Figure 4. Collagen uptake promotes survival and proline promotes both survival and proliferation.

(a) PK4A cell survival in indicated media supplemented with vehicle or water-soluble collagen IV alone or in combination with EIPA treatment (upper). After treatments, cells were submitted to crystal violet assay. (upper) Representative crystal violet images (n=3 independent experiments). (middle) Crystal violet intensity, expressed as mean±s.e.m. (n=3 independent experiments) is presented as fold-induction relative to intensity in glucose-free condition without EIPA and without col IV. ND: Value below detection level. **P<0.01, two-tailed unpaired Student’s t-test. (lower) Glutamine concentration in media of PK4A cells cultured in glucose-free medium supplemented or not with water-soluble collagen IV over 72 h. Data represent mean±s.e.m. (n=3 independent experiments). Two-way ANOVA followed by Bonferroni post-hoc test revealed no significant difference in PK4A glutamine uptake between the two culture conditions. (b) ERK1/2 and p70S6K phosphorylation status in PK4A cells cultured with or without water-soluble collagen IV in indicated media during 72 h. (c) Prolidase levels in supernatant from PK4A cells cultured with coated collagen I in indicated media. Total protein loading was determined by Ponceau red staining. (b,c) marker sizes are indicated in kDa. Photos are representative of n=2 (b) and n=3 (c) independent experiments. Uncropped images of blots are shown in Supplementary Fig. 10. (d) Survival of PK4A cells cultured under normoxic (21% O₂) or hypoxic (1% O₂) conditions (upper and lower charts, respectively) in indicated nutrient conditions, in media alone or supplemented with either DHP (1 mM) or with DHP and L-proline (500 μM). Living cells, determined 24 h after treatment, are expressed as percentage of untreated cells in complete media (mean±s.e.m., n=4 independent experiments). *P<0.05, **P<0.01, ***P<0.001; two-tailed unpaired Student's t-test. (e) Proliferation of PK4A cells cultured for 24–96 h under normoxia as in d. Data are mean±s.e.m., n=4 independent experiments. P value, is relative to corresponding untreated value at each time point.*P<0.05, **P<0.01, ***P<0.001; two-way ANOVA followed by Bonferroni post-hoc test. ANOVA, analysis of variance.