Fig 1.
a) MTT assay of NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 48 h. b) MTT assay of NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 72 h. “ns” indicates p> 0.05 was considered nonsignificant. Other groups had significant differences in mitochondrial metabolic activity.
Fig 2.
a) LDH release from NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 48 h. b) LDH release from NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 72 h. c) Cytoplasmic LDH content of NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 72 h. * indicates p<0.05, ** indicates p<0.01 and *** indicates p< 0.001.
Fig 3.
a) Intracellular ROS staining of NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 24 h. b) The percentage of intracellular ROS production from NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 6 and 72 h. c) The kinetics of intracellular ROS production in NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 72 h. Data were normalized against cell number. * indicates p<0.05, ** indicates p<0.01 and *** indicates p< 0.001.
Fig 4.
a) Annexin/PI graphs of NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 48 h. Early apoptosis is shown in the bottom right quadrant (Q3) for each panel, while late apoptosis (Annexin+/PI+) is shown in the upper right quadrant (Q2). Viable cells are represented in the lower left quadrant (Q4). Necrosis (Annexin −/PI+) is shown in the upper left quadrant (Q1). b) Annexin/PI staining data from NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 48 h. Viability was decreased and necrosis was increased in A, C, and C+A media compared to N media. Apoptosis-necrosis and necrosis were significantly observed in C+A cells compared to other cells. c) Annexin/PI staining data from NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 72 h. * indicates p value <0.05, ** indicates p value <0.01 and *** indicates p value < 0.001.
Fig 5.
a) Acridine orange staining of NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 72 h. b) Autophagosome percentage in NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 48 h. c) Autophagosome percentage in NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 72 h. d) Real-time PCR data related to p62 and LC3 gene analysis of NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 72 h. e) Western blotting of LC3-II/I protein of NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 7 days. * indicates p<0.05, ** indicates p<0.01 and *** indicates p< 0.001.
Fig 6.
a) Western blotting of Bcl2 protein. b) Bax. c) Bax/bcl2 ratio. d) Figures of beta-actin, bax and Bcl2 in NIH/3T3 cells treated with normal media (N), acidic media (A), conditioned media (C) and acidic plus conditioned media (C + A) after 7 days. * Mean p<0.05.
Fig 7.
DNA gas chromatography-mass spectrophotometry (GC–MS) to detect DNA methylation in CpG islands of a) NIH/3T3 cells treated with normal media (N), b) acidic media (A) and C) acidic plus conditioned media (C + A) at 7 days post-treatment.