, Fibronectin siRNA A GACUGGUGGUUACAUGUUAtt, Fibronectin siRNA B CGCAUCACUUGCACUUCUAtt, Fibronectin siRNA C GAUCCUGUCUACUUCACAAtt, Integrin α5 siRNA A GUCAGAAUUUCGAGACAAAtt, Integrin α5 siRNA B CCACUGACCAGAACUAGAAtt, Integrin β1 siRNA A GAGAUGAGGUUCAAUUUGAtt, Integrin β1 siRNA B GAUGAGGUUCAAUUUGAAAtt, (25nM, Santa Cruz), Integrin α5, Integrin β1, and PTEN (all 25nM, Cell Signaling Technologies), and BRAF (25nM, Thermo Scientific) siRNAs in complex with Lipofectamine 2000 (Invitrogen) were added. Scrambled, non-targeting siRNAs were used as controls. A final concentration of 5% FBS in RPMI was added the next day. For fibronectin knockdowns, final concentration 1% FBS in RPMI was added next day. Cells were transfected for 24-72 hours prior to treatment.
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2

Intracerebroventricular Drug Administration in Rats

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Intracerebroventricular (i.c.v) drug administration was performed as reported previously (Ma et al., 2017 (link); Tong et al., 2017 (link)). Briefly, rats were positioned prone in a stereotaxic apparatus under 2.5% isoflurane anesthesia. A burr hole was drilled into the skull according to the following coordinates relative to bregma: 0.92 mm posterior and 1.5 mm lateral. The needle of a 10 μL Hamilton syringe (Microliter 701; Hamilton Company, Reno, NV) was inserted into the left lateral ventricle through the burr hole 3.3 mm below the horizontal plane of bregma. Integrin β1 siRNA (10 μM/10 μL, Santa Cruz, USA) or scrambled siRNA (10 μM/10 μL, Santa Cruz, USA) was infused at the same rate 48 h before ICH induction.
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3

Gene Manipulation for Cell Line

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GLUT1 siRNA and control siRNA were purchased from Santa Cruz Biotechnology (CA, USA; catalog number: sc-35493), integrin β1 siRNA and control siRNA were purchased from Santa Cruz Biotechnology (CA, USA; catalog number: sc-35674), and GLUT1 plasmid were purchased from OriGene (Rockville, USA; catalog number: SC116011). They were transiently transfected to cell line with Lipofectamine 2000 (Invitrogen, Carlsbad, USA; catalog number: 11668030) according to the instruction of manufacturer.
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4

Silencing MLC20, Integrin β1, and MYH11

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For MLC20 and integrin β1 KD, control siRNA (SC-37007), MLC20 siRNA (SC-45414), and integrin β1 siRNA (SC-35674) were purchased from Santa Cruz Biotechnology. HASM cells were transfected with siRNA according to the manual of the manufacturer (Santa Cruz Biotechnology, Dallas, TX, USA). For MYH11 KD, control construct (sc-418922) and MYH11 CRISPR/Casp KO plasmids (sc-400695) were purchased from Santa Cruz Biotechnology. The experiments were performed according to the manual of the manufacturer.
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5

Integrin and Fibronectin Knockdown Assay

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Cells were plated and left to grow overnight in 5% FBS/RPMI. 5%FBS/RPMI media was replaced with Opti-MEM (Invitrogen). Fibronectin pool, Fibronectin siRNA A GACUGGUGGUUACAUGUUAtt, Fibronectin siRNA B CGCAUCACUUGCACUUCUAtt, Fibronectin siRNA C GAUCCUGUCUACUUCACAAtt, Integrin α5 siRNA A GUCAGAAUUUCGAGACAAAtt, Integrin α5 siRNA B CCACUGACCAGAACUAGAAtt, Integrin β1 siRNA A GAGAUGAGGUUCAAUUUGAtt, Integrin β1 siRNA B GAUGAGGUUCAAUUUGAAAtt, (25nM, Santa Cruz), Integrin α5, Integrin β1, and PTEN (all 25nM, Cell Signaling Technologies), and BRAF (25nM, Thermo Scientific) siRNAs in complex with Lipofectamine 2000 (Invitrogen) were added. Scrambled, non-targeting siRNAs were used as controls. A final concentration of 5% FBS in RPMI was added the next day. For fibronectin knockdowns, final concentration 1% FBS in RPMI was added next day. Cells were transfected for 24-72 hours prior to treatment.
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