research cell line source s wt hela  (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: Inhibiting PDH, GLS1 and Hsp90 by the combination of CPI‐613+BPTES+17‐AAG gave rise to enhanced senolysis on senescent fibroblasts as well as the therapy‐induced senescent tumor cells. (A, B) The effects of CPI‐613+BPTES+17‐AAG combination treatment on proliferating (A) and senescent (B) BJ cells. For the dose of each compound in use, see the results 2.7 section for more details. ** p < 0.01 by Student's t ‐test. (C, D) The effects of CPI‐613+BPTES+17‐AAG combination treatment on proliferating and Dox‐induced senescent lung adenocarcinoma A549 cells. *** p < 0.001 by Student's t ‐test. (E, F) The effects of CPI‐613+BPTES+17‐AAG combination treatment on proliferating and Dox‐induced senescent cervical carcinoma HeLa cells. *** p < 0.001 by Student's t ‐test. (G) The morphological changes of senescent BJ induced by IR, senescent A549 and HeLa cells induced by Dox at the indicated time of CPI‐613+BPTES+17‐AAG treatment under the light microscopy. The senescent cells without treatment were stained with SA‐β‐gal. Cells were imaged at magnification 200×. (H) The schematic summarization of our findings. The activities of TCA cycle and chaperones are reduced in DNA damage induced senescent cells. Co‐inhibiting Hsp90 and TCA cycle with 17‐AAG+CPI‐613+BPTES combination leads to enhanced selective elimination of senescent cells, hinting TCA cycle and glutaminolysis are novel and potent targets for senolysis.

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: Light Microscopy, Staining

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: Distinct proteomic and transcriptional signatures of metabolic enzymes and chaperones between senescent fibroblasts and the therapy‐induced senescent tumor cells. (A) The abundance of glycolysis‐related enzymes PFKP, ALDOA, PKM2, TCA cycle‐related PDHA, and glutaminolysis‐related GLS1 was decreased significantly in senescent BJ and IMR‐90 cells compared to their proliferating counterparts. (B) The protein levels of chaperones TCP1, Hsp70, and Hsp90 were decreased significantly in senescent BJ and IMR‐90 cells. (C) The abundance of those glycolysis‐related enzymes remained unchanged or even elevated in Dox‐induced senescent A549, HeLa, and U2OS tumor cells compared to their proliferating counterparts. (D) The abundance of these chaperone proteins in Dox‐induced senescent A549, HeLa, and U2OS tumor cells remained nearly unchanged. The relative abundance of each protein was quantified by signal density scanning on Western blots and normalized to the signal of β‐Actin or β‐tubulin. * p < 0.05, ** p < 0.01 tested by Student's t ‐test.

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: Western Blot

Journal: bioRxiv

Article Title: SLC33A1 exports oxidized glutathione to maintain endoplasmic reticulum redox homeostasis

doi: 10.64898/2026.02.01.703113

Figure Lengend Snippet: a. Top, schematic of engineered GshF construct targeted to ER ( ER-GshF ), with signal peptide (SP) from ERP44 at N-terminus, V5 tag and ER retention signal KDEL at C-terminus. Bottom left, schematic of GSH synthesis in ER catalyzed by ER-GshF. Bottom right, immunoblot analysis of ER-GshF expression from HEK293T cells expressing inducible ER-GshF (iER-GshF HEK293T) treated with 1 µg/ml doxycycline for 24 hours. b. Immunofluorescence analysis of ER-GshF (V5, red) and calnexin (CANX, green) in iER-GshF HEK293T cells treated with 1 µg/ml doxycycline for 48 hours. Micrographs are representative of two independent experiments. c. Percent labeled glutathione from HEK293T cells expressing a vector control or inducible ER-GshF pre-treated with or without BSO and doxycycline. Cells pretreated with or without 1 mM BSO and 1 µg/ml doxycycline for 24 hours, were switched to cystine free media with 200 µM isotope labeled cystine (³CL, ¹LNL) for 8 hours before harvesting the cells, BSO and doxycycline were kept the same as pretreatment during labeling. d. Schematic of the ER-focused CRISPR genetic screens in iER-GshF HEK293T cells cultured in the presence or absence of 1 µg/ml doxycycline for 14 doublings. e. Left, CRISPR gene scores in iER-GshF HEK293T cells cultured in the presence or absence of 1 µg/ml doxycycline for 14 doublings. Top scoring hits color-coded. Pearson correlation coefficient, two-sided. Right, differential gene score from iER-GshF HEK293T cells cultured with doxycycline compared to untreated. Top genes sensitizing iER-GshF HEK293T cells under doxycycline treatment are shown. f. Left, percent cell number from SLC33A1 knockout HEK293T cells expressing inducible ER-GshF complemented with a vector control or SLC33A1 cDNA under different concentrations of doxycycline for 4 days. Numbers under doxycycline treated are normalized to untreated. Right, representative images of the indicated cells. g. Left, volcano plot showing relative fold change (log 2 ) in ER metabolite abundance versus −log (P values) from SLC33A1 knockout ER-tag HEK293T cells expressing a vector control or SLC33A1 cDNA. Statistical significance was determined by multiple two-tailed unpaired t-tests. The dotted line represents P =0.01. Right, volcano plot showing relative fold change (log 2 ) in whole-cell metabolite abundance versus −log (P values) from SLC33A1 knockout ER-tag HEK293T cells expressing a vector control or SLC33A1 cDNA. Statistical significance was determined by multiple two-tailed unpaired t-tests. The dotted line represents P =0.01. h. Left, volcano plot showing relative fold change (log 2 ) in ER metabolite abundance versus −log (P values) from Slc33a1 knockout ER-tag KPK cells expressing a vector control or SLC33A1 cDNA. Statistical significance was determined by multiple two-tailed unpaired t-tests. The dotted line represents P =0.01. Right, volcano plot showing relative fold change (log 2 ) in whole-cell metabolite abundance versus −log (P values) from Slc33a1 knockout ER-tag KPK cells expressing a vector control or SLC33A1 cDNA. Statistical significance was determined by multiple two-tailed unpaired t-tests. The dotted line represents P =0.01. i. Relative metabolite abundance of indicated whole cell or ER metabolites from SLC33A1 knockout ER-tag HEK293T, HeLa and KPK cells expressing a vector control compared to those expressing SLC33A1 cDNA. ER UDP-GlcNAc abundance is shown to indicate ER amount.

Article Snippet: Human cells lines HeLa, HEK293T cells were purchased from the ATCC.

Techniques: Construct, Western Blot, Expressing, Immunofluorescence, Labeling, Plasmid Preparation, Control, CRISPR, Cell Culture, Knock-Out, Two Tailed Test