Figure 2.
MAP1B is necessary for efficient tumorigenesis and invasion in TNBC. (A) Immunoblotting of MAP1B-LC1 in MAP1B shRNA-based stable knockdown (KD) and CRISPR/Cas9-based knockout (KO) MDA-MB-231 cells. Upper panel: shRNA-based stable KD. Lower panel: CRISPR/Cas9-based KO. (B) In vitro cell growth of MAP1B KD and KO MDA-MB-231 cells. The growth rate was quantified by a MTT colorimetric assay. Parental MDA, n = 8 from three experiments; MAP1B shRNA and KO, n = 6 from three experiments. (C) Orthotopic xenograft tumorigenesis of MAP1B KO MDA-MB-231 cells stably expressing Luc2 in nude mice. Top: A representative bioluminescence image of xenografted mice on day 29. Bottom: Quantification of the tumor bioluminescence in the mice at day 36. n = 12 (groins) from six mice for each indicated cell clone. (D) Subcutaneous xenograft tumorigenesis of MAP1B KO MDA-MB-231 cells in nude mice. Left: Extirpated subcutaneous tumors at day 31. Right: Quantification of tumor volumes in mice. Parental MDA, n = 6 mice; MAP1B KO clones #1 and #2, n = 5 mice. (E) Cell morphology of MAP1B KO cells. Left: Representative phase-contrast images. The arrowheads indicate membrane ruffling. Scale bar, 50 μm. Right: Quantification of cells with membrane ruffling. Parental MDA, n = 4 experiments; MAP1B KO clones #1 and #2, n = 3 experiments. (F) Transwell migration (left) and invasion (right) assays of MAP1B KD cells. Migration: parental MDA and both KO cells, n = 12 fields from three independent experiments; invasion: parental MDA and both KO cells, n = 3 experiments. (G) Inverted invasion assays of MAP1B KO cells. Left: Representative images. Right: Quantification of cells invading over 30 μm. Parental MDA and KO #1 cells, n = 13 fields from four experiments; KO #2 cells, n = 14 fields from four independent experiments. (B–G) The values indicate the mean ± SEM. P values were determined using one-way ANOVA with Dunnett’s multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. Source data are available for this figure: SourceData F2.

MAP1B is necessary for efficient tumorigenesis and invasion in TNBC. (A) Immunoblotting of MAP1B-LC1 in MAP1B shRNA-based stable knockdown (KD) and CRISPR/Cas9-based knockout (KO) MDA-MB-231 cells. Upper panel: shRNA-based stable KD. Lower panel: CRISPR/Cas9-based KO. (B) In vitro cell growth of MAP1B KD and KO MDA-MB-231 cells. The growth rate was quantified by a MTT colorimetric assay. Parental MDA, n = 8 from three experiments; MAP1B shRNA and KO, n = 6 from three experiments. (C) Orthotopic xenograft tumorigenesis of MAP1B KO MDA-MB-231 cells stably expressing Luc2 in nude mice. Top: A representative bioluminescence image of xenografted mice on day 29. Bottom: Quantification of the tumor bioluminescence in the mice at day 36. n = 12 (groins) from six mice for each indicated cell clone. (D) Subcutaneous xenograft tumorigenesis of MAP1B KO MDA-MB-231 cells in nude mice. Left: Extirpated subcutaneous tumors at day 31. Right: Quantification of tumor volumes in mice. Parental MDA, n = 6 mice; MAP1B KO clones #1 and #2, n = 5 mice. (E) Cell morphology of MAP1B KO cells. Left: Representative phase-contrast images. The arrowheads indicate membrane ruffling. Scale bar, 50 μm. Right: Quantification of cells with membrane ruffling. Parental MDA, n = 4 experiments; MAP1B KO clones #1 and #2, n = 3 experiments. (F) Transwell migration (left) and invasion (right) assays of MAP1B KD cells. Migration: parental MDA and both KO cells, n = 12 fields from three independent experiments; invasion: parental MDA and both KO cells, n = 3 experiments. (G) Inverted invasion assays of MAP1B KO cells. Left: Representative images. Right: Quantification of cells invading over 30 μm. Parental MDA and KO #1 cells, n = 13 fields from four experiments; KO #2 cells, n = 14 fields from four independent experiments. (B–G) The values indicate the mean ± SEM. P values were determined using one-way ANOVA with Dunnett’s multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. Source data are available for this figure: SourceData F2.

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