Journal: Engineering in Life Sciences

Article Title: Overexpression of GRP78 enhances survival of CHO cells in response to serum deprivation and oxidative stress

doi: 10.1002/elsc.201500152

Figure Lengend Snippet: Viability of Chinese hamster ovary (CHO) cells in response to serum deprivation and oxidative stress. CHO cells were seeded in 12‐well plates at a density of 1.5 × 105 cells per well and grown for 24 h. Then cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, serum free medium with or without 200 μM H2O2. In order to ensure that other nutrient limitations were not experienced, cells were fed fresh medium every 48 h. Cells were taken every 2 days and analyzed for cell viability using Annexin V/PI double staining. Error bars represent SD for each measurement.

Article Snippet: The Chinese hamster ovary (CHO) cells, human stomach adenocarcinoma (AGS) cell line, and human hepatoma cell line HepG2 (China Center for Type Culture Collection, Wuhan, P. R. China) were cultured in Roswell Park Memorial Institute (RPMI) 1640 and DMEM medium (Gbico BRL, NY, USA) supplemented with 10% FBS (Sijiqing, Hangzhou, China) and 100 U/mL ampicillin, 100 mg/mL streptomycin in an incubator containing 5% CO 2 at 37°C (standard conditions).

Techniques: Cell Culture, Double Staining

Journal: Engineering in Life Sciences

Article Title: Overexpression of GRP78 enhances survival of CHO cells in response to serum deprivation and oxidative stress

doi: 10.1002/elsc.201500152

Figure Lengend Snippet: Glucose‐regulated protein 78 (GRP78) engineering protected Chinese hamster ovary (CHO) cells against injury induced by H2O2. (A) Cells synchronized in serum‐free medium were treated with H2O2 at increasing concentration for 4 h and then cultured with 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT; 5 mg/mL) for 4 h. Bar graph represented the percentage of viable cells assayed by MTT. (B) The level of cleaved caspase‐3 in H2O2‐treated cells was detected by western blot (upper). The lower left bar graph represented densitometric analysis of GRP78 levels of the western blots and the lower right of cleaved caspase‐3. Data were mean values ± SD, n > 3.*p < 0.05,**p < 0.01,***p < 0.001, NS, nonsignificant difference versus negative control.

Article Snippet: The Chinese hamster ovary (CHO) cells, human stomach adenocarcinoma (AGS) cell line, and human hepatoma cell line HepG2 (China Center for Type Culture Collection, Wuhan, P. R. China) were cultured in Roswell Park Memorial Institute (RPMI) 1640 and DMEM medium (Gbico BRL, NY, USA) supplemented with 10% FBS (Sijiqing, Hangzhou, China) and 100 U/mL ampicillin, 100 mg/mL streptomycin in an incubator containing 5% CO 2 at 37°C (standard conditions).

Techniques: Concentration Assay, Cell Culture, Western Blot, Negative Control

Journal: Engineering in Life Sciences

Article Title: Overexpression of GRP78 enhances survival of CHO cells in response to serum deprivation and oxidative stress

doi: 10.1002/elsc.201500152

Figure Lengend Snippet: Glucose‐regulated protein 78 (GRP78) engineering enhanced yields of TfR‐Ab and improved cell viability. Chinese hamster ovary (CHO) and CHO modified by GRP78 (CHO‐GRP78) cells were seeded in 12‐well plates at a density of 2 × 105 cells per well and transiently transfected with pOptiVEC™‐TOPO®/TfR‐Ab, followed by culturing with SFM4CHO™ medium. (A) The concentration of TfR‐Ab in the supernatant was detected by ELISA assay. (B) The quality of TfR‐Ab in the supernatant was evaluated by its binding ability with TfR+ HepG2 cells using flow cytometry (FCM). The bar graphs represented FCM analysis of mean fluorescence index (MFI). (C) Bar graph represented FCM analysis of the percentage viability of CHO and CHO‐GRP78 cells. (D) The viable cell numbers of CHO and CHO‐GRP78 cells were counted after Trypan blue staining. Data were mean values ± SD, n > 3.*p < 0.05,**p < 0.01,***p < 0.001 versus negative control.

Article Snippet: The Chinese hamster ovary (CHO) cells, human stomach adenocarcinoma (AGS) cell line, and human hepatoma cell line HepG2 (China Center for Type Culture Collection, Wuhan, P. R. China) were cultured in Roswell Park Memorial Institute (RPMI) 1640 and DMEM medium (Gbico BRL, NY, USA) supplemented with 10% FBS (Sijiqing, Hangzhou, China) and 100 U/mL ampicillin, 100 mg/mL streptomycin in an incubator containing 5% CO 2 at 37°C (standard conditions).

Techniques: Modification, Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry, Fluorescence, Staining, Negative Control

Journal: Pharmaceuticals

Article Title: UCHL1 Promotes Gastric Cancer Progression by Regulating CIP2A Degradation

doi: 10.3390/ph18101468

Figure Lengend Snippet: UCHL1 knockdown suppresses cell growth in gastric cancer cells. ( A ) The mRNA expression of UCHL1 was analyzed in multiple gastric cancer cell lines using the ShinyThor database. ( B ) Western blot analysis of UCHL1 in MKN1 and SNU484 cells at 48 h after transfection with two different siRNAs. ( C ) The effect of UCHL1 knockdown on cell viability in MKN1 and SNU484 cells. Both cells were seeded in 96-well white bottom dishes and monitored over 3 days using a Cell Titer-Glo luminescence assay ( n = 5, normalized to day 0). The error bar represents the SD value. The two-sided Student’s t -test was used for statistical analysis (* p < 0.05). (ns, not significant; * p < 0.05, ** p < 0.001). ( D ) Colony formation of MKN1 and SNU484 gastric cancer cells following the silencing of UCHL1. Transfected with or without cells using siUCHL1s were incubated for at least 7 days. The quantitative data was analyzed by a two-sided Student’s t -test (** p < 0.001, *** p < 0.0001). ( E ) The effects of the UCHL1 knockdown on the potential of migration by using a wound healing assay. Cell migration was assessed in control and UCHL1 -silenced cells at each time point. (24, 48, and 72 h) ( F ) The effect of knockdown of UCHL1 on the potential of migratory and invasive ability in MKN1 and SNU484 cells. Transwell inserts were covered with Matrigel-coated membranes in the case of the invasion assay. The migratory potential was assessed after a 36 h incubation, and the invasive potential was assessed after a 24 h incubation.

Article Snippet: GC cell lines MKN1 and SNU484 were acquired from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and maintained with a Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: Knockdown, Expressing, Western Blot, Transfection, Luminescence Assay, Incubation, Migration, Wound Healing Assay, Control, Invasion Assay

Journal: Pharmaceuticals

Article Title: UCHL1 Promotes Gastric Cancer Progression by Regulating CIP2A Degradation

doi: 10.3390/ph18101468

Figure Lengend Snippet: UCHL1 interacts with and deubiquitinates CIP2A. ( A ) Lysates from MKN1 and SNU484 cells were lysed and immunoprecipitated with control mouse IgG or antibodies against CIP2A. Western blot analysis was then performed using antibodies against UCHL1 and CIP2A. ( B ) Lysates from HEK293T cells, which were transfected with HA-CIP2A and MYC-UCHL1 plasmids, were lysed and immunoprecipitated with HA-antibody–conjugated beads. ( C ) HA-CIP2A protein levels increased in a dose-dependent manner upon MYC-UCHL1 overexpression. HEK 293T cells were transfected with a fixed amount (1 μg) of HA-CIP2A and a gradient (from 0.3 to 3 μg) of MYC-UCHL1 constructs. Lysates of HEK293T cells were lysed and immunoblotted with the indicated antibodies. ( D ) Western blot analysis of MKN1 and SNU484 cells following the silencing of UCHL1. ( E ) Cycloheximide (CHX) chase assays were performed in cells transfected with Control or siUCHL1 to analyze the half-life of CIP2A. ( F ) Lysates from HEK293T cells, which were transfected with HA-CIP2A, MYC-UCHL1 WT, and FLAG-ubiquitin plasmids. After 12 h of MG132 treatment prior to lysis, the cell lysates were lysed and immunoprecipitated with HA-antibody-conjugated beads. Western blot analysis was then performed using antibodies against the tag. ( G ) Lysates from HEK293T cells, which were transfected with the same amount of MYC-UCHL1 and MYC-UCHL1 C90S plasmids, were lysed and immunoprecipitated with HA-antibodies-conjugated beads. Western blot analysis was then performed using antibodies as shown.

Article Snippet: GC cell lines MKN1 and SNU484 were acquired from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and maintained with a Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: Immunoprecipitation, Control, Western Blot, Transfection, Over Expression, Construct, Ubiquitin Proteomics, Lysis

Journal: Pharmaceuticals

Article Title: UCHL1 Promotes Gastric Cancer Progression by Regulating CIP2A Degradation

doi: 10.3390/ph18101468

Figure Lengend Snippet: UCHL1 regulates cycling D1 through the CIP2A-mediated signaling pathway. ( A ) Western blot analysis of MKN1 following the silencing of UCHL1. ( B ) Western blot analysis of SNU484 cells following the silencing of UCHL1. ( C ) Effects of the UCHL1 knockdown on cell cycle regulatory protein following 16 h nocodazole treatment in MKN1 and SNU484 cells, after which the medium was replaced with complete growth medium to allow a cell cycle re-entry. ( D ) Flow cytometric analysis of the G1 phase cell cycle arrest following UCHL1 knockdown in MKN1 and SNU484 cells. The error bar represents SD. The two-sided Student’s t -test was used for statistical analysis (* p < 0.05; n = 4).

Article Snippet: GC cell lines MKN1 and SNU484 were acquired from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and maintained with a Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: Western Blot, Knockdown

Journal: Pharmaceuticals

Article Title: UCHL1 Promotes Gastric Cancer Progression by Regulating CIP2A Degradation

doi: 10.3390/ph18101468

Figure Lengend Snippet: LDN-57444 treatment resulted in cell cycle arrest in gastric cancer cells. ( A ) MKN1 and SNU484 cells were treated with LDN-57444 for 48 h and immunoblotted with the indicated antibodies. ( B ) The inhibition of UCHL1 with LDN-57444 on cell viability in MKN1 and SNU484. The error bar represents SD. The two-sided Student’s t -test was used for statistical analysis (ns, not significant, * p < 0.05; n = 5, with normalization at day 0). ( C ) Effects of UCHL1 inhibition by LDN-57444 on cell proliferation in MKN1 and SNU484 cells. Cells were treated with or without LDN-57444 for at least 7 days. The error bar represents SD. The two-sided Student’s t -test was used for statistical analysis (** p < 0.05; n = 3). ( D ) Analyzed the G1 phase cell cycle arrest induced by a UCHL1 inhibitor, LDN-57444, using flow cytometric analysis. The error bar represents SD. The two-sided Student’s t -test was used for statistical analysis (** p < 0.01; n = 4).

Article Snippet: GC cell lines MKN1 and SNU484 were acquired from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and maintained with a Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: Inhibition