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. 2020 Aug 21;9:e54693. doi: 10.7554/eLife.54693

Figure 5. Profile of SASP factors released from ex vivo human IVDs cultures following senolytic treatment.

Culture media was analyzed by a RayBio Human Cytokine Array. Relative mean densitometry units of the 80 factors were normalized to pre-treatment media of the same IVD. The percentage change (post/pre) of the 15 most affected factors are shown for RG-7112 and o-Vanillin (A) and vehicle (B) treated discs. (C) Heatmap displaying quantification of 19 selected cytokines (19-plex Luminex array). Each column represents one individual and each row represent expression of a single protein. Data shown are log2 (fold change) in pre- or post-treated disc media relative to their respective expression level average. (D) Nine analytes (INF-γ, TNF-α, CCL11, CCL24, CXCL1, CXCL9, CXCL10, Angiogenin and VEGF) displayed statistically significant differences when measured in post compared to pretreated disc media. Error bars represent mean ± SEM. Statistical significance when comparing pre and post discs groups was assessed by two-tailed Student's t-test (A, B and D): * Indicates p<0.05 and **indicates p<0.01. Data was analyzed by repeated measures Analysis of Variance (ANOVA) with Turkey’s post hoc test for multiple pairwise comparison (D) where # Indicates significant difference (p<0.05) between treated and untreated groups, (n = 4). The analyzed media were collected from same donors used in Figure 4.

Figure 5—source data 1. Excel file containing the meausres of the significantly changed SASP factors measured using cytokine array and Luminex analysis in vehicle, RG-7112 and o-Vanilin disc media before and after the injection.

Figure 5.

Figure 5—figure supplement 1. Complete profiling and measure of growth factors and cytokines in disc cultures media.

Figure 5—figure supplement 1.

Total cytokine/chemokine array quantification of inflammatory factors release in (A) Control, (B) o-Vanillin and (C) RG-7112 discs. Shown in the graphs is the ratio (% change) of the factors detected in the cultures media of post-treated and compared with their respective pre-treated cultures media. (D) Ten analytes in disc media (IL-1α, IL-1β, IL-6, IL-8, CCL5, CCL7, CCL26, CXCL5, CXCL11 and CX3CL1) displayed no statistically significant decrease when compared with their respective controls. Data is presented as mean ± SEM and was analyzed by t-tests for pre and post groups and by repeated measures Analysis of Variance (ANOVA) with Turkey’s post hoc test for multiple pairwise comparison (n = 4).
Figure 5—figure supplement 1—source data 1. Excel file containing the complete list of SASP factors measured by cytokine arrays and the non-significantly regulated factors measured by Luminex assay in the pre and post media of injected discs.