Fig 1.
iAF cells are resistant to TNFα induced senescence.
(A) Representative low and high magnification phase contrast images and quantification of senescence associated β-galactosidase staining of iAF cells cultured in the absence (control) or presence of TNFα. (B) Growth curve of iAF cells grown in the presence or absence of TNFα or serum starvation (control) at 24, 48, and 72 hours. (C) Representative images of Ki67 immunocytochemistry at the 24 hour time-point. (D) Quantification of percentage of Ki67 positive cells at 24, 48, and 72 hours post treatment. Media was not changed throughout the 72 hour time course. (E) Representative images of p16 and p21 immunostaining of iAF cells treated with TNFα. (F) Quantification of p16 and p21 immunostaining of iAF cells grown in the presence and absence (control) of TNFα, represented as percentage of total cells that were stained. Scale bars = 100μm. p <0.05 = *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****, N = 3 for immunostaining. Positive staining control images are shown in S4 Fig.
Fig 2.
iAF cells have a differential ROS response to TNFα compared with NP cells.
(A) Representative images of CellROX green staining in NP cells exposed to TNFα (40 ng/mL). (B) Quantification of CellROX green average fluorescence intensity per cell in NP cells exposed to TNFα. (C) AmplexRed assay quantification of H2O2 released from NP cells exposed to TNFα. (D) Representative images of CellROX green staining in iAF cells exposed to TNFα (40 ng/mL). (E) Quantification of CellROX green average fluorescence intensity per cell in iAF cells exposed to TNFα. (F) AmplexRed quantification of H2O2 released from iAF cells exposed to TNFα. (G) Gene expression analysis relative to 18s rRNA of ROS related genes in NP cells exposed to TNFα. 18S rRNA Ct value (mean±SD): Control = 9.56± 0.5, TNFα = 9.55±0.36. (H) Gene expression analysis relative to 18s rRNA of ROS related genes in iAF cells exposed to TNFα. 18S rRNA Ct value (mean±SD): Control = 8.93±0.80, TNFα = 9.86±0.36. (I) AmplexRed quantification of H2O2 released from NP cells exposed to TNFα pre-treated with the NOX-inhibitor diphenyleneiodonium chloride (DPI). (J) AmplexRed quantification of H2O2 released from iAF cells exposed to TNFα pre-treated with the NOX-inhibitor DPI. Scale bars = 100μm. p<0.05 = *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****, N = 3 for all experiments.
Fig 3.
(A) Gene expression was determined by real time reverse-transcription-PCR (RT-PCR). The results were normalized to 18S rRNA (ΔCt) and expressed as mean ± SD (N = 3). (B and C) Expression of TNF receptor 1 (TNFR1) by nucleus pulposus (NP) and inner annulus (iAF) cells were evaluated by Western blot analysis (B) and quantified by densitometry (C). The expression was normalized by the loading control, GAPDH. (N = 3; ** p<0.01 relative to NP cells).
Fig 4.
iAF cells undergo H2O2-induced senescence.
(A) Representative phase contrast images and quantification of senescence associated β-galactosidase staining of iAF cells treated with H2O2 (50 μM). (B) Representative images and quantification of p16 immunostaining of iAF cells treated with H2O2. (C) Representative images and quantification of p21 immunostaining of iAF cells treated with H2O2. (D) Gene expression analysis of iAF cells treated with H2O2. 18S rRNA Ct value (mean±SD): Control = 7.76± 0.16, H2O2 = 7.75± 0.18. (E) Representative images and quantification of intracellular ROS with CellROX green. (F) AmplexRed quantification of H2O2 released from iAF cells exposed to H2O2. (G) Representative images and quantification of JC-1 staining in iAF cells exposed to TNFα or H2O2. Scale bars = 100μm. p <0.05 = *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****, N = 3 for all experiments.
Fig 5.
TNFα treated but not H2O2-induced senescent iAF cells show signs of degeneration at 24hrs.
(A) Gene expression analysis of iAF cells treated with TNFα (40 ng/mL). 18S rRNA Ct value (mean±SD): Control = = 9.37± 0.28, TNFα = 9.72±0.21. (B) Representative images of COL1, COL2, and ACAN immunocytochemistry of iAF cells treated with TNFα. (C) Quantification of immunocytochemistry in B, presented as percentage of total cells stained. (D) Gene expression of iAF cells treated with H2O2. 18S rRNA Ct value (mean±SD): Control = 7.62± 0.16), H2O2 = 7.47± 0.28. (E) Representative images of COL1, COL2, and ACAN immunocytochemistry of iAF cells treated with H2O2. (F) Quantification of immunocytochemistry in E, presented as percentage of total cells stained. Scale bars = 100μm. p <0.05 = *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****, N = 3 for all experiments. Positive staining controls are shown in S4 Fig.
Fig 6.
iAF cells in 3D tissue sheets undergo senescence when exposed to H2O2 but not TNFα.
(A) Representative images of untreated iAF tissue sheets following immunohistochemical staining for COL1, COL2, or ACAN, or H&E staining. (B) Representative images of immunohistochemistry and quantification of MMP13 and p16 of iAF tissue sheets exposed to TNFα (40 ng/mL) or H2O2 (50 μM) for 24 hours. (C) Average cross-sectional thickness of iAF tissue sheets exposed to TNFα or H2O2. Scale bars = 100μm. p<0.05 = *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****, N = 3.
Fig 7.
iAF cells have an altered phenotype when co-cultured with senescent NP cells.
(A) Gene expression of iAF cells exposed to non-contact co-culture with TNFα or untreated NP cells for 24 hours. 18S rRNA Ct value (mean±SD): Control = 8.75±0.28, TNFα = 8.93±0.27. (B) Representative images and quantification of COL1, COL2, and ACAN immunostained iAF cells exposed to non-contact co-culture with NP cells. (C) Representative phase contrast images of senescence associated β-galactosidase staining and quantification of iAF cells exposed to non-contact co-culture with NP cells. (D) Representative images and quantification of p16 and p21 immunocytochemistry of iAF cells exposed to non-contact co-culture with NP cells. (E) Representative images and quantification of iAF cells in a contact co-culture with TNFα and untreated NP cells. Scale bars = 100μm. p<0.05 = *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****, N = 6 for collagen and aggrecan immunostaining, N = 3 for all other immunostaining, N = 4 for gene expression. TNFα-T-NP = TNFα treated NP cells.