![Figure 2. RAB5A is required for tumor dissemination and for the conversion from DCIS to infiltrating mammary carcinoma. (A and B) Doxycycline-inducible RAB5AS34N- and control MDA-MB-231 cells were injected into the mammary fat pads of NSG mice. After 3 wk, mice were fed with doxycycline. Metastases were analyzed 4 wk after removal of primary tumor. (A) Ipsilateral metastasis (arrowhead) of control or RAB5AS34N-MDA-MB-231 tumors. (A, right) Quantification (mean ± SEM [error bars]; n = 10 mice/group) of the number and size of disseminated tumors. (B) H&E of control and RAB5AS34N-MDA-MB-231 lung tissue sections (left). The size of metastatic nodules in lungs is the mean tumor area/lung area ± SEM (error bars; n = 9 mice/group repeated in three independent experiments). (C) Mixtures of CFSE-labeled (green) scramble-transfected control (Ctr) and eFluor 670–labeled (red) RAB5A,B,C-depleted MDA-MB-231 cells (0.5 × 106 each) were coinjected into the tail vein of NSG mice. (C, left) Images of lung tissue. (C, middle) The number (mean ± SEM [error bars]; n = 10) of labeled control and RAB5A,B,C-depleted cells/field of lung tissue at the indicated times. **, P < 0.005. (C, right) Efficacy of RAB5 gene silencing by QRT-PCR. **, P < 0.005. (D) Doxycycline-inducible RAB5AS34N- and control-MCF10.DCIS.com cells were injected subcutaneously into NSG mice. After 4 d, mice were fed with doxycycline. (D, left) IHC analyses of control (Ctr) and RAB5AS34N-MCF10.DCIS.com xenografts performed at 1 and 3 wk after doxycycline treatment. Representative images from three independent experiments are shown (n = 5 mice/experimental condition). (D, right) mRNA level of RAB5AS34N expression in control (Ctr) and RAB5AS34N tumors. Error bars indicate SEM. **, P < 0.005. (E) Control (Ctr) and RAB5A-MCF10.DCIS.com cells (5 × 105 each) were labeled with eFluor 670 and injected into NSG mice tail vein. (E, left) Images of lung tissue. (E, middle) The number (mean ± SEM [error bars]; n = 10) of labeled control and RAB5A-MCF10.DCIS.com cells per field at the indicated times. **, P < 0.005. (E, right) RAB5A expression by immunoblotting. Bars: (B) 2 µm; (C) 80 µm; (D) 100 µm; (E) 80 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/206/2/10.1083_jcb.201403127/6/jcb_201403127_fig2.jpeg?Expires=1771616514&Signature=aUAs3rBrMNc3DdMvbu3CdKCXfRYdVcfY6HN4ihlHdzjR8IplEB9pTNgMcbvaZWc2DoR-rLAoQV-6ndT4~nbAo4gxsZu9VPx64v0zVqFgdLEx2GlzbTqvJGgxL3wDKqbCN7h-wwmiGLL8VL0-8KZjz4oC1KzS6LI2cGBF2ZBpInAt880CS91J18IyeFvqo-1sY~0FhvUzcTtElichbuus9zH2VSYsAKMcmUqy6SDYakU~fBGe4aKpW0j2WgLVM9znyBTQeSZf8yAqVBSF9FzzoGEJq1rpuuW3noIilBRd7sxyktLdjLg8E2Vt2SnovD4ZnINUnlxVxtVyqxipkrXs6g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
RAB5A is required for tumor dissemination and for the conversion from DCIS to infiltrating mammary carcinoma. (A and B) Doxycycline-inducible RAB5AS34N- and control MDA-MB-231 cells were injected into the mammary fat pads of NSG mice. After 3 wk, mice were fed with doxycycline. Metastases were analyzed 4 wk after removal of primary tumor. (A) Ipsilateral metastasis (arrowhead) of control or RAB5AS34N-MDA-MB-231 tumors. (A, right) Quantification (mean ± SEM [error bars]; n = 10 mice/group) of the number and size of disseminated tumors. (B) H&E of control and RAB5AS34N-MDA-MB-231 lung tissue sections (left). The size of metastatic nodules in lungs is the mean tumor area/lung area ± SEM (error bars; n = 9 mice/group repeated in three independent experiments). (C) Mixtures of CFSE-labeled (green) scramble-transfected control (Ctr) and eFluor 670–labeled (red) RAB5A,B,C-depleted MDA-MB-231 cells (0.5 × 106 each) were coinjected into the tail vein of NSG mice. (C, left) Images of lung tissue. (C, middle) The number (mean ± SEM [error bars]; n = 10) of labeled control and RAB5A,B,C-depleted cells/field of lung tissue at the indicated times. **, P < 0.005. (C, right) Efficacy of RAB5 gene silencing by QRT-PCR. **, P < 0.005. (D) Doxycycline-inducible RAB5AS34N- and control-MCF10.DCIS.com cells were injected subcutaneously into NSG mice. After 4 d, mice were fed with doxycycline. (D, left) IHC analyses of control (Ctr) and RAB5AS34N-MCF10.DCIS.com xenografts performed at 1 and 3 wk after doxycycline treatment. Representative images from three independent experiments are shown (n = 5 mice/experimental condition). (D, right) mRNA level of RAB5AS34N expression in control (Ctr) and RAB5AS34N tumors. Error bars indicate SEM. **, P < 0.005. (E) Control (Ctr) and RAB5A-MCF10.DCIS.com cells (5 × 105 each) were labeled with eFluor 670 and injected into NSG mice tail vein. (E, left) Images of lung tissue. (E, middle) The number (mean ± SEM [error bars]; n = 10) of labeled control and RAB5A-MCF10.DCIS.com cells per field at the indicated times. **, P < 0.005. (E, right) RAB5A expression by immunoblotting. Bars: (B) 2 µm; (C) 80 µm; (D) 100 µm; (E) 80 µm.
