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: Stem Cell Reports

Article Title: The role of CDK8 in mesenchymal stem cells in controlling osteoclastogenesis and bone homeostasis

doi: 10.1016/j.stemcr.2022.06.001

Figure Lengend Snippet: CDK8 expression level is increased in MSCs and their progeny with aging (A) The t-SNE plot of color-coded clustering of 1-, 1.5-, 3-, and 16-month cells (top, n = 8,753 cells) and the violin plots of cell-specific marker gene expression (bottom). (B) CDK8 expression levels of each cluster by age ( ∗∗∗ p < 0.001). (C) Expression levels of transcription-related CDKs at the indicated time points of MSCs ( ∗∗∗ p < 0.001). (D) Expression levels of transcription-related CDKs expressed in 1-, 1.5-, 3-, and 16-month MSCs ( ∗∗∗ p < 0.001). (E) CDK8 expression levels of 1-, 1.5-, 3-, and 16-month datasets among each cluster ( ∗∗∗ p < 0.001). (F) Protein levels of CDK8, p-STAT1 Ser727 , STAT1, p-STAT3 Ser727 and STAT3 in MSCs, OBs, and CHs. β-Actin served as a loading control (n = 3 independent replicates, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (G and H) The enrichment plots for aging-related gene sets in MSCs, OBPs, and OBs. (I) CDK8 expression levels in PαS MSCs compared with juvenile (2-week-old) and aged (2-year-old) mice (n = 4, ∗ p < 0.05). (J) Protein levels of CDK8 in aged MSCs. β-Actin served as a loading control (n = 4 independent replicates, ∗∗ p < 0.01). (K) Flow cytometric analysis of cell size in aged MSCs (n = 4 independent replicates, ∗∗∗ p < 0.001) and protein levels of CDK8 normalized by cell size (n = 4 independent replicates, ∗∗ p < 0.01). Statistical significance was detected using the Steel test in (D). t-SNE, t-distributed stochastic neighbor embedding; MSC, mesenchymal stem cell; OBP, osteoblast progenitor; OB, osteoblast; OCYs, osteocytes; CHs, chondrocytes; PαS, PDGFRα + Sca-1 + ; FSC, forward scatter.

Article Snippet: Plasmids pLKO.1.sh CDK8 #1 (#19760, deposited by William Hahn), pLKO.1 puro (#8453, deposited by Bob Weinberg), and FLAG-STAT1 (#71454, deposited by George Stark) were obtained from Addgene. pLKO.1.sh CDK19 (#TRCN0000003140) was purchased from Sigma-Aldrich.

Techniques: Expressing, Marker, Gene Expression, Control

Journal: Stem Cell Reports

Article Title: The role of CDK8 in mesenchymal stem cells in controlling osteoclastogenesis and bone homeostasis

doi: 10.1016/j.stemcr.2022.06.001

Figure Lengend Snippet: CDK8 deficiency in MSCs suppresses osteoclastic activities, leading to high bone mass (A) Schematic diagram of generation of tissue-specific CDK8 knockout mice. Black arrows represent primer binding sites. (B) Deletion efficiency of CDK8 in the marrow-flushed bone of Prx1-Cre;CDK8 fl/fl at the genomic DNA and mRNA levels (n = 4 independent replicates, ∗ p < 0.05). (C and D) μCT (C) and von Kossa staining (D) and BV/TV measured by each method of femurs from CDK8 fl/fl and Prx1-Cre;CDK8 fl/fl mice (n = 6–7, ∗ p < 0.05). (E) Toluidine blue staining (left) and N.Ob/T.Ar of femurs from CDK8 fl/fl and Prx1-Cre;CDK8 fl/fl mice (n = 6–7). Arrowheads indicate osteoblasts. (F) Calcein labeling (left) and BFR/BS of femurs from CDK8 fl/fl and Prx1-Cre;CDK8 fl/fl mice (n = 6–7). (G) TRAP staining (left) and Oc.S/BS of femurs from CDK8 fl/fl and Prx1-Cre;CDK8 fl/fl mice (n = 6–7, ∗∗ p < 0.01). Arrowheads indicate TRAP-positive osteoclasts. (H) TRAP serum level in CDK8 fl/fl and Prx1-Cre;CDK8 fl/fl mice (n = 6–8, ∗ p < 0.05). (I) Deletion efficiency of CDK8 in the marrow-flushed bone of Col1a1-Cre;CDK8 fl/fl at the genomic DNA level and the mRNA level (n = 4, ∗ p < 0.05). (J) μCT analysis and BV/TV measurement of femurs from CDK8 fl/fl and Col1a1-Cre;CDK8 fl/fl mice (n = 6–7). (K–O) BV/TV measured by von Kossa staining (K), N.Ob/T.Ar (L), BFR/BS (M), Oc.S/BS (N), and N.Oc/B. Pm of femur from CDK8 fl/fl and Col1a1-Cre;CDK8 fl/fl mice (n = 6–7) (O). All of the mice used in this study were female. Scale bars, 1 mm (C, D, J, and K), 50 μm (G), and 10 μm (E and F). μCT, micro-computed tomography; BV/TV, bone volume/tissue volume; N.Ob/T.Ar, number of osteoblasts/tissue area; BFR/BS, bone formation rate/bone surface; TRAP, tartrate-resistant acid phosphatase; Oc.S/BS, osteoclast surface/bone surface; N.Oc/B.Pm, number of osteoclasts/bone perimeter; N.S., not significant.

Article Snippet: Plasmids pLKO.1.sh CDK8 #1 (#19760, deposited by William Hahn), pLKO.1 puro (#8453, deposited by Bob Weinberg), and FLAG-STAT1 (#71454, deposited by George Stark) were obtained from Addgene. pLKO.1.sh CDK19 (#TRCN0000003140) was purchased from Sigma-Aldrich.

Techniques: Knock-Out, Binding Assay, Staining, Labeling, Micro-CT

Journal: Stem Cell Reports

Article Title: The role of CDK8 in mesenchymal stem cells in controlling osteoclastogenesis and bone homeostasis

doi: 10.1016/j.stemcr.2022.06.001

Figure Lengend Snippet: CDK8 deficiency in MSCs attenuates osteoclastogenesis through STAT1-RANKL axis (A) Protein levels of CDK8, p-STAT1 Ser727 , and STAT1 in MSCs infected with sh CDK8 . β-Actin served as a loading control. (B) BMMs prepared from WT mice were co-cultured with MSCs infected with sh CDK8 , followed by TRAP staining (n = 4 independent replicates, ∗∗∗ p < 0.001). (C) mRNA levels of Tnfsf11 and Tnfrsf11b and Tnfsf11 / Tnfrsf11b ratio in MSCs infected with sh CDK8 (n = 4 independent replicates, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (D) BMMs prepared from WT mice were co-cultured with MSCs infected with sh CDK8 in combination with STAT1 expression vector, followed by TRAP staining (n = 5 independent replicates, ∗∗ p < 0.01 [sh Ctrl + E.V. versus sh CDK8 + E.V. ], # p < 0.05 [sh CDK8 + E.V. versus sh CDK8 + STAT1 ]). (E and F) MSCs were infected with sh CDK8 in combination with STAT1 expression vector, followed by determination of mRNA levels of Tnfsf11 (E) and Tnfrsf11b (F) (n = 4 independent replicates, ∗∗∗ p < 0.001 [sh Ctrl + E.V. versus sh CDK8 + E.V. ], # p < 0.05, ### p < 0.001 [sh CDK8 + E.V. versus sh CDK8 + STAT1 ]). (G) mRNA levels of STAT1-targeted genes in MSCs were infected with sh CDK8 (n = 4 independent replicates, ∗ p < 0.05, ∗∗ p < 0.01). (H) Tnfsf11 expression in CDK8 low (n = 1,185) and CDK8 high (n = 845) MSCs ( ∗ p < 0.05). (I) mRNA levels of STAT1-targeted genes of CDK8 low (n = 1,788) and CDK8 high (n = 1,164) MSCs ( ∗ p < 0.05, ∗∗∗ p < 0.001). (J) MSCs were infected with sh CDK8 , followed by determination of CFU-Fs and CFU-Obs (n = 3 independent replicates). Scale bars, 800 μm (B and D) and 2 mm (J). BMM, bone marrow macrophage; CFU-F, colony-forming unit fibroblast; CFU-Ob, colony-forming unit osteoblast; sh Ctrl , sh Control ; E.V., empty vector.

Article Snippet: Plasmids pLKO.1.sh CDK8 #1 (#19760, deposited by William Hahn), pLKO.1 puro (#8453, deposited by Bob Weinberg), and FLAG-STAT1 (#71454, deposited by George Stark) were obtained from Addgene. pLKO.1.sh CDK19 (#TRCN0000003140) was purchased from Sigma-Aldrich.

Techniques: Infection, Control, Cell Culture, Staining, Expressing, Plasmid Preparation

Journal: Stem Cell Reports

Article Title: The role of CDK8 in mesenchymal stem cells in controlling osteoclastogenesis and bone homeostasis

doi: 10.1016/j.stemcr.2022.06.001

Figure Lengend Snippet: CDK8 deficiency in MSCs corrects higher osteoclastogenesis-supporting activity associated with aging (A and B) Aged MSCs were cultured, followed by determination of (A) SA-β-gal activity (n = 4 independent replicates, ∗∗∗ p < 0.001) and (B) CFU-Fs and CFU-Obs (n = 3 independent replicates, ∗∗ p < 0.01). (C) BMMs prepared from WT mice were co-cultured with aged MSCs, followed by TRAP staining (n = 4 independent replicates, ∗ p < 0.05). (D) mRNA levels of Tnfsf11 and Tnfrsf11b and Tnfsf11 / Tnfrsf11b ratio in aged MSCs (n = 4 independent replicates, ∗ p < 0.05, ∗∗∗ p < 0.001). (E) Protein levels of CDK8, p-STAT1 Ser727 , and STAT1 in aged MSCs infected with sh CDK8 . β-Actin served as a loading control. (F) BMMs prepared from WT mice were co-cultured with aged MSCs infected with sh CDK8 , followed by TRAP staining (n = 4 independent replicates, ∗∗∗ p < 0.001). Scale bars, 800 μm (A, C, and F) and 2 mm (B). MSC, mesenchymal stem cell; SA-β-gal, senescence-associated β-galactosidase; CFU-F, colony-forming unit fibroblast; CFU-Ob, colony-forming unit osteoblast; BMM, bone marrow macrophage; sh Ctrl , sh Control .

Article Snippet: Plasmids pLKO.1.sh CDK8 #1 (#19760, deposited by William Hahn), pLKO.1 puro (#8453, deposited by Bob Weinberg), and FLAG-STAT1 (#71454, deposited by George Stark) were obtained from Addgene. pLKO.1.sh CDK19 (#TRCN0000003140) was purchased from Sigma-Aldrich.

Techniques: Activity Assay, Cell Culture, Staining, Infection, Control

Journal: Stem Cell Reports

Article Title: The role of CDK8 in mesenchymal stem cells in controlling osteoclastogenesis and bone homeostasis

doi: 10.1016/j.stemcr.2022.06.001

Figure Lengend Snippet: Pharmacological inhibition of CDK8 in MSCs decreases their osteoclastogenesis-supporting activity and protects against OVX-mediated bone loss (A and B) BMMs prepared from WT mice were stimulated with RANKL in the presence of 30 nM KY-065, followed by (A) TRAP staining (n = 3 independent replicates) and (B) determination of protein levels of p-STAT1 Ser727 and STAT1. β-Actin served as a loading control. (C–E) MSCs were treated with 30 nM KY-065, followed by determination of (C) CFU-Fs (n = 3 independent replicates), (D) CFU-Obs (n = 3 independent replicates), and (E) protein levels of p-STAT1 Ser727 and STAT1. β-Actin served as a loading control. (F) mRNA levels of Tnfsf11 and Tnfrsf11b and Tnfsf11 / Tnfrsf11b ratio in MSCs treated with 30 nM KY-065 (n = 4 independent replicates, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (G) BMMs prepared from WT mice were co-cultured with MSCs in the presence of 30 nM KY-065, followed by TRAP staining (n = 4 independent replicates, ∗∗∗ p < 0.001). (H) BV/TV measured by von Kossa staining, N.Ob/T. Ar determined by toluidine blue staining and BFR/BS determined by calcein labeling of vertebrae (n = 10, ∗ p < 0.05 [sham + vehicle versus OVX + vehicle], # p < 0.05 [OVX + vehicle vs OVX + KY-065]). (I) Oc.S/BS and N.Oc/B.Pm determined by TRAP staining of femur (n = 10, ∗∗ p < 0.01 [sham + vehicle versus OVX + vehicle], # p < 0.05 [OVX + vehicle versus OVX + KY-065]). Arrowheads indicate TRAP-positive osteoclasts. Scale bars, 800 μm (A and G), 2 mm (C and D), 500 μm (H), and 50 μm (I). OVX, ovariectomy.

Article Snippet: Plasmids pLKO.1.sh CDK8 #1 (#19760, deposited by William Hahn), pLKO.1 puro (#8453, deposited by Bob Weinberg), and FLAG-STAT1 (#71454, deposited by George Stark) were obtained from Addgene. pLKO.1.sh CDK19 (#TRCN0000003140) was purchased from Sigma-Aldrich.

Techniques: Inhibition, Activity Assay, Staining, Control, Cell Culture, Labeling