Journal: International Journal of Molecular Sciences

Article Title: Thrombospondin 1–CD47 Signalling Modulates Vascular Smooth Muscle Cell Senescence in Chronic Kidney Disease

doi: 10.3390/ijms27020755

Figure Lengend Snippet: TSP1 and indoxyl sulfate limit VSMC proliferation via CD47. hVSMC cell viability was measured by assessing reduction in tetrazolium salt sodium 3′- [1- [(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT) and measuring absorbance at 450 nm in cells after 48 h treatment with ( A ) TSP1 (0, 0.2, 2.2, 5, 10 nM) ( n = 4), ( B ) TSP1 2.2 nM ± pre-treatment with anti-CD47 antibody for 30 min ( n = 6), ( C ) IS (0, 1, 10, 100, 500 µM) ( n = 4), or ( D ) IS (100, 500 µM) ± pre-treatment with anti-CD47 antibody for 30 min ( n = 4–8). All data shown are mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 by one-way analysis of variance with Holm–Sidak post-hoc test ( A , C ) or Kruskal–Wallis test ( B , D ). Abbreviations: αCD47—anti-CD47 antibody; hVSMC—human aortic vascular smooth muscle cell; IS—indoxyl sulfate; TSP1—thrombospondin-1.

Article Snippet: Cell proliferation and senescence were measured using XTT Cell Viability Kit (#9095, Cell Signalling Technology, Danvers, MA, USA) and Mammalian β-Galactosidase Assay Kit (75707, Thermo Fisher Scientific), respectively.

Techniques:

Journal: Oncogene

Article Title: Wnt target IQGAP3 promotes Wnt signaling via disrupting Axin1-CK1α interaction

doi: 10.1038/s41388-025-03512-y

Figure Lengend Snippet: A Immunoblot of NUGC3 wild-type and IQGAP3 CRISPR knock-out clones. Samples were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. The relative immunoblot bands of β-catenin and β-actin (βcat/βact) were quantified by densitometry. B NUGC3 cells were lysed, and RNA collected were converted to cDNA and subjected to RT-PCR to quantify the amount of Wnt target genes CCND1 and MYC . Representative data were collected and are expressed as the mean ± SD from three independent experiments. Student’s t test was performed, with ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001. C XTT Cell Proliferation Assay of NUGC3 wild-type and IQGAP3 CRISPR knock-out clones. Representative data were collected and are expressed as the mean % growth ±SD from three independent experiments. Two-way ANOVA was performed, with ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001. D Clonogenic Assay of NUGC3 wild-type and IQGAP3 CRISPR knock-out clones. E Colony-Forming Efficiency (%) of NUGC3 wild-type and IQGAP3 CRISPR knock-out clones. F Immunoblot of AGS wild-type and IQGAP3 CRISPR knock-out clones. Image is best representative of three independent experiments. Samples were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. The relative immunoblot bands of β-catenin and α-Tubulin (βcat/αTub) were quantified by densitometry. G AGS cells were lysed, and RNA collected were converted to cDNA and subjected to RT-PCR to quantify the amount of Wnt target genes CCND1 and MYC . Representative data were collected and are expressed as the mean ± SD from three independent experiments. Student’s t test was performed, with ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001. H XTT Cell Proliferation Assay of AGS wild-type and IQGAP3 CRISPR knock-out clones. Representative data were collected and are expressed as the mean % growth ±SD from three independent experiments. Two-way ANOVA was performed, with ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001. I Clonogenic Assay of AGS wild-type and IQGAP3 CRISPR knock-out clones. J Colony-Forming Efficiency (%) of AGS wild-type and IQGAP3 CRISPR knock-out clones.

Article Snippet: Cell proliferation was determined using the TACS® XTT Cell Proliferation Assay (R&D Systems, Inc.), following the manufacturer’s instructions.

Techniques: Western Blot, CRISPR, Knock-Out, Clone Assay, SDS Page, Reverse Transcription Polymerase Chain Reaction, Proliferation Assay, Clonogenic Assay

Journal: PLOS ONE

Article Title: Mutation of SIVA , a candidate metastasis gene identified from clonally related bilateral breast cancers, promotes breast cancer cell spread in vitro and in vivo

doi: 10.1371/journal.pone.0302856

Figure Lengend Snippet: ( A ) Schematic of SIVA protein showing the position (red line in exon 4) of point mutation identified in the “breast to breast” spread in patient 8. Domains: Amphipathic helix (SAH), death domain homology region (DDHR), cysteine rich domains (Cys-rich). ( B ) Stable knockdown of SIVA protein expression by shRNA (KO) and reintroduction of SIVA-WT protein (WT*), compared to endogenous (end) SIVA expression in empty vector control cells (EV) was demonstrated by Western blot analysis. ( C) 231L cell migration (Boyden Chamber) and (D) 231L cell invasion (Matrigel matrix invasion) analyses were expressed as total cell counts in four fields of view (4 fov) normalized against EV control. Each bar represents the mean ± S.E.M (n = 3, total cells in 4 images/assay); One-way ANOVA ( C : p = 0.0035 and D : p = 0.0.0002) followed by Tukey’s post-hoc test. ( E ) Overexpression of SIVA-WT (WT) and mutated SIVA (D160N) protein, compared to endogenous SIVA levels in EV control 231L cells was demonstrated by Western blot analysis. ( F ) Cell migration and ( G ) cell invasion analyses of 231L cells overexpressing wild type SIVA (WT) and SIVA mutation ( D160N ) were expressed as in ( C , D ). One-way ANOVA ( F : p = 0.0002, G : p = 0.0005). ( H ) Proliferation XTT assays, each point represents the mean ± S.E.M (n = 2, 6 replicates/assay); simple linear regression ( p = 0.8788). ( I ) SIVA-D160N expressing 231L cells increased spread of breast cancer cells in immuno-deficient NSG mice. 25K cells were injected into the lateral tail veins of female NSG mice (n = 5), and the proliferation and spread monitored by serial in vivo imaging (IVIS). Exposure times are 60s and 3s for 7 days and 21 days respectively. Luminescence counts is indicated in colored bar on the y -axis. ( J ) Absolute luminescent intensity of photons emitted from spreading tumor cells in each animal in the IVIS images ( I ) was quantified. Each line represents the mean ± SEM (n = 5). Mixed-effects analysis ( p <0.0001) followed by Tukey’s post-hoc test. Post Test p values: * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: Cancer cell proliferation in vitro was determined by the Trevigen TACS®XTT Proliferation Assay (R&D Systems, Cat# 4891-025-K) according to manufacturer’s instructions.

Techniques: Mutagenesis, Expressing, shRNA, Plasmid Preparation, Western Blot, Migration, Over Expression, Injection, In Vivo Imaging