Extended Data Fig. 5: Creatine homeostasis facilitates collective migration of pancreatic cancer cells.
(a) CCr activity schematic. (b) Ccr uptake by KPC cells. Values in b and c are mean ± SD from 3 biological replicates within the same day and representative of 2 independent experiments. (c) Phosphocreatine circuit and ATP levels of (b). (d) ADP to ATP ratio of (b). Mean ± SD from 4 independent experiments. (e) Growth curves of (b). Mean ± SD from 3 independent experiments. (f) Left; CCr-treated cells. Scale bars, 100μm. Right; Magnification of dashed boxes. White arrows; protrusions. Scale bars, 50μm. (g) Average protrusion length from (f). Values are mean ± SD from n = 206 control, n = 145 5 mM and n = 142 10 mM CCr-treated cells. (h) Cell speed from (f). Mean ± SD from n = 263 control, n = 185 5 mM and n = 192 10 mM CCr-treated cells from 3 independent experiments. (i) Immunofluorescence of CCr-treated cells showing F-actin (magenta) and nuclei (blue). Scale bars, 20μm. (j, k) Cell area (μm2) (j) and circularity index (k) from (i). Mean ± SD from n = 60 control, n = 78 5 mM and n = 69 10 mM CCr-treated cells from 3 independent experiments. (l–n) Strain energy (l), maximum force (m) of CCr-treated cells and strain energy of blebbistatin treated cells (n). Mean ± SD of n = 33 control, n = 36 CCr-treated and n = 17 blebbistatin-treated cells from 3 independent experiments. Mann-Whitney U test. (o) Creatine and phosphocreatine levels of control or pCr supplemented cells. Values in o and p are mean ± SD from 3 biological replicates within the same day. (p) ADP/ATP ratio of (o). (q) Control (EV) or CKBCRISPR KO cells expressing GFP or CKB-GFP were immunoblotted for GFP and CKB. Pictures representative of 3 independent experiments. (r) Pictures of cells from (q) populating a wounded monolayer. Scale bars, 100μm. (s) Relative wound closure of control or CCr-treated wild-type (EV) cells. Values in s, t, u are mean ± SD from 3 independent experiments. (t–u) Relative wound closure (t) and relative closure at t1/2 of control (u) of control or CCr-treated CKBCRISPR KO cells. In d,s,u: two-tailed one-sample t-test on LN transformed values. In g,j,k: Kruskal-Wallis with Dunn’s multiple comparisons test.
