human colon cancer cell lines  (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: eLife

Article Title: Uev1A counteracts oncogenic Ras stimuli in both polyploid and diploid cells

doi: 10.7554/eLife.107104

Figure Lengend Snippet: ( A ) Kaplan-Meier analysis of relapse-free survival in KRAS -mutant colorectal cancer patients with high or low expression levels of UBE2V1 and UBE2V2. ( B and E ) The knockdown efficiency assays. The relative mRNA levels were normalized to GAPDH . ( C–G ) Assays to evaluate the effects of UBE2V1- and UBE2V2-RNAi on colony formation and cell viability in SW480 and HCT116 cells. In ( B, C, E, and F ), three independent replicates were conducted, and statistical significance was determined using t test. In ( D and G ), five ( D ) and six ( G ) independent replicates were conducted at each time point, and statistical significance was determined using two-way ANOVA with multiple comparisons. * (p<0.05), *** (p<0.001), and **** (p<0.0001).

Article Snippet: The human colon cancer cell lines SW480 and HCT116, as well as the human embryonic kidney cell line 293T, were purchased from the American Type Culture Collection (ATCC), authenticated by STR profiling, and tested negative for mycoplasma contamination.

Techniques: Mutagenesis, Expressing, Knockdown

Journal: eLife

Article Title: Uev1A counteracts oncogenic Ras stimuli in both polyploid and diploid cells

doi: 10.7554/eLife.107104

Figure Lengend Snippet: ( A and C ) The knockdown efficiency assays. The relative mRNA levels were normalized to GAPDH . Three independent replicates were conducted, and statistical significance was determined using one-way ANOVA. ( B and D ) Assays to evaluate the effects of UBE2V1- and UBE2V2-RNAi on colony formation and cell viability in SW480 cells. Six independent replicates were conducted at each time point, and statistical significance was determined using two-way ANOVA with multiple comparisons. n.s . (p>0.05), * (p<0.05), ** (p<0.01), *** (p<0.001), and **** (p<0.0001).

Article Snippet: The human colon cancer cell lines SW480 and HCT116, as well as the human embryonic kidney cell line 293T, were purchased from the American Type Culture Collection (ATCC), authenticated by STR profiling, and tested negative for mycoplasma contamination.

Techniques: Knockdown

Journal: eLife

Article Title: Uev1A counteracts oncogenic Ras stimuli in both polyploid and diploid cells

doi: 10.7554/eLife.107104

Figure Lengend Snippet: ( A ) Western blotting to confirm the transient overexpression of UBE2V1 and UBE2V2 in SW480 and HCT116 cell lines. β-Actin was used as the loading control. ( B ) Western blotting to confirm the stable overexpression of UBE2V1 and UBE2V2 in SW480 cells, with UBE2V1-OE #1 and UBE2V2 #3 cell lines utilized in subcutaneous tumorigenesis assays. α-Tubulin was used as the loading control. In both ( A ) and ( B ), cells transfected with an empty overexpression vector served as the control. Figure 9—figure supplement 1—source data 1. PDF files that contain original western blots indicating the relevant bands and treatments. Figure 9—figure supplement 1—source data 2. Original files for western blot analysis.

Article Snippet: The human colon cancer cell lines SW480 and HCT116, as well as the human embryonic kidney cell line 293T, were purchased from the American Type Culture Collection (ATCC), authenticated by STR profiling, and tested negative for mycoplasma contamination.

Techniques: Western Blot, Over Expression, Control, Transfection, Plasmid Preparation

Journal: eLife

Article Title: Uev1A counteracts oncogenic Ras stimuli in both polyploid and diploid cells

doi: 10.7554/eLife.107104

Figure Lengend Snippet: ( A and B ) 5-Ethynyl-2’-deoxyuridine (EdU) incorporation assays to assess the effects of UBE2V1 and UBE2V2 overexpression (OE) on cell proliferation in SW480 and HCT116 cells. Empty OE vector was used as the control. All images in ( A ) are of the same magnification. ( C–E ) Assays to evaluate the effects of UBE2V1- and UBE2V2-OE on colony formation and cell viability in SW480 and HCT116 cells. In ( B and D ), three independent replicates were conducted, and statistical significance was determined using one-way ANOVA. In ( E ), five independent replicates were conducted, and statistical significance was determined using two-way ANOVA with multiple comparisons. * (p<0.05), ** (p<0.01), and **** (p<0.0001).

Article Snippet: The human colon cancer cell lines SW480 and HCT116, as well as the human embryonic kidney cell line 293T, were purchased from the American Type Culture Collection (ATCC), authenticated by STR profiling, and tested negative for mycoplasma contamination.

Techniques: Over Expression, Plasmid Preparation, Control