flp in t rex u2os host cells  (ATCC)

Journal: Cell Death and Differentiation

Article Title: Cisplatin-induced cell death increases the degradation of the MRE11-RAD50-NBS1 complex through the autophagy/lysosomal pathway

doi: 10.1038/s41418-022-01100-1

Figure Lengend Snippet: A COS-7 cells were transiently transfected with pCDNA3 Flag FBXW7 and nuclear (NE) and cytosolic extracts (S100) immunoprecipitated with anti-Flag or normal mouse serum (IgG) as a control. Immunoprecipitated materials were analyzed by Western blotting. Inp: the input lane was loaded with 1/20 of the extract used in each assay. Data are representative of two independent experiments. B Similar to A, but using anti-MRE11, anti-NBS1, or anti-RAD50 antibodies. Data are representative of two independent experiments. C Nuclear extracts of U2OS cells were used to immunoprecipitate endogenous FBXW7, and the obtained complexes were analyzed by immunoblotting. IgG: immunoprecipitation using normal rabbit serum, as a control. Inp: the input lane was loaded with 1/20 of the nuclear extract. Western blot against PLK1 was used as a control of the efficiency of FBXW7 immunoprecipitation. Data are representative of three independent experiments. D U2OS cells were treated with Con A (for the indicated times), NH 4 Cl (24 h), rapamycin (Rapa, 16 h) or trehalose (Tre, 24 h), whole cell extracts or nuclear and cytosolic extracts (where indicated) prepared, and membranes immunoblotted with the indicated antibodies. C: extracts of untreated cells. Western blots are representative of at least three replicates. E Whole cell extracts from U2OS or transduced U2OS:: HA FBXW7 or U2OS:: HA FBXW7ΔF cells were electroblotted and probed with the indicated antibodies. Western blot against cyclin E was used as a control of the efficiency of FBXW7 or FBXW7ΔF expression. F U2OS:: HA FBXW7 and U2OS:: HA FBXW7ΔF cells were treated or not with Con A (8 h) and analyzed by immunoblotting. *non-specific band from a previous immunoblotting. Data from E and F are representative results from three independent experiments. Quantitative fold change in proteins was determined relative to the loading control (α actinin or β actin).

Article Snippet: A U2OS cell line with inducible expression of HA FBXW7ΔF was also constructed as previously described [ ] using pCDNA5 FRT/TO-HA FBXW7ΔF, pOG44 (Addgene) and the U2OS Flp In T-REx host cell line [ ].

Techniques: Transfection, Immunoprecipitation, Control, Western Blot, Expressing

Journal: Cell Death and Differentiation

Article Title: Cisplatin-induced cell death increases the degradation of the MRE11-RAD50-NBS1 complex through the autophagy/lysosomal pathway

doi: 10.1038/s41418-022-01100-1

Figure Lengend Snippet: A HEK293T cells were transfected with the indicated plasmids and treated with ammonium chloride 4 h before collection. Extracts were prepared and polyubiquitylated Flag MRE11, HA NBS1, or HA RAD50 visualized after Western blot analysis of the corresponding immunoprecipitations. Brackets mark a ladder of bands corresponding to polyubiquitylated proteins. B In vitro ubiquitylation assay of in vitro transcribed and translated MRE11, NBS1 or RAD50 labeled with [ 35 S] was carried out in the presence or absence of the following products: recombinant SCF (FBXW7) complex expressed in Sf21 insect cells, E1 (His 6 -E1), E2 (His 6 -UbcH3 and UbcH5a), and ubiquitin (Ub). The samples were incubated at 30 °C for 1 h and analyzed by SDS-PAGE and autoradiography. Polyubiquitylated proteins appear as a ladder of bands. C U2OS cells were treated or not with ammonium chloride for 24 hours prior to harvesting. NP40 extracts were used to immunoprecipitate endogenous MRE11, NBS1, or RAD50. Normal mouse or rabbit sera (IgG) were used as a control. Complexes were analyzed by immunoblotting. C: extracts from U2OS cells; NH 4 Cl: extracts from U2OS cells treated with ammonium chloride. *non-specific band. D U2OS cells were interfered with the indicated siRNAs, and whole cell extracts were blotted with the designated antibodies. The graphs show the quantification of protein levels using ImageJ software. Error bars represent the SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t -test).

Article Snippet: A U2OS cell line with inducible expression of HA FBXW7ΔF was also constructed as previously described [ ] using pCDNA5 FRT/TO-HA FBXW7ΔF, pOG44 (Addgene) and the U2OS Flp In T-REx host cell line [ ].

Techniques: Transfection, Western Blot, In Vitro, Ubiquitin Assay, Labeling, Recombinant, Ubiquitin Proteomics, Incubation, SDS Page, Autoradiography, Control, Software

Journal: Cell Death and Differentiation

Article Title: Cisplatin-induced cell death increases the degradation of the MRE11-RAD50-NBS1 complex through the autophagy/lysosomal pathway

doi: 10.1038/s41418-022-01100-1

Figure Lengend Snippet: A Cytosolic (S100) and nuclear (NE) extracts were prepared from U2OS cells treated with ammonium chloride. Immunoprecipitations with an anti-NBS1 rabbit monoclonal antibody or with normal rabbit serum were performed and complexes immunoblotted with the indicated antibodies. Inp1, 2: the input lanes were loaded with 1/20 of the extract used in each assay (Inp1: S100, Inp2: NE). B U2OS cells treated or not with Con A for 8 h were analyzed by light microscopy. Yellow arrows indicate magnified areas (x2). The bars represent 10 μm. C Similar to A, but using only nuclear extracts from the indicated cell lines treated with ammonium chloride.

Article Snippet: A U2OS cell line with inducible expression of HA FBXW7ΔF was also constructed as previously described [ ] using pCDNA5 FRT/TO-HA FBXW7ΔF, pOG44 (Addgene) and the U2OS Flp In T-REx host cell line [ ].

Techniques: Light Microscopy

Journal: Cell Death and Differentiation

Article Title: Cisplatin-induced cell death increases the degradation of the MRE11-RAD50-NBS1 complex through the autophagy/lysosomal pathway

doi: 10.1038/s41418-022-01100-1

Figure Lengend Snippet: A Subcellular fractions were prepared from HeLa cells treated or not with ammonium chloride for 24 h, as described in Materials and Methods. The amount of proteins used from the cytosolic, lysosomal, and nuclear fractions represented 5%, 30%, and 2.5% of the total proteins of each fraction, respectively. The extracts were immunoblotted with the indicated antibodies. The purity of the fractions was tested with anti-α Tubulin (cytosolic fraction), anti-Histone H1 (nuclear fraction), and anti-LAMP1 (lysosome-rich fraction). B The lysosomal fractions of HeLa, HeLa:: HA βTrCP , and HeLa:: HA FBXW7 treated or not with ammonium chloride for 24 h were prepared as in A. Equal amounts of protein were analyzed by SDS-PAGE and immunoblot. C . Similar to B using the indicated cell lines. *non-specific band. Quantitative fold change in proteins was determined relative to the LC3 protein. Data from A , B , and C are representative results from 1-3 independent experiments. D U2OS and U2OS:: HA FBXW7ΔF cells treated or not with Con A for 8 h were analyzed by light microscopy. Yellow arrows indicate magnified areas (x2). The bars represent 10 μm.

Article Snippet: A U2OS cell line with inducible expression of HA FBXW7ΔF was also constructed as previously described [ ] using pCDNA5 FRT/TO-HA FBXW7ΔF, pOG44 (Addgene) and the U2OS Flp In T-REx host cell line [ ].

Techniques: SDS Page, Western Blot, Light Microscopy

Journal: Cell Death and Differentiation

Article Title: Cisplatin-induced cell death increases the degradation of the MRE11-RAD50-NBS1 complex through the autophagy/lysosomal pathway

doi: 10.1038/s41418-022-01100-1

Figure Lengend Snippet: A HeLa cells were grown in serum containing complete medium (10% FCS) or in serum starved medium (0.15% FCS) treated or not with Con A 8 h before harvesting. Whole cell extracts were electroblotted and probed with different antibodies. B U2OS cells were incubated with different concentrations of cisplatin (CDDP) for 24 h. Whole cell extracts were analyzed by immunoblotting. C Percentage of annexin V positive U2OS cells treated as in B detected by flow cytometry. Error bars represent the SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t -test). D U2OS cells were treated or not with CDDP (50 μM) or with CDDP and ammonium chloride for 24 h and whole cell extracts analyzed by Western blotting. Unt: untreated cells. E U2OS cells transfected with siRNA FBXW7 (or siRNA EGFP as a control) were treated with CDDP (50 μM) for 24 h. Whole cell extracts were electroblotted and probed with the indicated antibodies. All of data are representative of at least two independent experiments. Quantitative fold change in proteins was determined relative to the loading control (α tubulin or β actin).

Article Snippet: A U2OS cell line with inducible expression of HA FBXW7ΔF was also constructed as previously described [ ] using pCDNA5 FRT/TO-HA FBXW7ΔF, pOG44 (Addgene) and the U2OS Flp In T-REx host cell line [ ].

Techniques: Incubation, Western Blot, Flow Cytometry, Transfection, Control

Journal: Cell Death and Differentiation

Article Title: Cisplatin-induced cell death increases the degradation of the MRE11-RAD50-NBS1 complex through the autophagy/lysosomal pathway

doi: 10.1038/s41418-022-01100-1

Figure Lengend Snippet: A U2OS and U2OS:: HA FBXW7ΔF cells treated or not with cisplatin (CDDP) for 24 h and/or Con A for 8 h were analyzed by light microscopy. Yellow arrows indicate magnified areas (x2). The bars represent 10 μm. B Percentage of U2OS versus U2OS:: HA FBXW7ΔF cells displaying NBS1 in lysosomes (visualized with anti-LAMP1) after cisplatin (CDDP) and Con A treatment. A minimum of 50 cells were analyzed in triplicate for each condition. Student’s t -test was used to determine statistical significance. **** p < 0.0001. C Colony assays showing cell viability for U2OS Flp In T-REx HA FBXW7ΔF cells induced or not with doxycycline (DOX) and treated with CDDP (30 μM) for 4 h. Colony forming units from initial 5 × 10 3 and 10 4 cells of each condition were quantified using ImageJ software, and represented in graphs. Error bars represent the SD ( n = 3). ** p < 0.01 (Student’s t -test).

Article Snippet: A U2OS cell line with inducible expression of HA FBXW7ΔF was also constructed as previously described [ ] using pCDNA5 FRT/TO-HA FBXW7ΔF, pOG44 (Addgene) and the U2OS Flp In T-REx host cell line [ ].

Techniques: Light Microscopy, Software