Journal: Cell reports
Article Title: Hypoxia restores the acidosis-induced inhibition of cancer cell dissemination
doi: 10.1016/j.celrep.2026.116970
Figure Lengend Snippet: (A) Western blot images of Akt and ERK from cells preconditioned at pH e = 7.4 or 6.4 for 24 h before blotting. (B) Confined migration velocity of cells preconditioned at pH e = 7.4 or 6.4 for 24 h in the absence or presence of LY294002 (10 μM, 24 h). Data represent mean ± SD of the average value of each biological repeat ( N = 4) each with n ≥ 24 cells. * p < 0.05, ** p < 0.01 by one-way ANOVA followed by Tukey’s test. (C–E) Western blot images (C) and quantification of NHE1 expression of wild-type (WT) cells (D) and NHE1 activity of pHRed-expressing cells (E) at pH e = 7.4 following 24-h incubation with vehicle control or LY294002 (LY; 10 μM, 24 h). Data represent mean ± SD from three experiments (D and E) using n ≥ 44 cells per condition (E). ** p < 0.01 and *** p < 0.001 by one-way ANOVA followed by Tukey’s test (D), * p < 0.05 by unpaired t test (E). (F and H) Immunofluorescence images of cells in 2D collagen I-coated substrates at prescribed pH e (F) or at pH e = 7.4 in the absence or presence of LY294002 (10 μM, 24 h) (H), and stained for YAP1 (red), actin (green), and Hoechst (blue). (G and I) Quantification of nuclear-to-cytosolic YAP1 ratio at prescribed pH e (G) or at pH e = 7.4 in the absence or presence of LY294002 (10 μM, 24 h) (I). Data represent mean ± SD of the average value of each biological repeat ( N = 3) each with n ≥ 37 cells. * p < 0.05, ** p < 0.01 by unpaired t test. (J and K) Western blot images (J) and migration velocity (K) following 24-h incubation with verteporfin (0.3 μM) at prescribed pH e . Data represent mean ± SD of the average value of each biological repeat ( N = 3) each with n ≥ 50 cells. ** p < 0.01, *** p < 0.001 by one-way ANOVA followed by Tukey’s test. (L) Time-lapse montage of a representative cell expressing opto-iSH2 and CAAX-CIBN-GFP and preconditioned at pH e = 6.4 before and after light stimulation at the cell leading edge in a region enclosed by the yellow box. Enrichment of activated Akt is shown by the yellow arrowheads, whereas white arrowheads denote the cell rear. (M) Migration velocity of WT and NHE1-KD cells before and after light stimulation. Cells were preconditioned at pH e = 6.4 for 3 h prior to the onset of the experiment. Data represent mean ± SD for n ≥ 12 cells from five experiments. *** p < 0.001 by paired t test. (N) Migration velocity of WT and Myr-Akt-expressing cells after preconditioning at pH e = 7.4 or 6.4 for 24 h. Data represent mean ± SD of the average value of each biological repeat ( N = 3) for n ≥ 14 cells per condition. * p < 0.05, ** p < 0.01 by one-way ANOVA followed by Tukey’s test. Scale bars: 10 μm. Cell model: MDA-MB-231.
Article Snippet: CAAX-CIBN-GFP was a kind gift from Dr. Xavier Trepat (Institute for Bioengineering of Catalonia)., iSH2 was amplified from mCherry-CRY2-iSH2 (plasmid 66839, Addgene) and then inserted into pLV-EF1a-Cry2-mCherry-puro to create Cry2-mCherry-iSH2 (opto-iSH2) in the lentiviral backbone.
Techniques: Western Blot, Migration, Expressing, Activity Assay, Incubation, Control, Immunofluorescence, Staining
