skim milk powder  (MedChemExpress)

Journal: iScience

Article Title: Promotion of breast cancer by the DNPH1 enzyme

doi: 10.1016/j.isci.2026.115227

Figure Lengend Snippet: Alterations in mammary tumors upon Dnph1 knockout (A) Examples of CD31 staining in mammary tumors from a MMTV-Her2/Dnph1 +/+ and a MMTV-Her2/Dnph1 −/− mouse. Right panels show ImageJ-mediated image modulation to enhance vessel structures, allowing the computer-mediated quantification of blood vessel density. Scale bars, 0.4 mm. (B) Corresponding quantification of vessel density. Tumors were age- and weight-matched; two-tailed, unpaired t test. (C) Tumor vessel density plotted against the weight of the tumor. These tumors were not weight- or age-matched, yet include those shown in panel B. Shown are linear regressions; while the slopes were not significantly different, the Y-intercepts were significantly different ( p = 0.0007). (D) Relative levels of AMP, GMP, and UMP in mammary tumors from MMTV-Her2/Dnph1 +/+ and MMTV-Her2/Dnph1 −/− mice; two-tailed, unpaired t test. (E and F) Immunohistochemical staining for phospho-AMPKα or phospho-YAP1, respectively, on age- and weight-matched tumors as in panel B. Examples of immunohistochemical staining are shown at the bottom. Scale bars, 0.4 mm. Means with standard deviation are shown in panels B and D-F.

Article Snippet: YAP1 phosphorylated on serine 127 , Cell Signaling , Cat# 4911.

Techniques: Knock-Out, Staining, Two Tailed Test, Immunohistochemical staining, Standard Deviation

Journal: iScience

Article Title: Promotion of breast cancer by the DNPH1 enzyme

doi: 10.1016/j.isci.2026.115227

Figure Lengend Snippet: Model how DNPH1 promotes breast tumor development and progression This model is centered around a DNPH1-YAP1 axis, yet DNPH1 may utilize to-be-identified effectors other than YAP1 to stimulate, e.g., stem cells or angiogenesis.

Article Snippet: YAP1 phosphorylated on serine 127 , Cell Signaling , Cat# 4911.

Techniques:

Journal: iScience

Article Title: Mesenchymal-epithelial transition reduces proliferation but increases immune evasion in tumor spheroids

doi: 10.1016/j.isci.2025.113023

Figure Lengend Snippet: Cell crowding causes cytoplasmic arrest of YAP1 and reduced proliferation in both mesenchymal and MET-induced MDA-MB-231 in 2D culture (A) Immunofluorescence images showing the intracellular distribution of YAP1 (green) in control (mesenchymal) and induced (epithelial) cells at 4 different seeding densities after 3 days of growth. Nuclei are stained by DAPI (blue). Scale bar = 50 μ m. (B) Nucleus-to-cytoplasm ratio of YAP1 intensity at different seeding densities corresponding to images shown in panel a. Average values of nucleus-to-cytoplasm ratios were calculated for each recorded frame, see panel a, giving rise to one data point in the statistics. For simplicity, the three-star significance between density 1 and 2, and density 2 and 3 were omitted in the chart. Details on the analysis can be found in the Section ‘ ’. (C) Fluorescence images of cells subject to an EdU proliferation assay at 4 different seeding densities in control (mesenchymal) and induced (epithelial) cells. Cells that have undergone S phase during the incubation time show EdU positive nuclei (red) while the rest of the nuclei are stained with only DAPI (blue). Scale bar = 50 μ m. (D) Percentage of EdU positive nuclei at different seeding densities for both conditions. For each recorded frame, fractions of EdU positive cells were determined. Correspondingly, each recorded frame gave rise to one data point, see Section “ ”. (E and F) Scatterplots showing YAP1 localization ratio and percentage of EdU positive cells, respectively, in control and MET-induced MDA-MB-231 with relation to cell density. Cell density is expressed in number of nuclei per area of a recorded frame. (G) Schematic representation of the micro-patterned surfaces used to constrain the area in which cells can adhere to the substrate on adhesive RGD squares of 20 μ m edge length. (H, H’) Representative images of cells seeded on unpatterned (top row) and micro-patterned surfaces to limit the cell spread (bottom row). Top (H): control, bottom (H’): MET-induced. Green: YAP1 immunostaining, blue: DAPI staining of cell nuclei. Scale bar = 20 μ m. (I and J) bar charts showing cellular spreading area (I) and YAP1 nuclear accumulation (J) for unconstrained cells and cells on micro-patterned surfaces. Each cell sampled constitutes a data point in the statistics. For panels B, D, I, and J, bar heights represent the mean and error bars show the corresponding standard error of the mean. Further, “n” shows the total number of technical replicates sampled from at least 2 independent biological replicates. Significance was tested using a two-tailed Mann-Whitney-U-Test and p ≤ 0.05. ∗ ≤ 0.05, ∗∗ ≤ 0.01 and ∗∗∗ ≤ 0.001. See also .

Article Snippet: Nitrocellulose membranes were blocked with 5% (w/v) skimmed milk powder (T145.1, Carl Roth, Karlsruhe, Germany) or 5% BSA for the case of phosphorylated proteins, both in TBST (20 mM Tris–HCl, 137 mM NaCl, 0.1% Tween 20 (pH 7.6)) for 1 h at room temperature followed by washing with TBST, and incubation at 4 ° C overnight with the corresponding primary antibody diluted 1:1000 E-cadherin (#60335, Proteintech), 1:10.000 Vimentin (#60330, Proteintech), 1:1000 ZEB1 (#21544-1-AP, Proteintech), 1:2000 YAP1 (#13584-1-AP, ProteinTech), 1:1000 phospho-YAP1 (Ser127) (#4911, Cell Signaling), 1:500 SNAI1 (#26183-1-AP, ProteinTech) and 1:5000 GAPDH (#ab9485, Abcam) in 5% (w/v) bovine serum albumin/TBST solution.

Techniques: Immunofluorescence, Control, Staining, Fluorescence, Proliferation Assay, Incubation, Adhesive, Immunostaining, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Mesenchymal-epithelial transition reduces proliferation but increases immune evasion in tumor spheroids

doi: 10.1016/j.isci.2025.113023

Figure Lengend Snippet: MET-induction reduces proliferative signaling in self-organized dense islands of MDA-MB-231 cells with open boundaries (A) Cells are seeded at high density in an isolated droplet of 10 μ L. After cell adhesion, additional medium is added to fill the well, see Section ‘ ’. (B) Low magnification images of cell islands after two days of growth showing YAP1 localization (green, top row) and proliferation indicated by EdU-staining (red, bottom row). Cell nuclei are in addition stained with DAPI (blue), scale bar = 500 μ m. (C) YAP1 nucleus-to-cytoplasm ratio in center and border regions of adhered cell islands. (D) Percentage of EdU-positive cell nuclei for both center and border regions. (C, D) For each cell island, 4–5 frames were recorded for both center and edge regions. (E) Cell nuclei density outside of cell islands. Each data point corresponds to cell counts in a peripheral region of on average 1 mm 2 in a low magnification image, see Section ‘ ’. (F–H) Actomyosin perturbation enhances crowding in the center of mesenchymal islands and reduces nuclear YAP1 and cell escape rates. Actomyosin was perturbed by the addition of: 2.5 μ M Blebbistatin or 2.5 μ M Y-27632 Rho Kinase inhibitor. For panels C, D, E, F, G, and H, each data point corresponds to one recorded frame. The total number of recorded frames (technical replicates) is given by “n”. Data were obtained from 2 independent biological replicates that contained each 2 islands per condition. The height of the bars represent the mean for each condition and error bars show the corresponding standard error of the mean. Significance was tested using Mann-Whitney-U-Test and p ≤ 0.05. ∗ ≤ 0.05, ∗∗ ≤ 0.01 and ∗∗∗ ≤ 0.001. See also .

Article Snippet: Nitrocellulose membranes were blocked with 5% (w/v) skimmed milk powder (T145.1, Carl Roth, Karlsruhe, Germany) or 5% BSA for the case of phosphorylated proteins, both in TBST (20 mM Tris–HCl, 137 mM NaCl, 0.1% Tween 20 (pH 7.6)) for 1 h at room temperature followed by washing with TBST, and incubation at 4 ° C overnight with the corresponding primary antibody diluted 1:1000 E-cadherin (#60335, Proteintech), 1:10.000 Vimentin (#60330, Proteintech), 1:1000 ZEB1 (#21544-1-AP, Proteintech), 1:2000 YAP1 (#13584-1-AP, ProteinTech), 1:1000 phospho-YAP1 (Ser127) (#4911, Cell Signaling), 1:500 SNAI1 (#26183-1-AP, ProteinTech) and 1:5000 GAPDH (#ab9485, Abcam) in 5% (w/v) bovine serum albumin/TBST solution.

Techniques: Isolation, Staining, MANN-WHITNEY