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) Measurements of NHE1 activity at prescribed pH e in the absence or presence of EIPA (40 μM) using NH 4 Cl prepulse technique by quantifying the pH i recovery of pHrodo-loaded cells. (B) ΔpH/min values were obtained after removal of NH 4 Cl medium and calculated by linear regression fitting of the initial recovery phase. Data represent mean ± SD of the individual averages from three experiments for n ≥ 51 cells. * p < 0.05, ** p < 0.01 by one-way ANOVA followed by Tukey’s test. (C) Immunofluorescence images of cells in confinement at pH e = 7.4 or 6.4, and stained for NHE1 (red), ezrin (green), and Hoechst (blue). (D) Percentage of cells displaying NHE1 and ezrin co-polarization at prescribed pH e . Data represent mean ± SD from three experiments. ** p < 0.01 by unpaired t test. (E) Western blot images and quantification of NHE1 expression. Data represent mean ± SD from four experiments. * p < 0.05 by unpaired t test. (F) Representative PLA images of NHE1-pAkt interaction after scramble control (shControl) or shNHE1 cell preconditioning at prescribed pH e for 24 h. (G) Quantification of PLA interaction normalized to total number of dots per nuclei. Data represent mean ± SD of multiple fields from four experiments. ** p < 0.01 and **** p < 0.0001 by one-way ANOVA followed by Tukey’s test after log transformation. (H and J) Western blot images of pAkt and tAkt (H), and NHE1 (J) before and after pH e change from 7.4 to 6.4 at prescribed time points (0.5, 1, and 2 h). (I) Quantification of pAkt and tAkt normalized by GAPDH (left y axis) and of pAkt normalized by tAkt (right y axis) from five experiments. * p < 0.05 and ** p < 0.01 by Kruskal-Wallis followed by Dunn’s. (K) Quantification of NHE1 expression from four experiments. (L) Western blot images of pAkt and tAkt at pH e of 7.4 before and after EIPA (40 μM) treatment for prescribed time points (0.5, 1, and 2 h). (M) Quantification of pAkt and tAkt, as described in (I), from three experiments before and after the addition of EIPA (40 μM). * p < 0.05 and ** p < 0.01 by one-way ANOVA followed by Tukey’s test. (N) Western blot images of pAkt and tAkt for SC and NHE1-KD cells. (O) Quantification of pAkt and tAkt normalized, as described in (I), from five experiments. * p < 0.05, ** p < 0.01 by unpaired t test. Data represent mean ± SD. Cell model: MDA-MB-231.

Article Snippet: Lentiviral shRNAs targeting NHE1 and ezrin were generated by subcloning the following sequences into pLKO.1 puro (plasmid #8453, Addgene) as previously described., non-targeting scramble control: 5′ -GCACTACCAGAGCTAACTCAGATAGTACT-3’. human shEZRIN: 5′ -CCGTGGGATGCTCAAAGATAA-3’. human sh1NHE1: 5′ -GACAAGCTCAACCGGTTTAAT-3’. human sh2NHE1: 5′ -CCAATCTTAGTTTCTAACCAA-3’.

Techniques: Activity Assay, Immunofluorescence, Staining, Western Blot, Expressing, Control, Transformation Assay

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–D) Western blot images (A and C) and quantification of ILK expression (B and D) in WT (B), SC and NHE1-KD (D) cells following preconditioning at pH e = 7.4 or 6.4 for 24 h. Data represent mean ± SD from N = 3 experiments. * p < 0.05 and ** p < 0.01 by unpaired t test (B) or by one-way ANOVA followed by Tukey’s test (D). (E–H) Western blot images (E and G) and quantification of ILK expression in cells preconditioned at pH e of 7.4 or 6.4 for 24 h in the presence of LY294002 (LY; 10 μM, 24 h) (E and F), verteporfin (Vert., 0.3 μM, 24 h) (G and H), or vehicle control. Data represent mean ± SD from N = 3 experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA followed by Tukey’s test. (I) Immunofluorescence staining of cells with NHE1 (magenta), ILK (white), and Hoechst (blue) following their exposure to pH e = 7.4 or 6.4 for 24 h. (J) Immunofluorescence staining with ezrin (green), ILK (white), β1-integrin (red), and Hoechst (blue) following their exposure to pH e = 7.4 or 6.4 for 24 h. (K) SC or NHE1-KD cell migration velocities on 2D following their preconditioning at prescribed pH e for 24 h in the presence of either the ILK inhibitor CPD22 (2.5 μM) or vehicle control (VC). Data represent mean ± SD of the average value of each biological repeat ( N = 3) each with n ≥ 17 cells. * p < 0.05 by one-way ANOVA followed by Tukey’s test. (L) Representative phase contrast images of cells dissociating from 3D breast cancer spheroids embedded in 3D collagen gels at the indicated time points in the presence of either CPD22 (2.5 μM) or VC, following cell preconditioning at pH e 7.4 or 6.4 for 24 h. (M) Number of dissociated cells from 3D spheroids embedded in 3D collagen gels at t = 22 h in the presence of CPD22 (2.5 μM) or VC at prescribed pH e . Data represent mean ± SD of the average value of each biological repeat ( N = 3) each with n ≥ 7 spheroids. * p < 0.05 by one-way ANOVA followed by Tukey’s test. Cell model: MDA-MB-231. Scale bars: 10 μm (I and J) or 100 μm (L).

Article Snippet: Lentiviral shRNAs targeting NHE1 and ezrin were generated by subcloning the following sequences into pLKO.1 puro (plasmid #8453, Addgene) as previously described., non-targeting scramble control: 5′ -GCACTACCAGAGCTAACTCAGATAGTACT-3’. human shEZRIN: 5′ -CCGTGGGATGCTCAAAGATAA-3’. human sh1NHE1: 5′ -GACAAGCTCAACCGGTTTAAT-3’. human sh2NHE1: 5′ -CCAATCTTAGTTTCTAACCAA-3’.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Migration