Journal: Experimental and Therapeutic Medicine

Article Title: MiR‑221 and miR‑222 regulate cell cycle progression and affect chemosensitivity in breast cancer by targeting ANXA3

doi: 10.3892/etm.2023.11826

Figure Lengend Snippet: Expression of ANXA3 is associated with chemotherapy sensitivity. (A) Transfection efficiency was assessed according to siANXA3 concentration. (B) Cell viability was evaluated in MDA-MB-231 cells treated with si-ANXA3 and adriamycin at different doses. (C) Percentage of viable MDA-MB-231 cells. Cells were treated with adriamycin (25 nM), miR-221/222 and siANXA3. (D) Cell cycle profile was analyzed using flow cytometry. Fluorescence-activated cell sorting analysis of cells transfected with control or siANXA3. (E) FACS analysis of cells transfected with adriamycin (50 nM) alone or a combination of adriamycin and siANXA3. (F) Expression of two cell cycle regulatory factors, cyclin A and cyclin B1, were evaluated using western blotting after treatment with adriamycin alone and a combination of adriamycin and siANXA3. (G) Flow cytometry and annexin V-FITC/PI labeling were used to examine apoptosis after treatment with adriamycin, siANXA3 and their combination. (H) Expression of apoptotic factors was evaluated by western blotting after treatment with adriamycin alone and a combination of siANXA3. (I) Overall survival was evaluated according to ANXA3 levels using the Kaplan-Meier Plotter software. * P<0.05, ** P<0.01 and *** P<0.001. ANXA3, annexin A3; FACS, fluorescence-activated cell sorting; Adr, adriamycin; SRC, scrambled negative control; siANXA3, small-interfering RNA targeting ANXA3; siCTL, small-interfering RNA negative control; Asy, asynchronous; PARP, poly (ADP-ribose) polymerase; anti-miR-CTL, anti-miR 221/222 negative control; miR, microRNA.

Article Snippet: The primary antibodies were: Anti-annexin A3 (1:1,000; cat. no. ab33068; Abcam); anti-cyclin D1 (1:500; cat. no. sc-20044; Santa Cruz Biotechnology, Inc.); anti-CDK4 (1:500; Santa Cruz Biotechnology, Inc.; cat. no. sc-56277); anti-cyclin B1 (1:500; cat. no. sc-245; Santa Cruz Biotechnology, Inc.); anti-cyclin A (1:500; cat. no. sc-274682; Santa Cruz Biotechnology, Inc.); anti-α tubulin (1:1,000; cat. no. sc-5286; Santa Cruz Biotechnology, Inc.); anti-β-Actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.); apoptosis western blot cocktail (1:250; cat. no. ab136812; Abcam); and total poly ADP ribose polymerase (1:1,000; cat. no. 9542; Cell Signaling Technology, Inc.).

Techniques: Expressing, Transfection, Concentration Assay, Flow Cytometry, Fluorescence, FACS, Western Blot, Labeling, Software, Negative Control, Small Interfering RNA

Journal: PLoS ONE

Article Title: Inhibition of p53-Dependent, but Not p53-Independent, Cell Death by U19 Protein from Human Herpesvirus 6B

doi: 10.1371/journal.pone.0059223

Figure Lengend Snippet: ( A & B ) Western blot analysis of PARP cleavage in cells either mock-treated or infected with HHV-6B for 48 hrs. The cells were subsequently treated with mock, leupeptin (10 µM), UV radiation (10 min exposure followed by 4 hrs of incubation), doxorubicin (0.2 µg/ml for 24 hrs), γ radiation (30 Gy followed by 24 hrs of incubation), or MG132 (10 µM). 7C7 was used as infection control and GAPDH was used as loading control. ( C & D ) Western blot analyses with antibodies against PARP, GAPDH, and 7C7 (infection control) on lysates from HCT116 (C) or MOLT3 (D) cells treated with varying doses of UV radiation (10, 20, 240, 600 J/cm 2 followed by 4 hrs of incubation). ( E ) ATP cell viability assay on MOLT3 cells with or without HHV-6B infection and treated with varying amounts of UV (0, 2.5, 5, 10, 15, 20, 40 and 60 J/cm 2 followed by 4 hrs of incubation) or γ radiation (0 and 30 Gy followed by 24 hrs of incubation). ( F ) Western blot analyses with antibodies against PARP, p53, PUMA, p21 or GAPDH (loading control) on lysates from HCT116 cells either mock-treated or infected with HHV-6B for 24 hrs followed by γ radiation (30 Gy) and 24 hrs of incubation. ( G ) Confocal microscopy images of HCT116 cells either mock-treated or infected with HHV-6B for 24 hrs, γ-irradiated (30 Gy) followed by 24 hrs of incubation. The cells were analyzed for cleaved caspase-3 (Alexa 488 green), DR6 (Alexa 546 red), and DAPI (blue).

Article Snippet: Proteins were separated on Criterion 12% BIS-TRIS gels (Bio-Rad, Copenhagen, Denmark), blotted to nitrocellulose membranes and visualized with the following antibodies: Rabbit anti-PARP mAb (1∶2000) (Cell Signaling), rabbit anti-GAPDH pAb (1∶1000) (Santa Cruz), mouse anti-RCC1 mAb (1∶1000) (Santa Cruz), mouse anti-p53 mAb (DO-7) (1∶1000) (Life Technologies), rabbit anti-PUMA pAb (1∶1000) (Abcam), mouse anti-p53-phospho-Ser15 (p-Ser15) mAb (1∶1000) (Cell Signaling), mouse anti-p53-phospho-Ser46 (p-Ser46) mAb (1∶1000) (Cell Signaling), mouse anti-HHV-6 mAb (7C7) (1∶1000) (Argene Biosoft, Verniolle, France).

Techniques: Western Blot, Infection, Incubation, Control, Viability Assay, Confocal Microscopy, Irradiation

Journal: PLoS ONE

Article Title: Inhibition of p53-Dependent, but Not p53-Independent, Cell Death by U19 Protein from Human Herpesvirus 6B

doi: 10.1371/journal.pone.0059223

Figure Lengend Snippet: ( A ) Western blot analyses with antibodies against p53, PUMA, and GAPDH on lysates from HCT116 cells transduced with an empty or a U19 lentivirus vector and treated with or without γ radiation (30 Gy) followed by incubation for 24 hrs. ( B ) Western blot analyses with antibodies against PARP and GAPDH on lysates from HCT116 cells transduced with an empty or a U19 lentivirus vector and treated with or without γ radiation (30 Gy) followed by incubation for 24 hrs. ( C ) Real-time PCR on mRNA from HCT116 wt, U19S and p53 −/− cells with PUMA or TBP specific primers. PUMA mRNA levels are represented relative to TBP . Measurements were performed in duplicate. Two independent experiments is shown. ( D ) Western blot analyses with antibodies against PARP and GAPDH on lysates from HCT116 cells transduced with an empty or a U19 lentivirus vector and treated with or without UV radiation (60 J/cm2) followed by 4 hrs incubation. ( E ) Western blot analyses with antibodies against PARP and GAPDH on lysates from HCT116 cells transduced with an empty or a U19 lentivirus vector and treated with or without MG132 for 24 hrs. ( F ) Western blot analyses with antibodies against p53 p-Ser15, p53 p-Ser46, and GAPDH on lysates from HCT116 cells transduced with an empty or a U19 lentivirus vector and treated with or without γ radiation (30 Gy) followed by 24 hrs of incubation. ( G & H ) HCT116 mock-LV (wt) and U19-LV (U19) cells where treated with or without zVAD, nec-1 or both for 1 hr followed by γ irradiation (30 Gy) or UV treatment (60 J/cm 2 ) for 24 or 48 hrs. Cell viability was assessed by measuring intracellular ATP-levels. Data are shown as % dead cells calculated relative to untreated cells. Measurements were performed in duplicate. An average of three independent experiments is shown. Error bars represent SD. ( I ) HCT116 p53 −/− cells were treated with or without γ radiation (30 Gy) or UV (60 J/cm 2 ) for 24 hrs followed by analysis of intracellular ATP levels. Measurements were performed in duplicate. An average of three independent experiments is shown. Error bars represent SD.

Article Snippet: Proteins were separated on Criterion 12% BIS-TRIS gels (Bio-Rad, Copenhagen, Denmark), blotted to nitrocellulose membranes and visualized with the following antibodies: Rabbit anti-PARP mAb (1∶2000) (Cell Signaling), rabbit anti-GAPDH pAb (1∶1000) (Santa Cruz), mouse anti-RCC1 mAb (1∶1000) (Santa Cruz), mouse anti-p53 mAb (DO-7) (1∶1000) (Life Technologies), rabbit anti-PUMA pAb (1∶1000) (Abcam), mouse anti-p53-phospho-Ser15 (p-Ser15) mAb (1∶1000) (Cell Signaling), mouse anti-p53-phospho-Ser46 (p-Ser46) mAb (1∶1000) (Cell Signaling), mouse anti-HHV-6 mAb (7C7) (1∶1000) (Argene Biosoft, Verniolle, France).

Techniques: Western Blot, Transduction, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Irradiation

Journal: Cell Communication and Signaling : CCS

Article Title: Ubiquitin-specific protease 22 is critical to in vivo angiogenesis, growth and metastasis of non-small cell lung cancer

doi: 10.1186/s12964-019-0480-x

Figure Lengend Snippet: USP22 knockout sensitizes NSCLC cells to irradiation. Representative flow cytometry profile of apoptotic cells in a . The parent (upper panel) and USP22−/− A549 (lower panel) cancer cells; and b . The parent (upper panel) and USP22−/− H1299 cancer cells (lower panel). Cells were first subjected to 5 or 10 Gy irradiation; apoptosis was measured at 48 h post-irradiation. Quantitative analysis of the experiments shows that apoptotic cells were significantly increased in both USP22−/− A549 ( a , right panel) and USP22−/− H1299 cancer cells ( b , right panel) compared with their parent cells (USP22+/+). The experiment was repeated three times and data represent the average of the early apoptotic and late apoptotic cells (** P < 0.01). The dynamics of γ-H2AX, H2Bub1, and apoptotic markers PARP cleaved product (c-PARP for cleaved protein) in c . the parent and USP22−/− A549 cancer cells, and d . the parent and USP22−/− H1299 cancer cells at 2 h, 48 h post-irradiation that analyzed by Western blot

Article Snippet: The mouse monoclonal antibodies against E-Cadherin (4A2), Vimentin (D21H3), Sirt1 (1F3), p16 INK4A (D7C1M), p53 (7F5), ALDH1A3 (ab12915), total and cleaved poly (ADP-ribose) polymerase (PARP) (Asp214, 19F4), Cyclin D1 (92G2), trimethylated H3K4 (C42D8) and H3K79 (Cat#: 4260) were purchased from Cell Signaling Technology (Beverly, CA USA) and Abcam.

Techniques: Knock-Out, Irradiation, Flow Cytometry, Western Blot

Journal:

Article Title: Inhibition of Focal Adhesion Kinase and Src Increases Detachment and Apoptosis in Human Neuroblastoma Cell Lines

doi: 10.1002/mc.20592

Figure Lengend Snippet: FAK inhibition with AdFAK-CD results in decreased proliferation and viability and increased apoptosis in neuroblastoma cell lines. Equal numbers of MYCN+ and MYCN− cells are plated and allowed to attach. MYCN+ and MYCN− neuroblastoma cells were then treated for 48 hours with nothing, AdLacZ, or AdFAK-CD. Experiments are repeated at least three times and data reported as mean ± SEM. A, Proliferation is measured with BrdU assay (Calbiochem) according to manufacture’s instructions. Data are reported as fold change in proliferation compared to untreated control cells. After treatment with AdFAK-CD, there is a significant decrease in proliferation in both cell lines, but the decrease is greater in the MYCN+ cells. Treatment with the control, AdLacZ, does not significantly affect proliferation in either cell line. B, Viability is measured with MTT assay. Data are reported as fold change in viability compared to untreated control cells. Findings are similar to the proliferation results, with significant decreases in viability after AdFAK-CD treatment in both the MYCN+ and MYCN− cell lines, but the change is more marked in the MYCN+ cells. Again, treatment with AdLacZ control does not significantly affect viability in either cell line. C, Apoptosis is measured with TUNEL staining by FACS analysis. The APO-BRDU™ kit (BD PharMingen) is a two color staining method for labeling DNA strand breaks that can then be detected by flow cytometry. After 48 hours, the treated cells are harvested and labeled according to manufacturer’s instructions and analyzed with a FACSCalibur™ machine gated to exclude cellular debris and evaluating 105 events. AdFAK-CD treatment leads to significant increases in apoptosis in both the MYCN+ and MYCN− cells, but as noted with the cell proliferation and viability experiments, the difference is more marked in the MYCN+ cells. D, Apoptosis is confirmed biochemically by detection of cleaved PARP products by Western blot. After 48 hours of treatment, the cells were harvested and cell lysates collected. Western blot was performed as described in the Methods section, utilizing an antibody that detects PARP cleavage products. There is a significant increase in cleaved PARP detected after treatment with AdFAK-CD, but again, the increase is more marked in the MYCN+ neuroblastoma cell line compared to that of the MYCN− cells.

Article Snippet: Monoclonal antibodies for total poly (ADP-ribose) polymerase (PARP, 611038) were obtained from BD Transduction Labs (BD PharMingen, San Jose, CA), for caspase 9 (9502), cleaved PARP (9532), AKT (9272), phospho-AKT (Ser 473, 9271S), ERK (9102) and phospho-ERK (43775) from Cell Signaling Technology, Inc. (Danvers, MA), and for phospho-Src (44–660G) from Biosource (Invitrogen).

Techniques: Inhibition, BrdU Staining, MTT Assay, TUNEL Assay, Staining, Labeling, Flow Cytometry, Western Blot

Journal:

Article Title: Inhibition of Focal Adhesion Kinase and Src Increases Detachment and Apoptosis in Human Neuroblastoma Cell Lines

doi: 10.1002/mc.20592

Figure Lengend Snippet: Dual inhibition of FAK and Src leads to increased detachment, decreased viability and increased apoptosis. Equal numbers of MYCN+ and MYCN− neuroblastoma cells were plated and then treated for 24 hours with nothing, AdLacZ, or AdFAK-CD, with or without the addition of PP2. Experiments are repeated at least three times and data reported as mean ± SEM. A, After treatment, floating and adherent cells are collected separately and counted. The percentage of detached cells is calculated by dividing the number of floating cells by the number of total cells (floating plus adherent cells). After just 24 hours of dual treatment with AdFAK-CD and PP2, significant detachment is seen in both cell lines. B, Cell viability was determined with trypan blue staining. After 24hours of dual inhibition with AdFAK-CD and PP2, all cells were collected, stained with trypan blue, and counted. Experiments were repeated at least three times, and data reported as fold change in viability. There was a significant increase in the non-viable cells in the MYCN+ and MYCN− cells as early as 24 hours after dual inhibition with AdFAK-CD and PP2, with non-viability increasing in the MYCN− cells over 3 times that of AdFAK-CD alone. C, After 24 hours treatment with dual inhibition using AdFAK-CD and PP2, cells were collected, fixed, stained with Hoechst, and counted. Apoptosis was measured by counting the number of stained cells. As early as 24 hours after initiating dual inhibition of FAK and Src, there is now significant apoptosis in the MYCN− as well as the MYCN+ isogenic cells lines. D, Apoptosis is detected biochemically by immunoblotting. After 24 hours of dual inhibition with AdFAK-CD and PP2, cell lysates are collected and Western blot performed (Materials and Methods) with antibodies that detect total PARP and total caspase 9. After 24 hours, treatment with AdFAK-CD leads to a loss of expression of total PARP and total caspase 9 in both cell lines, which becomes even more marked in the MYCN− cells with dual inhibition of Src and FAK after the addition of PP2 to the AdFAK-CD treatment (Fig. 5D, rows 1, 2, 3). Treatment with AdLacZ with or without PP2 did not affect total PARP or total caspase 9 in either cell line.

Article Snippet: Monoclonal antibodies for total poly (ADP-ribose) polymerase (PARP, 611038) were obtained from BD Transduction Labs (BD PharMingen, San Jose, CA), for caspase 9 (9502), cleaved PARP (9532), AKT (9272), phospho-AKT (Ser 473, 9271S), ERK (9102) and phospho-ERK (43775) from Cell Signaling Technology, Inc. (Danvers, MA), and for phospho-Src (44–660G) from Biosource (Invitrogen).

Techniques: Inhibition, Staining, Western Blot, Expressing