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. 2016 Jan 11;5:e10727. doi: 10.7554/eLife.10727

Figure 4. GLS2 inhibits migration and invasion of HCC cells through negative regulation of Rac1.

(A, B) Ectopic GLS2 expression inhibited the migration (A) and invasion (B) of different HCC cells as determined by trans-well assays. Upper panels: representative images of migrating (A) and invading (B) Huh-1 cells transduced with control (con) or GLS2-Flag vectors. Scale bars: 200 μm. Lower panels: quantification of average number of migrating (A) and invading (B) cells/field in different HCC cells transduced with control (con) or GLS2-Flag vectors. Data present mean ± SD (n=6). **p<0.001; Student’s t-test. (C, D) Knockdown of GLS2 by shRNA vectors promoted the migration (C) and invasion (D) of different HCC cells. Upper panels: representative images of migrating (C) and invading (D) Huh-1 cells with or without GLS2 knockdown. Scale bars, 200 μm. Data present mean ± SD (n=6). **p<0.001; Student’s t-test. (E) Ectopic GLS1 expression promoted the migration (left) and invasion (right) of Huh-1 and HepG2 cells as determined by trans-well assays. (F) Knockdown of GLS1 decreased the migration (left) and invasion (right) of Huh-1 and HepG2 cells. In E, F, data are presented as mean ± SD (n=6). **p<0.001; Student’s t-test. (G, H) Ectopic expression of DN Rac1-T17N largely abolished the promoting effects of GLS2 knockdown on migration (G) and invasion (H) of different HCC cells as measured by trans-well assays. Cells with knockdown of GLS2 by shRNA vectors were transduced with Rac1-T17N expression vectors for trans-well assays. Data present mean ± SD (n=6). **p<0.001; Student’s t-test. (I, J) Ectopic expression of the C-terminus of GLS2, GLS2-C139, inhibited migration (I) and invasion (J) of Huh-1 and HepG2 cells as measured by trans-well assays. Cells were transduced with different GLS2 expression vectors described in Figure 1E for assays. Upper panels in I: representative images of migrating Huh-1 cells. Data present mean ± SD (n=6). **p<0.001; Student’s t-test. GLS, glutaminase; HCC, hepatocellular carcinoma; shRNA, short hairpin RNA; WT, wild type.

DOI: http://dx.doi.org/10.7554/eLife.10727.009

Figure 4.

Figure 4—figure supplement 1. Rac1 promotes the migration and invasion of Huh-1 and HepG2 cells.

Figure 4—figure supplement 1.

(A) Ectopic expression of CA Myc-Rac1-G12V and DN Myc-Rac1-T17N in Huh-1 and HepG2 cells detected by western-blot assays. Cells were transduced with Myc-Rac1-G12V and Myc-Rac1-T17N vectors for assays. (B) Ectopic expression of CA Myc-Rac1-G12V promoted the migration of Huh-1 and HepG2 cells, whereas ectopic expression of DN Myc-Rac1-T17N inhibited the migration of Huh-1 and HepG2 cells. Upper panels: representative images of migrating Huh-1 cells. Scale bars: 200 μm. Lower panel: quantification of average number of migrating cells/field. Data are presented as mean ± SD (n=6). **p<0.001; Student’s t-test. (C) Ectopic expression of CA Myc-Rac1-G12V promoted the invasion of Huh-1 and HepG2 cells, whereas ectopic expression of DN Myc-Rac1-T17N inhibited the invasion of Huh-1 and HepG2 cells. Data are presented as mean ± SD (n=6). **p<0.001; Student’s t-test.
DOI: http://dx.doi.org/10.7554/eLife.10727.010
Figure 4—figure supplement 2. The viability and number of HCC cells with GLS2 overexpression or knockdown after being cultured in serum-free medium for 36 hr.

Figure 4—figure supplement 2.

(A, B) The viability of HCC cells with GLS2 overexpression (A) or knockdown (B) cultured in serum-free medium for 36 hr. (C, D) The relative number of HCC cells with GLS2 overexpression (C) or knockdown (D) cultured in serum-free medium for 36 hr. Same number of cells with GLS2 overexpression or knockdown and their control cells were cultured in serum-free medium for 36 hr before the the viability and number of cells was measured in a Vi-CELL Cell Viability Analyzer (Beckman Coulter, Indianapolis, IN). The viability of cells was analyzed by the trypan blue cell exclusion method. GLS, glutaminase; HCC, hepatocellular carcinoma
DOI: http://dx.doi.org/10.7554/eLife.10727.011
Figure 4—figure supplement 3. Knockdown of endogenous Rac1 largely abolishes the effect of GLS2 on migration and invasion of HCC cells.

Figure 4—figure supplement 3.

(A) The knockdown of Rac1 by 2 different shRNA vectors in Huh-1 and HepG2 cells measured by western blot assays. (B) Knockdown of endogenous Rac1 largely abolished the promoting effects of GLS2 knockdown on migration of Huh-1 and HepG2 cells as measured by trans-well assays. The endogenous Rac1 was knocked down by shRNA vectors in Huh-1 and HepG2 cells with GLS2 knockdown. Left panels: representative images of migrating Huh-1 cells. Scale bars, 200 μm. Right panels: quantification of number of migrating cells/field. Data are presented as mean ± SD (n=6). **p<0.001; Student’s t-test. (C) Knockdown of endogenous Rac1 largely abolished the promoting effects of GLS2 knockdown on invasion of Huh-1 and HepG2 cells as measured by trans-well assays. Data are presented as mean ± SD (n=4). **p<0.001; Student’s t-test. (D) The viability (left panel) and number (right panel) of HCC cells with GLS2 and/or Rac1 knockdown cultured in serum-free medium for 36 hr. Same number of cells were cultured in serum-free medium for 36 hr before the the viability and number of cells was measured in a Vi-CELL Cell Viability Analyzer (Beckman Coulter). The viability of cells was analyzed by the trypan blue cell exclusion method. GLS, glutaminase; HCC, hepatocellular carcinoma; shRNA, short hairpin RNA.
DOI: http://dx.doi.org/10.7554/eLife.10727.012