Fig 1: Spatial localization of perivascular populations in thyroid cancer.(A–E) Spatial analysis of representative PTC Thy15 showing (A) pathologist annotation of PTC and stromal spatial barcodes, (B) spatial feature plots of PTC and myCAF/pericyte RCTD scores, (C) violin plots of myCAF and pericyte RCTD scores stratified by pathologist annotation, (D) spatial feature plots and violin plots depicting myCAF (POSTN) and pericyte (RGS5) marker gene expression stratified by pathologist annotation, and (E) a heatmap of MERINGUE spatial cross-correlation of myCAF, PTC, and pericyte RCTD scores (ordered by hierarchical clustering). (F) Box plot showing pericyte spatial cross-correlation with myCAF and PTC across 12 PTC samples containing PTC and stromal regions. P value calculated with paired t test. (G) Pathologist annotation of representative PTC regions into predominantly tumor cell or a mix of tumor and fibrovascular cells. Left/middle: zoomed-in hematoxylin and eosin staining (left) with labeled spatial barcodes (middle). Right: zoomed-out hematoxylin and eosin staining with a sample of the spatial barcodes labeled. (H) Violin plots showing PTC and pericyte RCTD scores (left) and pericyte marker RGS5 expression (middle) stratified by pathologist annotation from G. Right: box plots depicting average pericyte RCTD deconvolution scores in PTC or PTC/fibrovascular component mix regions across five samples (Thy15-17, Peds07-08). P value calculated with paired t test. (I) Representative RGS5 IHC of PTC. (J) Pathologist spatial annotation of large perivascular areas (PVAs) (left) and spatial feature plot of vSMC RCTD scores (middle). Right: violin plot of vSMC RCTD score stratified by pathologist large PVA annotation. (K and L) Spatial feature plots (left) and associated violin plots (right) for vSMC marker genes (K) ACTA2 and (L) TAGLN. (M) Box plots showing average vSMC RCTD score (left) or vSMC marker gene expression (right) for 5 tumors stratified by pathologist annotation (Supplemental Figure 10F). P values calculated with paired t test. (N) IHC of αSMA in a representative PTC. Black arrow highlights a large PVA.
Fig 2: Demethoxycurcumin inhibits breast cancer angiogenesis through RGS5. (A) Schematic diagram illustrating the timeline for AAV virus injection in mice and drug administration for modeling. (B, C) Western blot analysis demonstrating the expression of RGS5 in tumor. (D, E, G)Images of breast cancer tumors in different mouse groups (n=3). (F, H) Immunofluorescence images of CD31 in tumor tissues of mice injected with AAV virus(n=6).(I, J) After transfection of HUVECs with the OE-RGS5 plasmid, flow cytometry was again used to detect apoptosis, showing significant results (n=3) .⁎⁎p < 0.01.
Fig 3: Demethoxycurcumin has the capacity to inhibit angiogenesis in tumors. (A, C) Immunofluorescence analysis shows the co-localization of CD31 and RGS5. (B, D) After a 48-hour period, chicken embryo chorioallantoic membranes (CAM) were collected to evaluate the level of angiogenesis (n = 3, compared to the control group). (E-H) Protein immunoblotting was employed to quantitatively analyze the expression levels of VEGF and RGS5 in tumor tissues (n = 3, compared to the control group). Results are expressed as the arithmetic mean ± SD from a minimum of three independent experiments. Compared to the control group, *p<0.05, ⁎⁎p<0.01.
Fig 4: Schematic diagram of demethoxylcurcumin molecule docking and CETSA validation. (A, B) Small molecule binding to protein in active Pocket 119, magnified schematic diagram of small molecule binding to protein in active pocket 119. (C) Schematic diagram of ligand-receptor non-bonding binding of small molecules to proteins. (D) According to OCASA's prediction, the more likely active pockets in human RGS5 are 119, 127, 210, 20 and 91. (E, F) Expression of RGS5 at different temperatures during the intervention process of demethoxylcurcumin (n=3). Compared to the Con-RGS5 group, *p<0.05, ⁎⁎p<0.01.
Fig 5: RGS5 is a key target of demethoxylcurcumin inhibiting angiogenesis in breast cancer.
Supplier Page from Abcam for Anti-RGS5 antibody [EPR28539-64]