(A) Representative images indicating how we deemed positive and negative p16INK4a (a), Ki-67 (d), and caspase-3 (g) staining. (b, e and h) Magnified images of (a, d and g). Arrow heads indicate …
Excel file containing the percentage of p16Ink4a, ki-67 and caspase three positive in NP and AF cells control and treated with RG-7112.
(A) Metabolic activity in control and treated NP and AF pellet cultures from degenerate IVDs (n = 6). (B) Representative photomicrographs of untreated (a–c) and RG-7112 treated (d–f) NP cells …
Excel file containing the fluorescence values for metabolic and caspase3/7 activity, the number of apoptotic cells, and the concentrations of sGAG in NP (and AF) cells treated and untreated with RG-7112.
(A) Venn diagrams of the differentially up and downregulated genes among the different groups. O-Vanillin, RG-7112-treated NP cells in pellets culture. For Upregulated genes odds ratio (OR) = 2.13 …
Excel file containing the list of Differentially Expressed genes significantly up- or downregulated at p < 0.05 in RG-7112 and o-Vanillin conditions and the raw values of the fold change in control and treated groups for all tested genes.
(A) 47 genes did not show significant expression change in RG-7112 (5 µM) and o-Vanillin (100 µM) treated NP cells when compared with control (CTRL). See methods for statistical calculation and …
Excel file containing the the list of Non-Differentially Expressed genes in RG-7112 and o-Vanillin conditions and the raw values of the fold change of the 91 genes tested in control and treated groups.
(A) Culture media was analyzed by a RayBio Human Cytokine Array. Relative mean densitometry units of the 80 factors were normalized to untreated controls with the most 25 downregulated SASP factors …
Excel file containing the meausres for the selected and significantly SASPs factors measured using cytokine array and Luminex analysis in NP pellets media following the treatment with RG-7112 and o-Vanilin.
(A) Schematic representation of the media analysis by cytokine arrays and Luminex assay. Cytokine/chemokine array quantification of the media in NP cell pellets treated with (B) o-Vanillin (100 μM) …
Excel file containing the complete list of SASP factors measured by cytokines arrays and the non-significantly regulated factors measured by Luminex assay in treated NP pellets media.
(A) Schematic of the ex vivo organ culture experiment. Lumbar spines from organ donors were assessed radiographically for signs of degeneration. Three discs per experiment were isolated from the …
Excel file containing the complete list of donors, slices and their respective voxel intensities, p16Ink4a and ki-67-positive cells in control and treated discs.
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 …
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.
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 …
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.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Rabbit polyclonal anti-caspase-3 | Sigma-Aldrich | C8487 RRID:AB_476884 | IF (1:500) IHC-Fr (1:500) |
Antibody | Rabbit polyclonal anti-Ki-67 | Novus Biologicals | NB500-170 RRID:AB_10001977 | IF (1:300) IHC-Fr (1:500) IHC-P (1:2000) |
Antibody | Mouse monoclonal Anti- p16Ink4a | Abcam | ab54210 RRID:AB_2059963 | IHC-P (1:500) |
Antibody | Polyclonal Alexa Fluor 488 (Donkey anti-Rabbit) | Invitrogen Thermo Fisher | A-21202 RRID:AB_141607 | IF (1:1000) |
Antibody | Polyclonal Alexa Fluor 555 (Donkey anti-Mouse) | Invitrogen Thermo Fisher | A-31570 RRID:AB_2536180 | IF (1:1000) |
Antibody Array | Human Cytokine Antibody Array C5 | RayBiotech, Inc | AAH-CYT-5–2 RRID:AB_10185250 | |
Chemical compound, drug | o-Vanillin | Sigma-Aldrich | 120804 | |
Chemical compound, drug | RG-7112 | Selleck Chemicals | S7030 | |
Chemical compound, drug | DMSO | Sigma-Aldrich | D8418 | |
Commercial Kit | Apoptosis detection | Abcam | ab176749 RRID:SCR_018379 | |
Commercial Kit | Caspase 3/7 activity | AAT Bioquest 13503 | Amplite Fluorimetric Catalog # 13503 | |
Commercial Kit | qScript cDNA Synthesis Kit | Quanta Biosciences | 95047–500 | |
Commercial Kit | Goat anti-Mouse and Rabbit specific HRP/DAB Detection IHC Kit | Abcam | ab64264 | |
Commercial Kit | Mouse monoclonal Anti- p16Ink4a | Roche, Ventana Laboratories | CINtec Plus (9531) RRID:SCR_018380 | Prediluted |
Commercial Kit | Mycoplasma PCR Detection Kit | ZmTech Scientific | M209001 | |
Commercial Assay | Custom Taqman (R) Gene Expression 96-Well Fast Plates | Thermo Fisher | 4413255 | See Supplementary file 1 for each gene product ID |
Commercial Assay | Luminex immunoassay 19-PLEX plate - Human Cytokine PROCARTAPLEX | Life Technologies | MXMFXHX | |
Software | ImageJ | ImageJ http://imagej.nih.gov/ij/ | RRID:SCR_003070 | |
Software | GraphPad Prism 8 | GraphPad Prism https://www.graphpad.com | RRID:SCR_015807 | |
Software | Ingenuity Pathway Analysis (IPA) | Ingenuity Systems | RRID:SCR_008653 | |
Software | R package ggplot2 | Wickham, 2016 | ISBN 978-3-319-24277-4 | |
Software | MIPAV | NIH Center for Information Technology | RRID:SCR_007371 | |
Software | Image Quant TL array analysis | GE Healthcare | RRID:SCR_018374 | |
Software | FlowCytomix Pro2.2.1 | eBioscience | RRID:SCR_018375 | |
Software | AxioVision LE64 | Zeiss | RRID:SCR_002677 | |
Other | DMMB | Sigma-Aldrich | 341088 | |
Other | Alamar Blue reagent | Thermo Fisher | DAL1025 | |
Other | DAPI stain | Invitrogen | D1306 | (1 µg/mL) |
Other | Safranin-O | Sigma-Aldrich | S2255 | |
Other | TaqMan Fast Universal PCR Master Mix (2×) | Thermo Fisher | 4366073 | |
Cell line | Primary Human Nucleus Pulposus (NP) and Annulus Fibrosis (AF) | Primary Cells | Human Biological samples. See Table 1 for demographics. | Cell line maintained in Haglund Lab. |
Donor ID | Age | Sex | Cause of death |
---|---|---|---|
1 | 53 | F | Anoxia |
2 | 53 | M | bone; cerebral aneurysm rupture |
3 | 76 | F | Anoxia |
4 | 52 | M | Ischemic cerebral vascular accident |
5 | 17 | M | Brain death |
6 | 73 | F | Cerebrovascular hemorrhage |
7 | 68 | F | Cerebral hemorrhage |
8 | 39 | M | Gunshot to the neck |
9 | 66 | F | Unknown |
10 | 67 | M | Cardiac arrest |
11 | 69 | M | Cerebral hemorrhage |
12 | 61 | M | Cardiac arrest |
13 | 40 | F | Brain hemorrhage |
14 | 53 | F | Medical suicide |
15 | 28 | M | Suicide (Hang) |
16 | 35 | M | Anoxia |
Donor ID | Discs | Thompson grade and Degeneration | Cell type | Type of culture | Type of assays | Results |
---|---|---|---|---|---|---|
6 | L1-L5 | 3 - (D) | NP & AF | Pellet | IHC Metabolic activity | Figure 1 (A–D) Donor # 6, 7, 10–15 Figure 1—figure supplement 1(A) Donor # 10–15 Figure 1—figure supplement 1(G) Donor # 10–15 |
7 | L2-L5 | 4 - (D) | ||||
8 | L3-S1 | 3 - (D) | ||||
10 | L4-L5 | 4 - (D) | ||||
11 | L5-S1 | 3 - (D) | ||||
12 | L5-S1 | 3 - (D) | ||||
13 | L4-S1 | 3 - (D) | ||||
14 | L5-S1 L1-L2 | 3 - (D) | ||||
15 | L5-S1 | 5 - (D) | ||||
11 | L5-S1 | 3 - (D) | NP | Pellet | RT-qPCR Cytokine array Luminex | Figure 2 (A–E) Figure 3 (A–E) Figure 2—figure supplement 1(A) Figure 3—figure supplement 1(A-C) All Donor # 11–15 |
12 | L5-S1 | 3 - (D) | ||||
13 | L4-S1 | 3 - (D) | ||||
14 | L5-S1 L1-L2 | 3 - (D) | ||||
15 | L5-S1 | 5 - (D) | ||||
9 | T11-L2 | Table 3 | Intact disc | Ex vivo | MRI Luminex IHC | Figure 4 (A–F) Figure 5 (A–D) Figure 5—figure supplement 1(A-D) Donor # 9, 11, 13 and 14 |
11 | L1-L4 | Table 3 | ||||
13 | L1-L4 | Table 3 | ||||
14 | L2-L5 | Table 3 | ||||
1 | L1-S1 | 4 - (D) | NP | Monolayer | ICC Metabolic activity Caspase 3/7 activity | Figure 1—figure supplement 1(B, C and D) Donor # 1–3 Figure 1—figure supplement 1(E and F) Donor # 1–3 and 11 (D) Donor # 4, 5, 10 and 11 (ND) |
2 | L4-S1 | 3 - (D) | ||||
3 | L2-S1 | 4 - (D) | ||||
4 | L1-L5 | 1 - (ND) | ||||
5 | T11-S1 | 1 - (ND) | ||||
10 | T12-L1 | 1 - (ND) | ||||
11 | T12-L1 L5-S1 | 1 - (ND) 3 - (D) | ||||
15 | T11-L5 L5-S1 | 1 - (ND) 5 - (D) | Intact disc | NP Area and position measure | Figure 2—figure supplement 1(B-C) Donor # 15 and 16 | |
16 | L1-S1 | 1 - (ND) |
Donor ID | 9 | 11 | 13 | 14 | |
---|---|---|---|---|---|
Age | 66 | 69 | 40 | 53 | |
Sex | F | M | F | F | |
CTRL | Disc Level | T12/L1 | L1/2 | L1/2 | L2/3 |
Grade | 2 | 2 | 2 | 2 | |
Disc Height (cm) | 0.84 | 0.92 | 0.92 | 0.88 | |
O-Vanillin | Disc Level | L1/2 | L2/3 | L3/4 | L4/5 |
Grade | 2 | 2 | 2 | 2 | |
Disc Height (cm) | 0.92 | 0.89 | 0.99 | 0.88 | |
RG-7112 | Disc Level | T11/T12 | L3/4 | L2/3 | L3/4 |
Grade | 2 | 2 | 2 | 2 | |
Disc Height (cm) | 0.82 | 0.9 | 0.93 | 0.99 |
Gene names (aliases) and primers.
List of cytokines and synonyms.
List of differentially and non-differentially expressed genes following treatment with o-Vanillin and RG-7112.
(a) Eight Differentially Expressed genes significantly up- or downregulated at p < 0.05 in RG-7112 condition. (b) Forty Differentially Expressed genes significantly up- or downregulated at p < 0.05 in o-Vanillin condition. (c) Non-Differentially Expressed genes in RG-7112. (d) Non-Differentially Expressed genes in o-Vanillin.
O-Vanillin and RG-7112 IPA network scores, molecules and top diseases.
(a) O-Vanillin network scores, molecules and top diseases. Physical function analysis using IPA of o-Vanillin treated NP pellets generated three networks which are ordered by a score denoting significance. The highest-scoring network, which comprises 37 proteins in our list, revealed significant changes in cell death and survival, neurological disease, organismal Injury and abnormalities. In addition, the other networks revealed changes in cancer, cellular movement, connective tissue development and function, cell cycle, organismal injury and abnormalities. (b) RG-7112 network scores, molecules and top diseases. Physical function analysis using IPA of RG-7112 treated NP pellets generated a single network which is ordered by a score denoting significance of 21. This network reveals significant changes in cell cycle, cell death and survival, connective tissue development and function.