Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in immortalized MEFs. (A) Cells (WT, MJI-53; KO, MJI-1) were treated for 24 h using 0 or 3 μM aphidicolin. (B) Cells (lane 1–3, WT, MJI-91; lanes 4–6, KO, MJI-186; lanes 7–9, WT, MJI-105; lanes 1–12, KO, MJI-84) were treated for 24 h using 0, 1 or 3 μM aphidicolin. Actin and 53BP1 serve as loading controls. DYNLL1 is used as a surrogate marker for loss of ASCIZ. (C) Quantification of western blot band intensities. An arbitrary unit of 100 represents the average band intensity for the respective phospho-protein in the 3 μM aphidicolin-treated wildtype samples on each membrane. Graphs indicate the mean ± standard error, n = 3. Additional loading controls for total KAP1, p53 and H2AX are shown in Supplementary Fig. S1A and B.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Marker, Western Blot, Membrane

Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in primary MEFs. Freshly prepared primary MEFs from a wildtype and an Asciz −/− embryo were treated for 24 h with 0, 1 or 3 μM aphidicolin. For comparison, cultures from the same embryos were also irradiated with 2-Gy and allowed to recover for 30 min. Similar results for another independent pair of wildtype and Asciz −/− primary MEFs are shown in Supplementary Fig. S1C, and additional loading controls for total KAP1, p53 and H2AX are shown in Supplementary Fig. S1D.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Comparison, Irradiation

Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in human ASCIZ/ATMIN knockout cells. (A, B) BL30 cells were cultured as indicated in the presence or absence of doxycycline to induce a non-targeting control guide RNA or the guide RNA against ASCIZ , and aphidicolin to elicit prolonged DNA replication stress as indicated. For comparison, control guide RNA transduced cells were also irradiated for canonical ATM activation without doxycycline or aphidicolin treatment in panel A. Panel B shows a blot of the same samples except the irradiated control. Additional loading controls for total ATM, KAP1 and p53 are shown in Supplementary Fig. S1E.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Knock-Out, Cell Culture, Control, Comparison, Irradiation, Activation Assay

Journal: Oncology reports

Article Title: TRIM28 promotes cervical cancer growth through the mTOR signaling pathway.

doi: 10.3892/or.2018.6235

Figure Lengend Snippet: Figure 1. TRIM28 expression is elevated in cervical cancer tissues and cell lines. (A) Analysis from the Oncomine database showing that TRIM28 mRNA expression is significantly elevated in cervical cancer tissues (n=40) compared with that in normal tissues (n=5). The data were obtained from a previous study (35). (B) TRIM28 mRNA levels in 20 paired cervical cancer samples and adjacent normal tissues examined by qRT‑PCR. (C) Immunohistochemical staining of normal and cervical cancer tissues with anti-TRIM28 antibody. The expression levels of TRIM28 in human cervical cancer cells and normal human cervical tissues were measured by qRT‑PCR (D) and western blotting (E). Data are presented as means ± SD. *P<0.05.

Article Snippet: After blocking the endogenous peroxidase with 3% hydrogen peroxide, the sections were washed with Tris-buffered saline (TBS) and then incubated with a primary antibody targeting TRIM28 (Proteintech, Hubei, China) for 1 h at 37 ̊C.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot

Journal: Oncology reports

Article Title: TRIM28 promotes cervical cancer growth through the mTOR signaling pathway.

doi: 10.3892/or.2018.6235

Figure Lengend Snippet: Figure 3. TRIM28 enhances cervical cancer cell tumorigenicity. (A) Tumor volumes were measured on the indicated days. (B) At the end of the experiment, the weights were measured. (C) The proliferation index (right) was determined based on the percentage of Ki67-positive cells (left). (D) Western blot analysis of TRIM28 and p-mTOR protein expression in indicated tumor xenografts. Data are presented as means ± SD. *P<0.05.

Article Snippet: After blocking the endogenous peroxidase with 3% hydrogen peroxide, the sections were washed with Tris-buffered saline (TBS) and then incubated with a primary antibody targeting TRIM28 (Proteintech, Hubei, China) for 1 h at 37 ̊C.

Techniques: Western Blot, Expressing

Journal: Oncology reports

Article Title: TRIM28 promotes cervical cancer growth through the mTOR signaling pathway.

doi: 10.3892/or.2018.6235

Figure Lengend Snippet: Figure 2. TRIM28 promotes cervical cancer cell proliferation by accelerating the cell cycle. (A) Western blot analysis of TRIM28 protein expression in TRIM28-infected cells and TRIM28 shRNA-infected cells. GAPDH was used as the loading control. (B) Effects of TRIM28 upregulation or downregulation on the proliferation of SiHa or CaSki cells analyzed by the MTT assay. (C) Representative micrographs (left) and quantification (right) of colonies formed by the indicated cervical cancer cell lines. (D) Cell cycle progression of the indicated cervical cancer cell lines analyzed by flow cytometry. (E) Representative images (left) and quantification (right) of BrdU-positive cells in the indicated cervical cancer cell lines. Data are presented as means ± SD. *P<0.05.

Article Snippet: After blocking the endogenous peroxidase with 3% hydrogen peroxide, the sections were washed with Tris-buffered saline (TBS) and then incubated with a primary antibody targeting TRIM28 (Proteintech, Hubei, China) for 1 h at 37 ̊C.

Techniques: Western Blot, Expressing, Infection, shRNA, Control, MTT Assay, Flow Cytometry

Journal: Oncology reports

Article Title: TRIM28 promotes cervical cancer growth through the mTOR signaling pathway.

doi: 10.3892/or.2018.6235

Figure Lengend Snippet: Figure 4. TRIM28 promotes cervical cancer cell proliferation via the mTOR signaling pathway. (A) Western blot analysis of TRIM28, p-mTOR(S2448), mTOR, p-S6K1(T389), and S6K1 in TRIM28 shRNA-infected CaSki cells and TRIM28-infected SiHa cells in the presence or absence of 20 nM evero- limus (EVE) for 48 h. GAPDH served as the loading control. (B-D) Cell proliferation, cell cycle, and BrdU incorporation were analyzed in TRIM28-infected SiHa cells in the presence or absence of EVE for 48 h. Data are presented as means ± SD. *P<0.05.

Article Snippet: After blocking the endogenous peroxidase with 3% hydrogen peroxide, the sections were washed with Tris-buffered saline (TBS) and then incubated with a primary antibody targeting TRIM28 (Proteintech, Hubei, China) for 1 h at 37 ̊C.

Techniques: Western Blot, shRNA, Infection, Control, BrdU Incorporation Assay