Journal: Biomaterials advances

Article Title: Towards modular engineering of cell signalling: Topographically-textured microparticles induce osteogenesis via activation of canonical hedgehog signalling.

doi: 10.1016/j.bioadv.2023.213652

Figure Lengend Snippet: Fig. 7. Hedgehog activation and topographically induced osteogenesis is Smo-dependant. (A) Representative Western blot (I) and densitometry analysis (II) showing GLI1 expression levels in C3H10T1/2 cells after 3 days of 300 μM KAAD-cyclopamine treatment, normalised to GAPDH (n = 3). (B–D) Relative qPCR analysis of (B) Gli1, (C) Runx2 and (D, E) Bglap2 after 3 (D) and 7 (E) days in culture, represented as fold change normalised to Gapdh and relative to 2D vehicle-only controls. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. Values are means ± standard deviation for three independent biological repeats. (**p < 0.01, ***p < 0.001, ****p < 0.0001, ns = no statistically significant difference). Abbreviations: D-KAAD and S-KAAD, 300 μM KAAD-cyclopamine treated cells cultured on dimpled and smooth microparticles, respectively: D-DMSO and S-DMSO, 0.06 % DMSO-treated cells cultured on dimpled and smooth microparticles, respectively; Gli1, glioma associated oncogene homolog 1; Runx2, Runt-related tran scription factor2; Bglap2, bone gamma-carboxyglutamate protein 2; DMSO, dimethyl sulphoxide; KAAD-cyclo, KAAD-cyclopamine; GAPDH, Glyceraldehyde-3- Phosphate Dehydrogenase.

Article Snippet: The following primary antibodies were used: anti-ADP-ribosylation factor-like protein (ARL13B) mouse monoclonal antibody (1:2000; clone N295B/66, NeuroMab, RRID: AB_2877361), human/mouse GLI-1 affinity purified polyclonal (1:100; AF3455, R&D systems, RRID: AB_2247710), RUNX2 (27-K) antibody (1:500; sc-101145, Santa Cruz Biotechnology, RRID: AB_1128251), anti-osteocalcin (OCN) antibody (1:75; AB10911, Millipore, RRID: AB_1587337), and monoclonal antiVinculin (1:70; V4505, Sigma-Aldrich, RRID: AB_477617).

Techniques: Activation Assay, Western Blot, Expressing, Standard Deviation, Cell Culture

Journal: Biomaterials advances

Article Title: Towards modular engineering of cell signalling: Topographically-textured microparticles induce osteogenesis via activation of canonical hedgehog signalling.

doi: 10.1016/j.bioadv.2023.213652

Figure Lengend Snippet: Fig. 8. Schematic depicting the proposed mechanism underpinning the osteoinductive influence of 3D dimpled surface topographical features of PLA microparticles on C3H10T1/2 cells. Dimpled topographies are sensed by C3H10T1/2 cells, which leads to changes in cytoskeleton organisation and the formation of primary cilia. This facilitates the activation of the Hedgehog signalling pathway and translocation of active Gli1 into the nucleus, where it upregulates the expression of Hh-and osteogenic-related genes, leading to the expression of early and late markers of osteogenesis. Image was created using Biorender.com. Abbreviations: Hh, Hedgehog signalling pathway; Gli1, glioma associated oncogene homolog 1; Runx2, Runt-related transcription factor2; Ptch1, Patched 1.

Article Snippet: The following primary antibodies were used: anti-ADP-ribosylation factor-like protein (ARL13B) mouse monoclonal antibody (1:2000; clone N295B/66, NeuroMab, RRID: AB_2877361), human/mouse GLI-1 affinity purified polyclonal (1:100; AF3455, R&D systems, RRID: AB_2247710), RUNX2 (27-K) antibody (1:500; sc-101145, Santa Cruz Biotechnology, RRID: AB_1128251), anti-osteocalcin (OCN) antibody (1:75; AB10911, Millipore, RRID: AB_1587337), and monoclonal antiVinculin (1:70; V4505, Sigma-Aldrich, RRID: AB_477617).

Techniques: Activation Assay, Translocation Assay, Expressing

Journal: Cell systems

Article Title: Before and After: A Comparison of Legacy and Harmonized TCGA Data at the Genomic Data Commons

doi: 10.1016/j.cels.2019.06.006

Figure Lengend Snippet: A) Summary of HM27/HM450 processing differences between legacy (hg19, GDCv1–3) and current (hg38, GDCv4–12) versions, and an upcoming version available for manual download in the GDC Community Tools repository (see supplement for details). B) Associating array features with genes in the hg19 and hg38 pipelines: hg19 used the RefSeq version 40 annotations from the Illumina HM450 manifest, and only associated probes within 1,500 bp upstream of a transcript start site (“TSS -1500”); hg38 used GENCODE 22 annotations, and includes distance from the nearest TSS, which can be used to associate probes both upstream or downstream from a TSS (“TSS +/−1500”). GENCODE 22 often includes additional alternative promoters for the same gene. C) Number of Strong Negative Correlations (SNCs) between DNA methylation beta value and RNA expression, using different associations: “Legacy -1500” used hg19 associations, “hg38 – 1500” used hg38 annotations but only upstream associations, and “hg38 +/−1500” used the same annotations but both upstream and downstream associations. The number of SNCs increased for all transcript types (only three shown here). D) Example of a new alternative promoter for PAX8 present in hg38 annotations but not hg19, which also coincided with an SNC identified in the hg38 but not hg19 version. E) Methylation vs. expression for this SNC (cg07772999-PAX8) across all TCGA-CHOL samples - about 50% of tumors are demethylated at this alternative promoter and overexpress PAX8. See also Figure S2, Tables S2–S5.

Article Snippet: The DNA methylation data in this study were derived from one of two array-based platforms: the Infinium Human Methylation 27k (HM27) or Infinium Human Methylation 450k (HM450).

Techniques: DNA Methylation Assay, RNA Expression, Methylation, Expressing