Fig. 2: Depletion of abLIM1 results in blebbing during cell spreading. | Cell Discovery

Fig. 2: Depletion of abLIM1 results in blebbing during cell spreading.

From: abLIM1 constructs non-erythroid cortical actin networks to prevent mechanical tension-induced blebbing

Fig. 2

Pooled data are presented as mean ± s.d. Student’s t test: *P < 0.05; **P < 0.01; ***P < 0.001. a Efficient knockdown of abLIM1 by two different siRNAs (abL1-i1 and -i2) in RPE1 cells. Ctrl-i is a control siRNA. α-tubulin served as loading control. b abLIM1-depleted cells displayed repetitive protrusion-blebbing-retraction cycles during spreading and migration. The images were from Supplementary Video 1. RPE1 cells transfected for 48 h were re-plated at 10% confluency and imaged for 10 h at 6-min intervals. The arrowheads indicate the blebbing stage of the cell. c Quantification results from three independent experiments. Micrographs at 4 and 8 h of the live imaging as in (b) were used for the analysis. At least 60 cells were scored in each experiment and condition. d Scanning EM images for cells showing different extent of spreading. RPE1 cells transfected for 48 h were re-plated at 10% confluency for 4 h and processed for the EM. The arrowhead indicates a bleb in control cell. e Quantification results from three independent experiments as in (d). At least 60 cells were scored in each experiment and condition. f Representative frames of a typical blebbing cell. RPE1 cells stably expressing GFP-F and RFP-Utrch to label plasma membrane and F-actin, respectively, were transfected for 48 h with abL1-i1 and re-plated at 10% confluency. After 4 h of culture, the cells were imaged at 10-sec intervals. The frames were from Supplementary Video 2. The magnified (2×) views show a typical blebbing process. Other nascent blebs are indicated by arrows. g Expression levels of RNAi-insensitive abLIM1R and luciferase (Luc) in stable cells for rescue experiments. RPE1 cells infected with lentivirus were sorted out by FACS, based on GFP expressed from the lentiviral constructs through an internal ribosome entry site (IRES), and maintained as stable cell lines. α-tubulin served as loading control. h Depletion of endogenous abLIM1 by RNAi in the stable RPE1 cells. GAPDH served as loading control. NS, non-specific band. i abLIM1R repressed the blebbing of the abLIM1-depleted cells. The stable RPE1 cells transfected for 48 h were re-plated at ~10% confluency. The spreading cells were then imaged for at least 240 min at 6-min intervals. The statistical results, from three independent experiments, were obtained using the micrographs at 240 min. Approximately 100 cells were scored in each experiment and condition

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