Extended Data Figure 2. Pyruvate depletion does not affect collagen synthesis.

(a) Total collagen + protein synthesis (left panel) as well as protein synthesis (right panel) in human and mouse breast cancer spheroids with and without pyruvate. Total collagen + protein synthesis was assessed by incorporation of radioactive proline into collagen and protein, while sole protein synthesis was assessed by fluorescent labeled methionine incorporation into protein. The latter is more specific towards protein synthesis since methionine is only to a minor extent incorporated into collagen. Changes that occur in both parameters indicate alterations in protein synthesis, while changes that occur only in total collagen + protein synthesis indicate alterations in collagen synthesis. Our data indicate that collagen synthesis was not altered by pyruvate depletion, because either total collagen + protein synthesis is not altered or both parameters are altered to a similar extent in the tested cell lines. n=3.

(b) Relative collagen I and III abundance and representative 3D reconstruction in human MCF10A H-RAS^V12 and mouse 4T1 breast cancer spheroids with and without pyruvate measured by immunofluorescence. n=5. Collagen I/III are major collagen species in breast cancer²². Blue indicates DAPI-stained nuclei, red indicates collagen I, and green indicates collagen III. The total fluorescence intensity was measured in each microscopy field and normalized over cell number, scored as number of DAPI-stained nuclei. Five microscopy fields were averaged for each sample. Relative fluorescence intensities per cell are depicted, normalized to control condition. Solid line indicates median, the box extends are the 25th to 75th percentiles, the whiskers span between the smallest and the largest value.

Error bars represent SD of mean from biological independent samples unless otherwise noted. Two-tailed unpaired student’s T-test.