plasmids containing the puromycin-resistant gene specifically targeting cnot7  (Genechem)

Journal: FEBS Open Bio

Article Title: CNOT7 depletion reverses natural killer cell resistance by modulating the tumor immune microenvironment of hepatocellular carcinoma

doi: 10.1002/2211-5463.12836

Figure Lengend Snippet: Changes in CNOT7, STAT1, TGF‐β1 and IFN‐γ levels in patients with HCCBC. (A) Correlation of CNOT7 expression level on HCC patient survival. Red lines represent the high expression of CNOT7 , and blue lines represent low expression. (B) Quantitative data (mean ± SD) of ELISAs were shown. Multiple comparisons were made by the Student‐Newman‐Keuls q (SNK‐ q ) method. * P < 0.05, one‐way ANOVA. Adjacent: adjacent liver tissue; normal: healthy liver tissue; tumor: tumor tissue. (C) Immunohistochemical analysis of CNOT7 and STAT1 expression in HCCBC and cirrhotic liver tissues [original magnification, ×100 and ×400 (insets)]. Scale bars: 100 µm; 25 µm (insets). Cells with yellow‐brown staining are immune positive. Normal: cirrhotic liver tissue; tumor: tumor tissue. Values with different numbers of asterisks differ significantly ( P < 0.05)

Article Snippet: Four shRNAs were encoded by plasmids containing the puromycin‐resistant gene specifically targeting CNOT7 (shCNOT7 #1, 5′‐TACTAACAACATCTGGTAT‐3′; #2, 5′‐TGACTATCAATACCAACTA‐3′; #3, 5′‐TGACTATCAATACCAACTA‐3′; and #4, 5′‐GTGTAATGTAGACTTGTTA‐3′), and nonspecific shRNAs as a control (5′‐TTCTCCGAACGTGTCACGT‐3′) were purchased from GeneChem (Shanghai, China).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: FEBS Open Bio

Article Title: CNOT7 depletion reverses natural killer cell resistance by modulating the tumor immune microenvironment of hepatocellular carcinoma

doi: 10.1002/2211-5463.12836

Figure Lengend Snippet: CNOT7 and STAT1 expression levels in tumor and normal liver tissues. High, high expression; low, low expression; normal, healthy liver tissues; tumor, tumor tissues.

Article Snippet: Four shRNAs were encoded by plasmids containing the puromycin‐resistant gene specifically targeting CNOT7 (shCNOT7 #1, 5′‐TACTAACAACATCTGGTAT‐3′; #2, 5′‐TGACTATCAATACCAACTA‐3′; #3, 5′‐TGACTATCAATACCAACTA‐3′; and #4, 5′‐GTGTAATGTAGACTTGTTA‐3′), and nonspecific shRNAs as a control (5′‐TTCTCCGAACGTGTCACGT‐3′) were purchased from GeneChem (Shanghai, China).

Techniques: Expressing

Journal: FEBS Open Bio

Article Title: CNOT7 depletion reverses natural killer cell resistance by modulating the tumor immune microenvironment of hepatocellular carcinoma

doi: 10.1002/2211-5463.12836

Figure Lengend Snippet: Resistance of HCC cell lines to NK cells. (A) Four HCC cell lines were cocultured with or without NK‐92MI cells at 1 : 10 ratio for 12 h. MTT assay was applied for cell proliferation detection. Quantitative data (mean ± SD) of individual groups of cells were shown. Experiments were conducted in triplicate; * P < 0.05, Student’s t ‐test. Con, cultured without NK‐92MI cells; +NK, cocultured with NK‐92MI cells. (B) Quantitative real‐time PCR analysis of transcript levels of CNOT7 and STAT1 in the four HCC cell lines and L02 cells. The GAPDH mRNA level was used for normalization. Transcript levels in L02 cells were used as reference. CNOT7 and STAT1 proteins were determined by western blotting, using GAPDH for normalization. Data were expressed as the mean ± SD of individual groups of cells from three separate experiments. Multiple comparisons were made by the SNK‐ q method. * P < 0.05, one‐way ANOVA. (C) TGF‐β1 production in the four HCC cell lines and L02 cells was analyzed by ELISA. Transcript levels of TGF‐β1 were detected by quantitative real‐time PCR. Data were expressed as mean ± SD of individual groups of cells from three separate experiments. Multiple comparisons were made by the SNK‐ q method. * P < 0.05, one‐way ANOVA. Values with different numbers of asterisks differ significantly ( P < 0.05).

Article Snippet: Four shRNAs were encoded by plasmids containing the puromycin‐resistant gene specifically targeting CNOT7 (shCNOT7 #1, 5′‐TACTAACAACATCTGGTAT‐3′; #2, 5′‐TGACTATCAATACCAACTA‐3′; #3, 5′‐TGACTATCAATACCAACTA‐3′; and #4, 5′‐GTGTAATGTAGACTTGTTA‐3′), and nonspecific shRNAs as a control (5′‐TTCTCCGAACGTGTCACGT‐3′) were purchased from GeneChem (Shanghai, China).

Techniques: MTT Assay, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: FEBS Open Bio

Article Title: CNOT7 depletion reverses natural killer cell resistance by modulating the tumor immune microenvironment of hepatocellular carcinoma

doi: 10.1002/2211-5463.12836

Figure Lengend Snippet: Modulation of CNOT7 expression alters TGF‐β1 secretion in HCC and IFN‐γ production in NK cells. (A) TGF‐β1 production in HepG2, HepG2 ns and HepG2 shCNOT7 cells was analyzed by ELISA. Transcript levels of TGF‐β1 were detected by quantitative real‐time PCR. Data were expressed as the mean ± SD of individual groups of cells from three separate experiments. Multiple comparisons were made by the SNK‐ q method. * P < 0.05, one‐way ANOVA. (B) IFN‐γ production by NK‐92MI cells was determined by ELISA. NK ns , cultured in HepG2 ns cell supernatant; NK shCNOT7 , cultured in HepG2 shCNOT7 cell supernatant. Data were expressed as mean ± SD of individual groups of cells from three separate experiments. Multiple comparisons were made by the SNK‐ q method. * P < 0.05, one‐way ANOVA. (C) STAT1 and NF‐κB p65 protein levels were analyzed by western blotting, using GAPDH for normalization. Data were expressed as mean ± SD of individual groups of cells from three separate experiments. Multiple comparisons were made by the SNK‐ q method. * P < 0.05, one‐way ANOVA. (D) STAT1 and NF‐κB p65 interaction. Extracts from HepG2 cells transfected with shCNOT7 and nonspecific shRNA were immunoprecipitated with IgG or anti‐STAT1 antibody. Immunoprecipitates were then analyzed by immunoblotting with anti‐p65 antibody. 1: HepG2 ns ; 2: HepG2 shCNOT7 . The experiments were conducted in triplicate.

Article Snippet: Four shRNAs were encoded by plasmids containing the puromycin‐resistant gene specifically targeting CNOT7 (shCNOT7 #1, 5′‐TACTAACAACATCTGGTAT‐3′; #2, 5′‐TGACTATCAATACCAACTA‐3′; #3, 5′‐TGACTATCAATACCAACTA‐3′; and #4, 5′‐GTGTAATGTAGACTTGTTA‐3′), and nonspecific shRNAs as a control (5′‐TTCTCCGAACGTGTCACGT‐3′) were purchased from GeneChem (Shanghai, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Cell Culture, Western Blot, Transfection, shRNA, Immunoprecipitation

Journal: FEBS Open Bio

Article Title: CNOT7 depletion reverses natural killer cell resistance by modulating the tumor immune microenvironment of hepatocellular carcinoma

doi: 10.1002/2211-5463.12836

Figure Lengend Snippet: CNOT7 protein depletion reverses NK cell resistance in HepG2 cells. (A) HepG2, HepG2 ns and HepG2 shCNOT7 cells were cocultured with or without NK‐92MI cells at a 1 : 10 ratio for 12 h. MTT assay was applied for cell proliferation detection. Quantitative data (mean ± SD) of individual groups of cells were shown. Experiments were conducted in triplicate; * P < 0.05, Student’s t ‐test. (B) Cytotoxicity was measured at various E:T ratios by flow cytometry and CFSE/7‐AAD staining. The right upper quadrant represents apoptotic cells. The experiments were performed in triplicate. Data were expressed as mean ± SD of individual groups of cells from three separate experiments. Multiple comparisons were made by the SNK‐ q method. * P < 0.05, one‐way ANOVA.

Article Snippet: Four shRNAs were encoded by plasmids containing the puromycin‐resistant gene specifically targeting CNOT7 (shCNOT7 #1, 5′‐TACTAACAACATCTGGTAT‐3′; #2, 5′‐TGACTATCAATACCAACTA‐3′; #3, 5′‐TGACTATCAATACCAACTA‐3′; and #4, 5′‐GTGTAATGTAGACTTGTTA‐3′), and nonspecific shRNAs as a control (5′‐TTCTCCGAACGTGTCACGT‐3′) were purchased from GeneChem (Shanghai, China).

Techniques: MTT Assay, Flow Cytometry, Staining

Journal: FEBS Open Bio

Article Title: CNOT7 depletion reverses natural killer cell resistance by modulating the tumor immune microenvironment of hepatocellular carcinoma

doi: 10.1002/2211-5463.12836

Figure Lengend Snippet: NK cell cytotoxic immune functions are altered upon coculture with CNOT7‐deficient HepG2 cells. (A) To evaluate the cytotoxic immune function, we cocultured NK‐92MI cells with target tumor cells at a 1 : 10 ratio for 3.5 h. CD107a expression of NK‐92MI cells was analyzed by flow cytometry. The upper quadrant indicates that cells showed CD107a expression. Data were expressed as mean ± SD of individual groups of cells from three separate experiments. Multiple comparisons were made by the SNK‐ q method. * P < 0.05, one‐way ANOVA. (B) HepG2 shCNOT7 and HepG2 ns cells were cocultured with or without NK‐92MI cells at a 1 : 10 ratio for 12 h. Caspase‐3 expression was measured by western blotting and quantitative real‐time PCR. Plus signs (+) indicate cultured with NK‐92MI cells; minus signs (–) indicate cultured without NK‐92MI cells. Quantitative data (mean ± SD) of individual groups of cells were shown. Experiments were conducted in triplicate; * P < 0.05, Student’s t ‐test. Values with different numbers of asterisks differ significantly ( P < 0.05)

Article Snippet: Four shRNAs were encoded by plasmids containing the puromycin‐resistant gene specifically targeting CNOT7 (shCNOT7 #1, 5′‐TACTAACAACATCTGGTAT‐3′; #2, 5′‐TGACTATCAATACCAACTA‐3′; #3, 5′‐TGACTATCAATACCAACTA‐3′; and #4, 5′‐GTGTAATGTAGACTTGTTA‐3′), and nonspecific shRNAs as a control (5′‐TTCTCCGAACGTGTCACGT‐3′) were purchased from GeneChem (Shanghai, China).

Techniques: Expressing, Flow Cytometry, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture