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. 2004 Jul 19;166(2):261–272. doi: 10.1083/jcb.200401034

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Figure 1. — In vitro experiments with anti-mAChR siRNA. (A) Representative results of Western blot analysis of the effects of siRNA-M₃ and siRNA-M₄ on M₃ and M₄ mAChR expression in human KCs transfected with anti-mAChR siRNAs or negative control (NC) siRNA. On the third day after transfection, the cellular proteins were analyzed by Western blotting. The results were compared with those obtained with control (C) KCs exposed to TransIT-TKO (Mirus Corp.) transfection reagent mixed in Opti-MEM medium (GIBCO BRL) alone. The numbers underneath the bands are ratios of the densitometry value of each mAChR receptor to that of β-actin compared with the values obtained in control KCs (taken as 1). (B) IF analysis of the effects of anti-M₃ and anti-M₄ siRNAs on the expression of the M₃ and M₄ mAChR subtypes on the cell membrane of human KCs. The cells were cultured and treated with the siRNA-M₃ (black bars), siRNA-M₄ (white bars), or negative control siRNA (not depicted) as described in A, fixed to avoid cell membrane permeabilization, and stained with anti-M₃ or anti-M₄ antibodies. (C) Dose-dependent effects of receptor-selective siRNA on KC migration. Second passage human KCs were loaded into AGKOS plates, transfected with 1.2, 12, or 120 nM of negative control M₃ or M₄ siRNA, or exposed to TransIT-TKO transfection reagent mixed in Opti-MEM medium alone (control), and then used in AGKOS assay. (D) Opposing alterations in KC crawling locomotion due to M₃ and M₄ receptor gene silencing. (B–D) Asterisk indicates significant (P < 0.05) differences from untreated control. NC, negative control siRNA.