Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Selective inhibition of carbonic anhydrase-IX by sulphonamide derivatives induces pH and reactive oxygen species-mediated apoptosis in cervical cancer HeLa cells

doi: 10.1080/14756366.2018.1481403

Figure Lengend Snippet: Representative DNA damage microscopy images from HeLa cell treated with sulphonamide 1 . A fragment of each cell was fixed and processed for γ-H2AX immunofluorescent staining (IFA). γ-H2AX staining is green; nuclei are stained with DAPI blue.

Article Snippet: They were washed with PBS, and 1% BSA-containing PBS was then added, and incubation with primary mouse monoclonal anti-γ-H2AX antibody (Cell Signaling Technology, Danvers, MA) was applied at 37 °C for 1 h. The cells were washed with PBS for 5 min three times.

Techniques: Microscopy, Staining

Journal: bioRxiv

Article Title: VCP/p97 extracts DNA polymerase α/Primase from chromatin to limit the activation of the replication stress response

doi: 10.1101/2022.07.25.501345

Figure Lengend Snippet: (A-B) Flow cytometry analysis of DNA content (DAPI) and DNA replication (EdU) in HCT116 cells synchronized in S phase with a single thymidine block, released for 3 hrs and treated with 2 and 5 µM NMS873 for 3 hrs (A) or with 5 µM NMS873 (VCPi) for 3 or 6 hrs (B). The experiments were repeated twice and one representative experiment is shown. (C-H) Stretched DNA fiber analysis in HCT116 cells synchronized with a single thymidine block, released for 2 hrs and treated for 2 hrs with 10 µM NMS873 (VCPi) or DMSO as a control. (C) Scheme of labeling for fork rate and origin firing measures (1 st label origin). Cells were sequentially incubated with CldU and IdU for 20 min. The fork rate (FR) (D) and percentage of origins fired during the first labelling time (1 st label origins) (E) were measured and quantified after the different treatments. All the experiments were repeated three times; the pool of the three experiments (fork rate, bars represent the median of the pooled data) or the average (1 st label origins, %) is shown. (F) Scheme of labeling for fork asymmetry (CldU/IdU) and origin asymmetry (long/short) measures. Cells were sequentially incubated with CldU for 10 min and IdU for 30 min. The ratio CldU/IdU (CldU/IdU ratio) is shown in (G) and the ratio between the long and the short IdU track arising from the same origin (long/short ration) is shown in (H) (individual data are shown in ). The experiments were repeated twice and the pool of both experiments is shown. (I) Western blot analysis of whole cell extracts obtained from HCT116 cells synchronized by a single thymidine block, released for 2 hrs and treated with 2 mM hydroxyurea (HU), 10 µM NMS873 (VCPi), a combination of both (HU-VCPi) or DMSO as a control (lane 1, C). The levels of total and phosphorylated Chk1 (S345), total and phosphorylated RPA2 (S4/S8), phosphorylated H2AX (γH2AX), total histone H2A and PCNA were measured with specific antibodies. One of two repetitions is shown.

Article Snippet: The antibodies against USP7 (Bethyl A300-033A), VCP (Bethyl A300-589A), SUMO2/3 (MBL, M114-3 and University of Iowa, clone 8A2), PCNA (Santa Cruz, sc-56), Chk1 (Novocastra and Cell Signalling #2360), Chk1-S345P (Cell Signaling #2348), RPA2 (Abcam ab2175), RPA2-S4/S8P (Bethyl, A300-245A), H2A (Cell Signaling #3636), γH2AX (Millipore, 05-636), Ubiquitin (Cell Signaling #3933), MCM2 (custom made, Juan Méndez ), MCM2-S40P (Abcam, ab133243), MCM2-S53P (Abcam, ab70367), POLA1 (Abcam, ab31777), PRIM2 (Invitrogen PA5-88189), TOPBP1 (Bethyl, A300-111), Vinculin (Sigma-Aldrich V9264), UFD1L (Abcam, ab155003), CDC25A (Santa Cruz Biotechnology sc-7389) were used for Western Blot and immunofluorescence.

Techniques: Flow Cytometry, Blocking Assay, Labeling, Incubation, Western Blot

Journal: bioRxiv

Article Title: VCP/p97 extracts DNA polymerase α/Primase from chromatin to limit the activation of the replication stress response

doi: 10.1101/2022.07.25.501345

Figure Lengend Snippet: (A-F) Stretched DNA fiber analysis in HCT116 cells synchronized with a single thymidine block, released for 2 hrs and treated for 2 hrs with: 10 µM NMS873 (VCPi), 150 µM nucleosides (dNTP), a combination of both or DMSO as a control (A-B); 10 µM NMS873 (VCPi), 20 µM XL413 (CDC7i), a combination of both (VCPi-CDC7i) or DMSO as a control (C-D); 10 µM NMS873 (VCPi), 0.5 µM adarotene (POLAi), a combination of both (VCPi-POLAi) or DMSO as a control (E-F). Cells were sequentially incubated with CldU and IdU for 20 min. The fork rate (FR) (A, C, E) and percentage of origins fired during the first labelling time (1 st label origins) (B, D, F) were measured and quantified after the different treatments. All the experiments were repeated twice and the pool of the two experiments (FR, bars represent the median of the pooled data) or the average (1 st label origins) is shown. (G-H) Stretched DNA fiber analysis in HCT116 cells synchronized with a single thymidine block, released for 2 hrs and treated for 2 hrs with: 10 µM NMS873 (VCPi), 0.5 µM adarotene (POLAi), a combination of both or DMSO as a control. Cells were sequentially incubated with CldU for 10 min and IdU for 30 min. The ratio CldU/IdU (CldU/IdU ratio) is shown in (G) and the ratio between the long and the short IdU track arising from the same origin (long/short ratio) is shown in (H) (individual data are shown in ). The experiments were repeated twice and the pool of both experiments is shown. (I-J) Stretched DNA fiber analysis in HCT116 cells synchronized with a single thymidine block, released for 2 hrs and treated for 2 hrs with 0.5 µM adarotene (POLAi), 20 µM XL413 (CDC7i), a combination of both or DMSO as a control. Cells were sequentially incubated with CldU and IdU for 20 min. The fork rate (FR) (I) and percentage of origins fired during the first labelling time (1 st label origins) (J) were measured and quantified after the different treatments. All the experiments were repeated twice and the pool of the two experiments (FR, bars represent the median of the pooled data) or the average (1 st label origins) is shown. (K) Western blot analysis of whole cell extracts obtained from HCT116 cells synchronized by a single thymidine block, released for 2 hrs and treated with increasing concentrations of adarotene (0.1, 0.2, 0.5, 1 µM) (POLAi) alone or in combination with 10 µM NMS873 (VCPi). The levels of total and phosphorylated Chk1 (S345), total and phosphorylated RPA2 (S4/S8), phosphorylated H2AX (γH2AX) and total histone H2A were measured with specific antibodies. The experiment was repeated three times and one representative result is shown.

Article Snippet: The antibodies against USP7 (Bethyl A300-033A), VCP (Bethyl A300-589A), SUMO2/3 (MBL, M114-3 and University of Iowa, clone 8A2), PCNA (Santa Cruz, sc-56), Chk1 (Novocastra and Cell Signalling #2360), Chk1-S345P (Cell Signaling #2348), RPA2 (Abcam ab2175), RPA2-S4/S8P (Bethyl, A300-245A), H2A (Cell Signaling #3636), γH2AX (Millipore, 05-636), Ubiquitin (Cell Signaling #3933), MCM2 (custom made, Juan Méndez ), MCM2-S40P (Abcam, ab133243), MCM2-S53P (Abcam, ab70367), POLA1 (Abcam, ab31777), PRIM2 (Invitrogen PA5-88189), TOPBP1 (Bethyl, A300-111), Vinculin (Sigma-Aldrich V9264), UFD1L (Abcam, ab155003), CDC25A (Santa Cruz Biotechnology sc-7389) were used for Western Blot and immunofluorescence.

Techniques: Blocking Assay, Incubation, Western Blot

Journal: bioRxiv

Article Title: VCP/p97 extracts DNA polymerase α/Primase from chromatin to limit the activation of the replication stress response

doi: 10.1101/2022.07.25.501345

Figure Lengend Snippet: (A) WB analysis of the immunoprecipitation of VCP after cross-linking in HCT116 cells synchronized with a single thymidine block, released for 2 hrs and treated for 3 hrs with DMSO (marked as C), 5 µM NMS873 (VCPi) or 0.5 µM adarotene (POLAi) for 2 hrs. Pull-down with a non-specific IgG was used as control and 2% of the input material is shown. VCP/p97 and POLA1 were detected using specific antibodies and histone H2A was used as control. One representative experiment out of three repetitions is shown. (B) Western blot analysis of the immunoprecipitation of POLA1 from whole nuclear extracts obtained from HCT116 cells synchronized with a single thymidine block, released for 2 hrs and treated for 3 hrs with 5 µM NMS873 (VCPi), 0.5 µM Adarotene (POLAi) or DMSO as a control (marked as C). 2% of the input material is shown together with the pull-down using a non-specific IgG as a negative control. (C) Flow cytometry analysis of DNA content (DAPI) and DNA replication (EdU) in HCT116 cells wild-type and PRIMPOL knockout synchronized in S phase with a single thymidine block, released for 2 hrs and treated with DMSO (marked as C), 5 µM NMS873 (VCPi), 0.5 µM adarotene (POLAi) or a combination of both (VCPi-POLAi) for 2 hrs. The experiment was repeated twice and one representative experiment is shown. (D) Flow cytometry analysis of DNA content (DAPI) and DNA replication (EdU) in HCT116 cells synchronized in S phase with a single thymidine block, released for 2 hrs and treated with DMSO (marked as C), 5 µM NMS873 (VCPi), 0.5 µM adarotene (POLAi), 20 µM XL413 (CDC7i) or the indicated combinations for 2 hrs. The experiment was repeated three times and one representative experiment is shown. A bar is added as a reference.

Article Snippet: The antibodies against USP7 (Bethyl A300-033A), VCP (Bethyl A300-589A), SUMO2/3 (MBL, M114-3 and University of Iowa, clone 8A2), PCNA (Santa Cruz, sc-56), Chk1 (Novocastra and Cell Signalling #2360), Chk1-S345P (Cell Signaling #2348), RPA2 (Abcam ab2175), RPA2-S4/S8P (Bethyl, A300-245A), H2A (Cell Signaling #3636), γH2AX (Millipore, 05-636), Ubiquitin (Cell Signaling #3933), MCM2 (custom made, Juan Méndez ), MCM2-S40P (Abcam, ab133243), MCM2-S53P (Abcam, ab70367), POLA1 (Abcam, ab31777), PRIM2 (Invitrogen PA5-88189), TOPBP1 (Bethyl, A300-111), Vinculin (Sigma-Aldrich V9264), UFD1L (Abcam, ab155003), CDC25A (Santa Cruz Biotechnology sc-7389) were used for Western Blot and immunofluorescence.

Techniques: Immunoprecipitation, Blocking Assay, Western Blot, Negative Control, Flow Cytometry, Knock-Out

Journal: bioRxiv

Article Title: VCP/p97 extracts DNA polymerase α/Primase from chromatin to limit the activation of the replication stress response

doi: 10.1101/2022.07.25.501345

Figure Lengend Snippet: (A) Western blot analysis of the immunoprecipitation of PRIM2 and VCP/p97 from whole nuclear extracts obtained from HCT116 cells synchronized with a single thymidine block, released for 2 hrs and treated for 3 hrs with 5 µM NMS873 (VCPi), 0.5 µM Adarotene (POLAi) or DMSO as a control (C). 2% of the input material is shown together with the pull-down using a non-specific IgG as a negative control. A saturated exposure is included for VCP/p97 to show the material pulled-down by PRIM2. (B) Western blot analysis of the chromatin fraction purified from HCT116 cells that were synchronized with a single thymidine block, released for 2 hrs and treated for 3 hrs with 5 µM NMS873 (VCPi) or DMSO as a control (marked as C). The levels of POLA1, PRIM2 and VCP/p97 were analyzed with specific antibodies; PCNA and histone H2A were used as controls. (C) Western blot analysis of the chromatin and nuclear soluble fractions purified from HCT116 cells synchronized with a single thymidine block, released for 2 hrs and treated for 2 hrs with 5 µM NMS873 (VCPi), 0.5 µM adarotene (POLAi), 20 µM XL413 (CDC7i), combinations of these drugs or DMSO as a control (marked as C). The levels of POLA1, PRIM2 and total RPA2 were analyzed in chromatin using histone H2A as a control and the levels of total and phosphorylated CHK1 (S345) were analyzed in the nuclear soluble fraction using USP7 as a control. Experiments in (A-C) were repeated at least three times and one representative result is shown. (D) U2OS cells were transfected with a non-specific siRNA or siRNA pools directed against TOPBP1 or ETAA1. 72 hrs after the transfection the cells were treated with 10 µM NMS873 (VCPi), 0.5 µM adarotene (POLAi) or a combination of both using DMSO as a control. Western blot analysis of whole cell extracts with antibodies against TOPBP1, total and phosphorylated CHK1 (S345) using vinculin as a control. The experiment was performed twice with similar results.

Article Snippet: The antibodies against USP7 (Bethyl A300-033A), VCP (Bethyl A300-589A), SUMO2/3 (MBL, M114-3 and University of Iowa, clone 8A2), PCNA (Santa Cruz, sc-56), Chk1 (Novocastra and Cell Signalling #2360), Chk1-S345P (Cell Signaling #2348), RPA2 (Abcam ab2175), RPA2-S4/S8P (Bethyl, A300-245A), H2A (Cell Signaling #3636), γH2AX (Millipore, 05-636), Ubiquitin (Cell Signaling #3933), MCM2 (custom made, Juan Méndez ), MCM2-S40P (Abcam, ab133243), MCM2-S53P (Abcam, ab70367), POLA1 (Abcam, ab31777), PRIM2 (Invitrogen PA5-88189), TOPBP1 (Bethyl, A300-111), Vinculin (Sigma-Aldrich V9264), UFD1L (Abcam, ab155003), CDC25A (Santa Cruz Biotechnology sc-7389) were used for Western Blot and immunofluorescence.

Techniques: Western Blot, Immunoprecipitation, Blocking Assay, Negative Control, Purification, Transfection

Journal: bioRxiv

Article Title: VCP/p97 extracts DNA polymerase α/Primase from chromatin to limit the activation of the replication stress response

doi: 10.1101/2022.07.25.501345

Figure Lengend Snippet: HCT116 cells were transfected with a non-specific siRNA (siC) or with siRNA pools directed against POLA1, POLA2, PRIM1 or PRIM2. 24 hrs after transfection cells were synchronized with a single thymidine block, released for 2 hrs and treated with 2 or 10 µM NMS873 (VCPi) for 2 hrs or DMSO as control. (A-B) Western blot analysis of whole cell extracts measuring the levels of total and phosphorylated CHK1 (S345), total and phosphorylated RPA2 (S4/S8), phosphorylated H2AX (γH2AX), VCP/p97 and histone H2A and PCNA as controls. The experiments were repeated three times and one representative result is shown. (C-F) Stretched DNA fiber analysis of HCT116 cells depleted in POLA1 or PRIM2. (C-D) Cells were sequentially incubated with CldU and IdU for 20 min. The fork rate (FR) (C) and percentage of origins fired during the first labelling time (1 st label origins) (D) were measured and quantified after the different treatments. All the experiments were repeated twice and the pool of the two experiments (FR; bars represent the median of the pooled data) or the average (1 st label origins) is shown.

Article Snippet: The antibodies against USP7 (Bethyl A300-033A), VCP (Bethyl A300-589A), SUMO2/3 (MBL, M114-3 and University of Iowa, clone 8A2), PCNA (Santa Cruz, sc-56), Chk1 (Novocastra and Cell Signalling #2360), Chk1-S345P (Cell Signaling #2348), RPA2 (Abcam ab2175), RPA2-S4/S8P (Bethyl, A300-245A), H2A (Cell Signaling #3636), γH2AX (Millipore, 05-636), Ubiquitin (Cell Signaling #3933), MCM2 (custom made, Juan Méndez ), MCM2-S40P (Abcam, ab133243), MCM2-S53P (Abcam, ab70367), POLA1 (Abcam, ab31777), PRIM2 (Invitrogen PA5-88189), TOPBP1 (Bethyl, A300-111), Vinculin (Sigma-Aldrich V9264), UFD1L (Abcam, ab155003), CDC25A (Santa Cruz Biotechnology sc-7389) were used for Western Blot and immunofluorescence.

Techniques: Transfection, Blocking Assay, Western Blot, Incubation

Journal: bioRxiv

Article Title: VCP/p97 extracts DNA polymerase α/Primase from chromatin to limit the activation of the replication stress response

doi: 10.1101/2022.07.25.501345

Figure Lengend Snippet: (A) Model for the action of VCP/p97 during an unperturbed S phase. POLA/PRIM is loaded to chromatin upon origin firing and generates primed DNA that activates ATR in a TOPBP1 dependent manner. VCP/p97 extracts POLA/PRIM to limit the activation of CHK1. (B) Flow cytometry analysis of the cell cycle progression showing histograms for DNA content by DAPI staining in HCT116 cells arrested with a single thymidine block, released for 3 hrs and treated for 9 hrs with 10 µM NMS873 (VCPi), 2.5 µM LY2603618 (CHK1i), a combination of both (VCPi-CHK1i) or DMSO as a control. (C) Western blot analysis in whole cell extracts in HCT116 cells treated as in (B). The levels of total and phosphorylated CHK1 (S345), total and phosphorylated RPA2 (S4/S8), CDC25A, phosphorylated H2AX (γH2AX), histone H2A and PCNA were measured using specific antibodies. The experiments were repeated twice with equivalent results.

Article Snippet: The antibodies against USP7 (Bethyl A300-033A), VCP (Bethyl A300-589A), SUMO2/3 (MBL, M114-3 and University of Iowa, clone 8A2), PCNA (Santa Cruz, sc-56), Chk1 (Novocastra and Cell Signalling #2360), Chk1-S345P (Cell Signaling #2348), RPA2 (Abcam ab2175), RPA2-S4/S8P (Bethyl, A300-245A), H2A (Cell Signaling #3636), γH2AX (Millipore, 05-636), Ubiquitin (Cell Signaling #3933), MCM2 (custom made, Juan Méndez ), MCM2-S40P (Abcam, ab133243), MCM2-S53P (Abcam, ab70367), POLA1 (Abcam, ab31777), PRIM2 (Invitrogen PA5-88189), TOPBP1 (Bethyl, A300-111), Vinculin (Sigma-Aldrich V9264), UFD1L (Abcam, ab155003), CDC25A (Santa Cruz Biotechnology sc-7389) were used for Western Blot and immunofluorescence.

Techniques: Activation Assay, Flow Cytometry, Staining, Blocking Assay, Western Blot

Journal: Journal of Cardiovascular Pharmacology

Article Title: CFTR Suppresses Neointimal Formation Through Attenuating Proliferation and Migration of Aortic Smooth Muscle Cells

doi: 10.1097/FJC.0000000000001257

Figure Lengend Snippet: CFTR suppresses balloon injury–induced and PDGF-BB–stimulated VSMC proliferation. A, Protein expression of PCNA in injured arteries transfected with Ad-CFTR or Ad-GFP alone. n = 5, # P < 0.01 versus Sham, ***P < 0.001 versus Injury. B, VSMCs were treated with Ad-CFTR, Ad-GFP, or Ad-shCFTR and then exposed to PDGF-BB (20 ng/mL) for 24 hours. Cell viability was quantified by the MTT assay. n = 6, #### P < 0.0001 versus control cells. ** P < 0.01, ****P < 0.0001 versus PDGF-BB–stimulated cells. (C, D), Protein expression of PCNA in VSMCs infected with Ad-GFP, Ad-shCFTR, or Ad-CFTR, respectively. n = 6, ### P < 0.001, #### P < 0.0001 versus control cells. * P < 0.05 versus PDGF-BB–stimulated cells. (E, F), Expression of Ki67 in PDGF-BB–stimulated VSMCs infected Ad-GFP, Ad-shCFTR, or Ad-CFTR, respectively. n = 5, * P < 0.05 versus PDGF-BB–stimulated cells. n = 5, ### P < 0.001, #### P < 0.0001 versus control cells,* P < 0.05 versus PDGF-BB–stimulated cells.

Article Snippet: The immunodetection was processed using the following primary antibodies: CFTR (168 kDa, Cat. No. NB300-511, 1:1000; NOVUS Biologicals), Ki67 (358 kDa, Cat. No. ab 16667, 1:1000; Abcam), PCNA (36 kDa, Cat. No. 2586, 1:1000; Cell Signaling Technology), PDGF receptor β (190 kDa, Cat. No. 3162, 1:1000; Cell Signaling Technology), p-PDGF receptor β (190 kDa, Cat. No. 3166, 1:1000; Cell Signaling Technology), p38 (38 kDa, Cat. No.9212, 1:1000; Cell Signaling Technology), p-p38 (38 kDa, Cat. No. 9211, 1:1000; Cell Signaling Technology), SGK1 (55 kDa, Cat. No. S5188, 1:1000; Merck), p-SGK1 (48 kDa, Cat. No. SAB4503834, 1:1000; Merck), JNK (56/42 kDa, Cat. No. sc-7345, 1:500; Santa Cruz Biotechnology), p-JNK (56/42 kDa, Cat. No. sc-6254, 1:500; Santa Cruz Biotechnology), ERK (44/42 kDa, Cat. No. 9102, 1:1000; Cell Signaling Technology), and p-ERK (44/42 kDa, Cat. No. 4370, 1:1000; Cell Signaling Technology).

Techniques: Expressing, Transfection, MTT Assay, Infection

Journal: Scientific Reports

Article Title: Interferon-γ increases sensitivity to chemotherapy and provides immunotherapy targets in models of metastatic castration-resistant prostate cancer

doi: 10.1038/s41598-022-10724-9

Figure Lengend Snippet: IFNγ treatment alters the expression of MHC-I, PD-L1 and E-cadherin in mCRPC cells. ( A ) Geometric Mean Fluorescence Intensity (GMFI) of E-cadherin, MHC-I, and PD-L1 total expression in DU-H and DU-L cells after treatment with control or IFNγ (5 ng/mL) for 48 h, determined by flow cytometry. ( B ) Immonoblot of E-cadherin, PD-L1, and MHC-I in DU-H and DU-L cells after control or IFNγ (5 ng/mL) treatment for 48 h, with GAPDH as loading control. Uncropped immunoblot images are located in supplementary figures (Fig. ). ( C ) Quantification of immunoblots with fold change compared with control. Data shown as mean ± SD of three independent experiments. Student t-test * p < 0.05, ** p < 0.005, *** p < 0.001 **** p < 0.0001, ns, not significant. ( D ) Representative immunofluorescence (IF) images of staining MHC-I (red), PD-L1 (green), E-cadherin (red), and Hoechst 33342 (blue) in DU-H and DU-L. Cells were treated with control or IFNγ (5 ng/mL) for 48 h. All scale bars, 50 μm.

Article Snippet: The following antibodies were used in this study: E-cadherin (24E10, Cell Signaling Technology and clone 36, BD), PD-L1 (E1L3N, Cell Signaling Technology), HLA Class 1 ABC (EMR8-5, Abcam), and GAPDH (D16H11, Cell Signaling Technology).

Techniques: Expressing, Fluorescence, Flow Cytometry, Western Blot, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Interferon-γ increases sensitivity to chemotherapy and provides immunotherapy targets in models of metastatic castration-resistant prostate cancer

doi: 10.1038/s41598-022-10724-9

Figure Lengend Snippet: IFNγ influences the response of mCRPC cells to chemotherapy in vitro. ( A ) Representative IF images of co-staining E-cadherin (red), cleaved caspase 3 (green), and nucleus (Hoechst 33342, blue) in DU-H, DU-L, PC3-H, and PC3-L. Cells were treated with 1 μM camptothecin and 100 ng/ml TRAIL (CPT-TRAIL) for 4 h after 48 h of control or IFNγ (5 ng/mL) treatment. All scale bars, 50 μm. ( B ) GMFI of cleaved caspase 3 and membranous E-cadherin expression in DU-H, DU-L, PC3-H and PC3-L cells determined by flow cytometry. Data shown as mean ± SD. Student t-test, * p < 0.05, ** p < 0.005, ***, p < 0.001, **** p < 0.0001.

Article Snippet: The following antibodies were used in this study: E-cadherin (24E10, Cell Signaling Technology and clone 36, BD), PD-L1 (E1L3N, Cell Signaling Technology), HLA Class 1 ABC (EMR8-5, Abcam), and GAPDH (D16H11, Cell Signaling Technology).

Techniques: In Vitro, Staining, Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: Interferon-γ increases sensitivity to chemotherapy and provides immunotherapy targets in models of metastatic castration-resistant prostate cancer

doi: 10.1038/s41598-022-10724-9

Figure Lengend Snippet: Alterations in HLA-A and E-cadherin expression and chemosensitivity of mCRPC after IFNγ pretreatment in vivo. ( A ) Experimental outline to test the effect of IFNγ pretreatment on HLA-A and E-cadherin expression, as well as sensitivity to chemotherapy in DU-L cells in vivo. ( B ) Limited liver metastatic tumor growth of DU-L prostate cancer cells in IFNγ and paclitaxel combination group. H&E images at 200 × magnification and all others at 400 × magnification. Tumor is not outlined where the vast majority of the captured image is tumor. ( C ) Enumeration of liver metastasis in each mouse. ( D ) Representative H&E, HLA-A, E-cadherin and TUNEL staining in the metastatic tumors at completion of the study. ( E ) Quantification of HLA-A and E-cadherin expression, and TUNEL assays. Data shown as mean ± SD. Analysis of variance (ANOVA) or Kruskal–Wallis with Dunn's multiple comparisons test after determination of normality based on Shapiro–Wilk test, * p < 0.05, ** p < 0.005.

Article Snippet: The following antibodies were used in this study: E-cadherin (24E10, Cell Signaling Technology and clone 36, BD), PD-L1 (E1L3N, Cell Signaling Technology), HLA Class 1 ABC (EMR8-5, Abcam), and GAPDH (D16H11, Cell Signaling Technology).

Techniques: Expressing, In Vivo, TUNEL Assay, Staining

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Enrichment of Wee1/CDC2 and NF-κB Signaling Pathway Constituents Mutually Contributes to CDDP Resistance in Human Osteosarcoma

doi: 10.4143/crt.2021.320

Figure Lengend Snippet: Inhibition of Wee1 restores the effects of cisplatin (CDDP) on cell proliferation and activation of cell death-related molecular pathways in osteosarcoma cells with high tolerance to CDDP. In the Saos2 cell proliferation experiment (A), there was no significant difference in the cell optical density (OD) value between the tumor necrosis factor α (TNFα) and CDDP treatment groups and the control group. Compared with the control and CDDP treatment groups, the AZD1775 treatment and siRNA-Wee1 treatment groups showed significantly smaller OD values (p < 0.05). Compared with the TNFα group, the AZD1775+TNFα group also showed significantly lower OD values (p < 0.05). Compared with the CDDP treatment group, the CDDP+AZD1775 treatment group and CDDP+si-Wee1 treatment group both had the most significant decreases in OD values (p < 0.01). In the PDX-OS3 cell proliferation experiment (B), similar to those in Saos2 cells, the OD value was significantly lower in the AZD1775 and siRNA-Wee1 treatment groups than in the control group (p < 0.05) and was significantly lower in the AZD1775+TNFα treatment group than in the TNFα treatment group (p < 0.05). Compared with the CDDP treatment group, the CDDP+AZD1775 treatment group and CDDP+si-Wee1 treatment group both had the most significant decreases in the OD value (p < 0.01). In the Saos2 western blot assay (C), compared with the control group, the CDDP treatment group showed a significant increase in the levels of the NF-κB downstream molecules Bcl-2 and CCND1 (p < 0.05), while the differences in the expression of the apoptosis-related proteins cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase-3 were not statistically significant. Compared with the control group, the AZD1775 or siRNA-Wee1 treatment group showed significantly lower Bcl-2 and CCND1 expression levels (p < 0.05) and significantly higher cleaved PARP and cleaved caspase-3 levels (p < 0.05). Moreover, Bcl-2 and CCND1 levels were significantly decreased in the CDDP+AZD1775 group and CDDP+si-Wee1 group (p < 0.01), which were accompanied by simultaneous increases in the apoptosis-related proteins cleaved PARP and cleaved caspase-3 (p < 0.01). In PDX-OS3 cells (D), consistent with the results in Saos2 cells, Wee1 inhibition treatment significantly decreased Bcl-2 and CCND1 expression and simultaneously increased cleaved PARP and cleaved caspase-3 levels, especially in the CDDP combined with Wee1 inhibition group.

Article Snippet: The primary antibodies used in this study targeted the following proteins: Wee1 (95 kD, #5285, Santa Cruz Biotechnology, Inc.), CDC2 (34 kD, #9116S, Cell Signaling Technology, Inc., Danvers, MA), pCDC2Y15 (34 kD, #8242S, Cell Signaling Technology, Inc.), IKKα (85 kD, #1930S, Cell Signaling Technology, Inc.), IKKβ (87 kD, #8943, Cell Signaling Technology, Inc.), RelA (65 kD, #8242S, Cell Signaling Technology, Inc.), pRelAS536 (65 kD, #3033S, Cell Signaling Technology, Inc.), IκB (39 kD, #9247, Cell Signaling Technology, Inc.), cyclin D1 (36 kD, #2922, Cell Signaling Technology, Inc.), Bcl-2 (26 kD, #15071, Cell Signaling Technology, Inc.), PARP (116 kD, #9532, Cell Signaling Technology, Inc.), cleaved caspase-3 (17 kD, #9662, Cell Signaling Technology, Inc.), and β-actin (42 kD, #ab8226, Abcam, Inc., MA).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing