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

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Basal cells of the human airways acquire mesenchymal traits in idiopathic pulmonary fibrosis and in culture.

doi: 10.1038/labinvest.2015.114

Figure Lengend Snippet: Figure 1 Basal cell reactivity, hyperplasia and upregulation of EMT markers adjacent to fibroblastic foci. Epithelium adjacent to fibroblastic foci shows increased reactivity and mesenchymal phenotype. Sparse staining of CK14 is seen in control samples, whereas CK14 is strongly expressed in epithelium overlying fibroblastic foci. P63, a restricted transcription factor for basal cells is expressed in epithelial cells located both basally and in the top layer indicating formation of metaplasia in IPF samples. E-cadherin is abundant in epithelium both in control and IPF samples. In addition, E-cadherin is present in cells within the foci. N-cadherin is expressed in lower basal epithelial cells in IPF samples whereas control samples are negative. Vimentin is expressed in both epithelium and mesenchyme in IPF samples. Bar 100 μm.

Article Snippet: Lentiviral transduction was performed as previously described.28 In short, plasmids containing scrambled hairpin (pLKO.1 shSCR; Addgene plasmid 17920)29 or shRNA against p63 (shp63alpha pLKO.1 puro; Addgene plasmid 19120)30 were used with packaging plasmids (psPAX2 and pMD2.G) (Addgene plasmids 12260 and 12259, respectively) to generate viral titer in HEK-293 T cells using Arrest-in (Open Biosystems).

Techniques: Staining, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Basal cells of the human airways acquire mesenchymal traits in idiopathic pulmonary fibrosis and in culture.

doi: 10.1038/labinvest.2015.114

Figure Lengend Snippet: Figure 5 Knockdown (KD) of p63 abrogates UG-induced EMT of VA10 cells. p63 KD cells are unable to undergo UG-induced EMT. The expression of the mesenchymal markers N-cadherin and Thy-1 is abrogated in treated KD cells, whereas scrambled control cells show a division into two cellular sub-populations much like the mother cell line VA10. The expression of Vimentin can still be detected in treated KD cells, although to a lesser extent than in treated scrambled cells. Bars 100 μm.

Article Snippet: Lentiviral transduction was performed as previously described.28 In short, plasmids containing scrambled hairpin (pLKO.1 shSCR; Addgene plasmid 17920)29 or shRNA against p63 (shp63alpha pLKO.1 puro; Addgene plasmid 19120)30 were used with packaging plasmids (psPAX2 and pMD2.G) (Addgene plasmids 12260 and 12259, respectively) to generate viral titer in HEK-293 T cells using Arrest-in (Open Biosystems).

Techniques: Knockdown, Expressing, Control

Journal: Developmental cell

Article Title: Tumor Suppressor Inactivation of GDF11 Occurs by Precursor Sequestration in Triple-Negative Breast Cancer

doi: 10.1016/j.devcel.2017.10.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TET-pLKO.1 puro shID2 #1 , This paper , Addgene #83086.

Techniques: Virus, In Situ, Recombinant, Enzyme-linked Immunosorbent Assay, Mutagenesis, RNA Amplification, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Software

Journal: Molecular Therapy

Article Title: Identification of Epigenetic Regulators of DUX4-fl for Targeted Therapy of Facioscapulohumeral Muscular Dystrophy

doi: 10.1016/j.ymthe.2018.04.019

Figure Lengend Snippet: Transcriptional Repression of Epigenetic Regulators by dCas9-KRAB Reduces Expression of DUX4-fl in FSHD Myocytes (A–D) Differentiated FSHD myocytes were subjected to four serial co-infections with combinations of lentiviral supernatants expressing either dCas9-KRAB or individual sgRNAs (g1–2 for each target gene) targeting BAZ1A (A), BRD2 (B), KDM4C (C), or SMARCA5 (D). Cells were harvested ∼72 hr later for analysis of gene expression by qRT-PCR (as in Figure 1 ). Data are plotted as the mean + SD value of three independent experiments, with relative mRNA expression for cells infected with dCas9-KRAB alone set to 1. *p < 0.05 and **p < 0.01 are from comparing sgRNAs to dCas9-KRAB alone. Refer to the , Figure S3 , and and for additional details.

Article Snippet: The pHAGE EF1-dCas9-KRAB (Addgene plasmid 50919) and pLKO.1-puro U6 sgRNA BfuAI stuffer lentiviral plasmids were developed by Rene Maehr and Scot Wolfe (Addgene plasmid 50920).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Infection