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. Author manuscript; available in PMC: 2023 Jan 3.
Published in final edited form as: Cell Rep. 2022 Nov 29;41(9):111744. doi: 10.1016/j.celrep.2022.111744

Figure 5. JNK regulates GLS and suppresses mitochondrial function in senescent cells.

Figure 5.

Senescent MSCs were treated with SP600125 (5 μM), and (A) GLS1 expression was measured via quantitative RT-PCR normalized to S and internally normalized to RPL32 cycle number; data shown as mean ± SD.

(B) Immunostaining for GLS1; scale bar represents 100 μm.

(C) Quantification of GLS1 intensity per cell, normalized to S; data shown as mean ± 95% CI for >200 cells.

(D) Glutaminase activity measurement in Y, S, and S cells treated with SP600125, normalized to S; data shown as mean ± SD.

(E) Intracellular urea concentration, normalized to S; data shown as mean ± SD.

(F) Western blot for COX IV and quantification, normalized to S (n = 3 independent experiments). GAPDH served as loading control.

(G) Live stain images of Mitotracker Red showing upregulation of mitochondrial membrane potential in senescent cells after knocking down JNK1 or JNK2; scale bar represents 100 μm.

(H) Quantification of Mitotracker Red intensity per cell; data shown as mean ± 95% CI for >100 cells.

(I) Measurement of oxygen consumption (OCR) using Seahorse extracellular flux analyzer in Y, S, shJNK1, and shJNK2 cells.

(J–M) Basal respiration (J), ATP-linked respiration (K), maximal respiration (L), and spare respiratory capacity (M) rates calculated from the OCR data in (I).