Fig 2: Temperature stress affects adhesion and migration of MSCs. Inflammation hallmark, febrile temperature stress led to alteration in cell morphology and fundamental properties of MSCs, such as adhesion and migration. Corresponding to these changes, expression of adhesion and migration related gene expression also got affected. Inhibition of NF-?ß pathway resulted in reversal of altered physiological traits as well as gene expression of MMP-1 via p53/p21, acting through JNK pathway.

Fig 3: Reversal of cell spread area, de-adhesion dynamics and migration following NF-?ß pathway inhibition. WJ-MSCs were treated with 700 nM PDTC at 40 °C, cell area was determined, and plotted against corresponding control cultures. A sample size of 150 total cells from three different biological samples was used. A representative Gaussian distribution plot from 50 cells to depict cell area comparison is shown, ***represents p < 0.0001 (a). Similarly, cell area was determined for WJ-MSCs transfected with p65-targeted esiRNA or NC siRNA at 40 °C for 48 h and plotted against control WJ-MSC samples. A sample size of 200 total cells, from four different biological samples, was used, ***represents p < 0.0001. A representative Gaussian distribution plot from 50 cells to depict cell area comparison is shown. (b). De-adhesion dynamics of p65 esiRNA transfected WJ-MSCs. Differences in t1 and t2 between negative control siRNA and p65 esiRNA treated cells were significant, ***represents p < 0.0001 (c). Comparison of scratch wound- induced migration between control WJ-MSCs and WJ-MSCs transfected with NC siRNA or p65-targeted esiRNA at 40 °C was undertaken. Representative images are shown (n = 3) (d). Wound closure rate in µm/h was estimated and compared between the three conditions (n = 3), *represents p < 0.05, **represents p < 0.01 (e). There was a reduction in p65 protein level on transfecting WJ-MSCs with p65-targeted esiRNA as detected by Western blot. Reduced expression of p53 and p21 proteins was noted too in the p65 esiRNA targeted WJ-MSC samples. GAPDH was used as a loading control (f). Band intensities were quantified relative to GAPDH and plotted (n = 4), *represents p < 0.05, **represents p < 0.01, *** represents p < 0.001 (g). Full length blots are presented in Supplementary Figure S3.

Fig 4: Inhibition of ROS decreases cellular senescence of Ndufs6-/--BM-MSCs.A The mitochondrial ROS of Ndufs6-/--BM-MSCs and Mito-TEMPO-treated Ndufs6-/--BM-MSCs was measured by Mito-Sox staining. B The mitochondrial ROS of Ndufs6-/--BM-MSCs and Mito-TEMPO-treated Ndufs6-/--BM-MSCs was statistically quantified. C SA-ß-gal staining showing ß-gal-positive cells in Ndufs6-/--BM-MSCs and Mito-TEMPO-treated Ndufs6-/--BM-MSCs. D The ß-gal positivity in Ndufs6-/--BM-MSCs and Mito-TEMPO-treated Ndufs6-/--BM-MSCs was quantified by calculating the ratio of stained cells to total cells. E The proliferation of Ndufs6-/--BM-MSCs and Mito-TEMPO-treated Ndufs6-/--BM-MSCs was assessed by Ki-67 staining. F The Ki-67 positivity in Ndufs6-/--BM-MSCs and Mito-TEMPO-treated Ndufs6-/--BM-MSCs was quantified by calculating the ratio of stained cells to total cells. G The mRNA level of p53 and p21 was assessed in Ndufs6-/--BM-MSCs and Mito-TEMPO-treated Ndufs6-/--BM-MSCs. H The protein level of p53 and p21 was assessed in Ndufs6-/--BM-MSCs and Mito-TEMPO-treated Ndufs6-/--BM-MSCs. Results are presented as mean ± SD. n = 3. **p < 0.01.

Fig 5: DCM fraction treatment induces S-phase arrest in HT-29 cells. (A) HT-29 cells were incubated with various concentrations of the DCM fraction for 20 h. Flow cytometry was used to determine the percentages of cells in each phase of the cell cycle. The letters displayed above the concentrations represent significant differences (P<0.05) as determined by Bonferroni's multiple comparisons test; groups with different letters are significantly different to one another, whereas those with the same letter are not. (B) Protein expression levels of S-phase-related proteins cyclin A, CDK2, Cdc25A and p21 were measured using western blotting. All results were normalized to the untreated control (0 µg/ml). Data are presented as the mean ± SD of three independent experiments. *P<0.05 vs. 0 µg/ml DCM. DCM, dichloromethane; CDK2, cyclin-dependent kinase 2; Cdc25A, cell division cycle 25 homolog A; p21, cyclin dependent kinase inhibitor 1.