wi 38  (ATCC)

Journal: Aging Cell

Article Title: Decreased Glucose Metabolism and Declined Chaperones Are Unique Features Required for the Survival of Senescent Fibroblasts and Pyruvate Dehydrogenase Is a Potent Senolytic Target

doi: 10.1111/acel.70434

Figure Lengend Snippet: Glucose consumption, ATP and alpha‐KG production, and NAD+/NADH ratio are reduced in senescent fibroblasts, inhibiting TCA cycle and glutaminolysis lead to the selective killing of senescent fibroblasts. (A) The glucose consumption rate is much lower in senescent fibroblasts compared to their proliferating counterparts. The media were collected from 1 × 10 6 cells cultured for 48 h, glucose concentration was measured by the enzyme mediated colorimetric glucose assay kit and normalized by the fresh medium and cell numbers at 48 h of culture. The consumption rate difference between growing and senescent cells was compared by Student's t ‐test, *** depicts p < 0.001. (B) ATP production was reduced in senescent BJ, WI‐38, and IMR‐90 cells compared to their proliferating counterparts. ATP concentration was measured by ATP luminometric assays, presented as means ± SD from three independent experiments. * p < 0.05 by Student's t ‐test. (C) Alpha‐KG level was reduced in senescent BJ, WI‐38, and IMR‐90 cells compared to their proliferating counterparts. Alpha‐KG was measured by α‐Ketoglutarate Assay Kit, presented as means ± SD from three independent experiments. *** p < 0.001 by Student's t ‐test. (D) The cytosolic NAD+/NADH ratios in senescent BJ, WI‐38, and IMR‐90 cells are decreased compared to their proliferating counterparts. * p < 0.05 or ** p < 0.01 by Student's t ‐test. (E) CPI‐613+BPTES combination treatment enhanced the decrease of cytosolic NAD+/NADH ratios in senescent BJ, WI‐38, and IMR‐90 cells compared to their untreated counterparts. The measurement was done at 48 h post treatment. * p < 0.05 by Student's t ‐test. (F) CPI‐613+BPTES combination treatment enhanced the decrease of alpha‐KG levels in senescent BJ, WI‐38, and IMR‐90 cells compared to their untreated counterparts. * p < 0.05 or ** p < 0.01 by Student's t ‐test. The measurement was performed at 48 h after treatment. (G) CPI‐613+BPTES combination treatment enhanced the decrease of ATP production in senescent BJ, WI‐38, and IMR‐90 cells compared to their untreated counterparts. * p < 0.05 or ** p < 0.01 by Student's t ‐test. The measurement was performed at 48 h after treatment. (H) Inhibition of glycolysis by 2‐DG treatment led to decreased cell survival in both proliferating and senescent BJ cells in a dose dependent manner. Cell viability at 72 h of treatment was determined by the CCK8 assay and normalized by the untreated cells. (I) The dose–response curves of CPI‐613 on proliferating and senescent BJ cells. * p < 0.05 by one‐way ANOVA. (J) The dose–response curves of BPTES on proliferating and senescent BJ cells. (K, L) Co‐inhibition of the PDH complex and glutaminolysis by CPI‐613+BPTES enhanced the selective killing of senescent BJ cells. ** p < 0.01 at 72 h of treatment tested by Student's t ‐test. (M) The morphological changes of growing and senescent BJ cells at the indicated time of treatment under the light microscopy. Senescent BJ cells before CPI‐613+ BPTES treatment were stained with SA‐β‐gal. Cells were imaged at magnification 200×.

Article Snippet: Human fibroblasts BJ (RRID: CVCL_3653; ATCC Cat#CRL‐2522), IMR‐90 (RRID: CVCL_0347; ATCC Cat#CCL‐186), WI‐38 (RRID: CVCL_0579; ATCC Cat#CCL‐75) and human cancer cell lines HeLa (RRID: CVCL_0030; ATCC Cat#CRM‐CCL‐2), A549 (RRID: CVCL_0023; ATCC Cat#CRM‐CCL‐185), and U2OS (RRID: CVCL_0042; ATCC Cat#HTB‐96) were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin at 37°C under 5% CO 2 .

Techniques: Cell Culture, Concentration Assay, Glucose Assay, Inhibition, CCK-8 Assay, Light Microscopy, Staining