wi38  (ATCC)


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    Structured Review

    ATCC wi38
    Cytotoxicity of Micrococcus lylae MW407006 echinenone ( a ) and nano-echinenone ( b ) on <t>WI38,</t> Vero, Caco-2, and Hep-G2.
    Wi38, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wi38/product/ATCC
    Average 97 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    wi38 - by Bioz Stars, 2022-12
    97/100 stars

    Images

    1) Product Images from "Micrococcus lylae MW407006 Pigment: Production, Optimization, Nano-Pigment Synthesis, and Biological Activities"

    Article Title: Micrococcus lylae MW407006 Pigment: Production, Optimization, Nano-Pigment Synthesis, and Biological Activities

    Journal: Biology

    doi: 10.3390/biology11081171

    Cytotoxicity of Micrococcus lylae MW407006 echinenone ( a ) and nano-echinenone ( b ) on WI38, Vero, Caco-2, and Hep-G2.
    Figure Legend Snippet: Cytotoxicity of Micrococcus lylae MW407006 echinenone ( a ) and nano-echinenone ( b ) on WI38, Vero, Caco-2, and Hep-G2.

    Techniques Used:

    2) Product Images from "Quantitative Comparison of HSF1 Activators"

    Article Title: Quantitative Comparison of HSF1 Activators

    Journal: Molecular Biotechnology

    doi: 10.1007/s12033-022-00467-3

    HSF1 activation with geldanamycin. X8-12H and X9-12H ( a and b ) or WI38-rep2-12H ( d ) cells were incubated with increasing concentrations of geldanamycin and Nluc activity was measured after 6 h ( a ) or 24 h ( b and d ) incubation time. Y -axis shows relative luciferase activity as Nluc signal of treated cells divided by untreated control cells. For Western blot analysis, ( c ) HeLa (left) and WI38 (right) cells were incubated with different concentrations of geldanamycin and whole cell protein extracts were taken after 6 h (upper panel) or 24 h (lower panel). Control cells were untreated. Western blot was performed with primary antibodies against Hsp72 and GAPDH. For luciferase experiments, all values show means of at least three independent experiments, and error bars indicate SEM
    Figure Legend Snippet: HSF1 activation with geldanamycin. X8-12H and X9-12H ( a and b ) or WI38-rep2-12H ( d ) cells were incubated with increasing concentrations of geldanamycin and Nluc activity was measured after 6 h ( a ) or 24 h ( b and d ) incubation time. Y -axis shows relative luciferase activity as Nluc signal of treated cells divided by untreated control cells. For Western blot analysis, ( c ) HeLa (left) and WI38 (right) cells were incubated with different concentrations of geldanamycin and whole cell protein extracts were taken after 6 h (upper panel) or 24 h (lower panel). Control cells were untreated. Western blot was performed with primary antibodies against Hsp72 and GAPDH. For luciferase experiments, all values show means of at least three independent experiments, and error bars indicate SEM

    Techniques Used: Activation Assay, Incubation, Activity Assay, Luciferase, Western Blot

    Analysis of potential HSR co-inducers. X9-12H cells were treated with different concentrations of geranylgeranylacetone (GGA, a ), arimoclomol ( c ), and BGP-15 ( d ) for 1 h before a 1 h heat treatment at 42 °C was performed (HS) followed by 24 h recovery at 37 °C or a continuous cultivation at 37 °C (37 °C) for 24 h before luciferase and fluorescence measurement ( a , c, and d ). Left Y -axes ( a , c and d ) show relative luciferase activity as Nluc signal compared to cells without co-inducer. Right X -axes ( a , c and d ) show viability as relative fluorescence of resazurin compared to cells without co-inducer. All values are means of at least three independent experiments, error bars indicate SEM, Student’s t test was applied to estimate significance (see text). For Western blot analysis ( b ), WI38 cells were used and treated with GGA under identical conditions as in ( a ). Primary antibodies targeting Hsp72, Hsp25, Hsp90, and GAPDH were used
    Figure Legend Snippet: Analysis of potential HSR co-inducers. X9-12H cells were treated with different concentrations of geranylgeranylacetone (GGA, a ), arimoclomol ( c ), and BGP-15 ( d ) for 1 h before a 1 h heat treatment at 42 °C was performed (HS) followed by 24 h recovery at 37 °C or a continuous cultivation at 37 °C (37 °C) for 24 h before luciferase and fluorescence measurement ( a , c, and d ). Left Y -axes ( a , c and d ) show relative luciferase activity as Nluc signal compared to cells without co-inducer. Right X -axes ( a , c and d ) show viability as relative fluorescence of resazurin compared to cells without co-inducer. All values are means of at least three independent experiments, error bars indicate SEM, Student’s t test was applied to estimate significance (see text). For Western blot analysis ( b ), WI38 cells were used and treated with GGA under identical conditions as in ( a ). Primary antibodies targeting Hsp72, Hsp25, Hsp90, and GAPDH were used

    Techniques Used: Luciferase, Fluorescence, Activity Assay, Western Blot

    3) Product Images from "Small-Molecule Ferroptotic Agents with Potential to Selectively Target Cancer Stem Cells"

    Article Title: Small-Molecule Ferroptotic Agents with Potential to Selectively Target Cancer Stem Cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-42251-5

    Drug sensitivity in various cell types. Cells were treated as indicated and viability determined by methylene blue staining. Bars represent averages and associated standard deviations. ( A ) Drug sensitivity in cancer cell lines differing in p53 status. Parental HCT116 with wild-type p53 were compared to p53-null HCT116 cells. Cells were exposed to 10 μM erastin, 10 μM compound 4 , or 300 μM sulfasalazine for 3 days before viability was determined. 4 was tested in parallel on NCI-H522 to confirm activity. 4 toxicity was also assessed in parental HT1080 cells with wild-type p53, and cells overexpressing a dominant negative p53 fragment, GSE56. Cells were exposed to 10 μM compound 4 in the presence or absence of βME and viability determined. ( B ) Effect of Liproxstatin. NCI-H522 and HCT116 cells were exposed to 10 μM of either compound 4 or Erastin in the presence or absence of 2.5 μM liproxstatin. Viability was determined 3 days later. Excess glutamate (5 mM) had no effect on cell viability. ( C ) NCI-H522 cells depend on external cystine. Cells were grown in medium lacking cystine supplemented with dialyzed fetal bovine serum for 3 days. Additional compounds were added and viability determined. ( D ) Dose response of external cystine. NCI-H522 cells were grown for 3 days in the indicated concentrations of cystine and viability determined. ( E ) Effects of 1 and analogues on cell viability. The cell types indicated were exposed to the indicated analogues for 2 days and viability determined. MDAH041 are a spontaneously immortalized p53-null human fibroblast cell line 51 , 52 . NCI-H522 were exposed to classical chemotherapy drugs for 2 days (HU: 2 mM, TX: 10 μM, AD: 0.2 μg/ml). ( F ) Effects of various compounds on WI38, MDAH041 and retinal pigmented epithelial (RPE) cells. Cells were exposed 4 , sulfasalazine (300 μM) or erastin (10 μM) 3 days and viability determined. (SSZ: sulfasalazine; βME: β-mercaptoethanol; LIP: liproxstatin; ERA: erastin; GLU: glutamate; CPO: ciclopirox olamine; TRO: trolox; FER: ferrostatin).
    Figure Legend Snippet: Drug sensitivity in various cell types. Cells were treated as indicated and viability determined by methylene blue staining. Bars represent averages and associated standard deviations. ( A ) Drug sensitivity in cancer cell lines differing in p53 status. Parental HCT116 with wild-type p53 were compared to p53-null HCT116 cells. Cells were exposed to 10 μM erastin, 10 μM compound 4 , or 300 μM sulfasalazine for 3 days before viability was determined. 4 was tested in parallel on NCI-H522 to confirm activity. 4 toxicity was also assessed in parental HT1080 cells with wild-type p53, and cells overexpressing a dominant negative p53 fragment, GSE56. Cells were exposed to 10 μM compound 4 in the presence or absence of βME and viability determined. ( B ) Effect of Liproxstatin. NCI-H522 and HCT116 cells were exposed to 10 μM of either compound 4 or Erastin in the presence or absence of 2.5 μM liproxstatin. Viability was determined 3 days later. Excess glutamate (5 mM) had no effect on cell viability. ( C ) NCI-H522 cells depend on external cystine. Cells were grown in medium lacking cystine supplemented with dialyzed fetal bovine serum for 3 days. Additional compounds were added and viability determined. ( D ) Dose response of external cystine. NCI-H522 cells were grown for 3 days in the indicated concentrations of cystine and viability determined. ( E ) Effects of 1 and analogues on cell viability. The cell types indicated were exposed to the indicated analogues for 2 days and viability determined. MDAH041 are a spontaneously immortalized p53-null human fibroblast cell line 51 , 52 . NCI-H522 were exposed to classical chemotherapy drugs for 2 days (HU: 2 mM, TX: 10 μM, AD: 0.2 μg/ml). ( F ) Effects of various compounds on WI38, MDAH041 and retinal pigmented epithelial (RPE) cells. Cells were exposed 4 , sulfasalazine (300 μM) or erastin (10 μM) 3 days and viability determined. (SSZ: sulfasalazine; βME: β-mercaptoethanol; LIP: liproxstatin; ERA: erastin; GLU: glutamate; CPO: ciclopirox olamine; TRO: trolox; FER: ferrostatin).

    Techniques Used: Staining, Activity Assay, Dominant Negative Mutation

    4) Product Images from "Amentoflavone Induces Autophagy and Modulates p53"

    Article Title: Amentoflavone Induces Autophagy and Modulates p53

    Journal: Cell Journal (Yakhteh)

    doi: 10.22074/cellj.2019.5717

    Effect of amentoflavone on cell viability. A. Amentoflavone was treated to A549 cells and B. Amentoflavone was treated to WI38 cells. The cells were treated with amentoflavone at the indicated concentration and the cell viability was determined by MTT assay after 48 hours. Data are presented as the mean of values ± SD obtained from three independent experiments. The level of significance was identified statistically (*; P
    Figure Legend Snippet: Effect of amentoflavone on cell viability. A. Amentoflavone was treated to A549 cells and B. Amentoflavone was treated to WI38 cells. The cells were treated with amentoflavone at the indicated concentration and the cell viability was determined by MTT assay after 48 hours. Data are presented as the mean of values ± SD obtained from three independent experiments. The level of significance was identified statistically (*; P

    Techniques Used: Concentration Assay, MTT Assay

    5) Product Images from "CB002, a novel p53 tumor suppressor pathway-restoring small molecule induces tumor cell death through the pro-apoptotic protein NOXA"

    Article Title: CB002, a novel p53 tumor suppressor pathway-restoring small molecule induces tumor cell death through the pro-apoptotic protein NOXA

    Journal: Cell Cycle

    doi: 10.1080/15384101.2017.1346762

    CB002 treatment of 48 hrs increases apoptotic cells as indicated by the sub-G1 content in SW480 cancer cells but not in normal WI38 cells. Two-way ANOVA statistical analysis, p ≤ 0.001 against DMSO vehicle control. Three replicates were performed, and a representative histogram is shown.
    Figure Legend Snippet: CB002 treatment of 48 hrs increases apoptotic cells as indicated by the sub-G1 content in SW480 cancer cells but not in normal WI38 cells. Two-way ANOVA statistical analysis, p ≤ 0.001 against DMSO vehicle control. Three replicates were performed, and a representative histogram is shown.

    Techniques Used:

    6) Product Images from "Merkel cell polyomavirus small T antigen induces genome instability by E3 ubiquitin ligase targeting"

    Article Title: Merkel cell polyomavirus small T antigen induces genome instability by E3 ubiquitin ligase targeting

    Journal: Oncogene

    doi: 10.1038/onc.2017.277

    sT expression results in increased chromosomal instability in WI38 cells. Karyotyping and G-banding analysis were performed on cells at 2 days post transduction with indicated lentivirus constructs. Representative karyotypes are shown for each condition, and chromosome breaks are indicated by red arrows. The greatest amount of breakage was seen in cells that express WT MCV sT, which is also missing an X chromosome.
    Figure Legend Snippet: sT expression results in increased chromosomal instability in WI38 cells. Karyotyping and G-banding analysis were performed on cells at 2 days post transduction with indicated lentivirus constructs. Representative karyotypes are shown for each condition, and chromosome breaks are indicated by red arrows. The greatest amount of breakage was seen in cells that express WT MCV sT, which is also missing an X chromosome.

    Techniques Used: Expressing, Transduction, Construct

    sT increases aneuploidy and number of centrosomes in human diploid WI38 cells. ( a ) Ploidy analysis. Cells transiently transduced with WT sT, L142A sT, LSDm sT or empty expression vectors were stained for DNA content by propidium iodide and analyzed by flow cytometry. Representative cell cycle profiles are shown for cells isolated at 2 days post transduction, with 2N, 4N and > 4N populations annotated. The population of cells with > 4N DNA content is magnified as respective inserts, and is most elevated for WT sT- and L142A sT-expressing cells. ( b ) Expression of sT. Immunoblotting was simultaneously performed on a portion of the cells used for flow cytometry to validate sT expression using mouse monoclonal, anti-sT antibody (CM8E6). Results are representative of expression at 2 days post transduction from four independent experiments. ( c ) Fold changes in the percentage of cells with > 4N content were calculated for 2, 5, 7 and 10 days after transduction in each condition. A single asterisk denotes a significant increase over empty vector control cells ( P
    Figure Legend Snippet: sT increases aneuploidy and number of centrosomes in human diploid WI38 cells. ( a ) Ploidy analysis. Cells transiently transduced with WT sT, L142A sT, LSDm sT or empty expression vectors were stained for DNA content by propidium iodide and analyzed by flow cytometry. Representative cell cycle profiles are shown for cells isolated at 2 days post transduction, with 2N, 4N and > 4N populations annotated. The population of cells with > 4N DNA content is magnified as respective inserts, and is most elevated for WT sT- and L142A sT-expressing cells. ( b ) Expression of sT. Immunoblotting was simultaneously performed on a portion of the cells used for flow cytometry to validate sT expression using mouse monoclonal, anti-sT antibody (CM8E6). Results are representative of expression at 2 days post transduction from four independent experiments. ( c ) Fold changes in the percentage of cells with > 4N content were calculated for 2, 5, 7 and 10 days after transduction in each condition. A single asterisk denotes a significant increase over empty vector control cells ( P

    Techniques Used: Transduction, Expressing, Staining, Flow Cytometry, Cytometry, Isolation, Plasmid Preparation

    7) Product Images from "Histone deacetylase inhibitor thailandepsin-A activates Notch signaling and suppresses neuroendocrine cancer cell growth in vivo"

    Article Title: Histone deacetylase inhibitor thailandepsin-A activates Notch signaling and suppresses neuroendocrine cancer cell growth in vivo

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19993

    Effect of thailandepsin A (TDP-A) on neuroendocrine (NE) cancer cell viability (A) The IC 50 value for TDP-A treated BON (pancreatic neuroendocrine), MZ (medullary thyroid), and TT (medullary thyroid) cancer cells was determined to be at low nanomolar concentrations (4.6, 4.5, and 6.0 nM, respectively) measured with the MTT assay after treatment for 48 hrs, while not affecting the viability of WI38 cells (lung fibroblasts). (B-D) TDP-A treatment reduced cell viability in a dose and time dependent manner in all three cell lines. Data is presented as mean percentage viability ± SEM normalized to DMSO vehicle control.
    Figure Legend Snippet: Effect of thailandepsin A (TDP-A) on neuroendocrine (NE) cancer cell viability (A) The IC 50 value for TDP-A treated BON (pancreatic neuroendocrine), MZ (medullary thyroid), and TT (medullary thyroid) cancer cells was determined to be at low nanomolar concentrations (4.6, 4.5, and 6.0 nM, respectively) measured with the MTT assay after treatment for 48 hrs, while not affecting the viability of WI38 cells (lung fibroblasts). (B-D) TDP-A treatment reduced cell viability in a dose and time dependent manner in all three cell lines. Data is presented as mean percentage viability ± SEM normalized to DMSO vehicle control.

    Techniques Used: MTT Assay

    8) Product Images from "Targeting the tumor-promoting microenvironment in MET-amplified NSCLC cells with a novel inhibitor of pro-HGF activation"

    Article Title: Targeting the tumor-promoting microenvironment in MET-amplified NSCLC cells with a novel inhibitor of pro-HGF activation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18260

    Inhibition of pro-HGF activation overcomes fibroblast-mediated resistance to MET tyrosine kinase inhibition ( A) EBC-1 cells were treated with JNJ38877605 (25 nM) alone or in the presence of conditioned medium (CM) from WI38 fibroblasts (FIB). CM was also prepared from fibroblasts with silenced HGF (HGF −/− FIB), or from fibroblasts cultured with HGF neutralizing antibody (α-HGF Ab) or SRI31215 (10 μM) as indicated. Cell viability was determined by CellTiter Glo ® 72 h after treatment. (B) H1993 cells were treated with JNJ38877605 (25 nM), fibroblast CM (FIB) and SRI31215 (10 μM) as indicated and cell viability was determined after 72 h. (C) Serum-starved EBC-1 cells were treated with JNJ38877605, recombinant HGF (100 nM), FIB and SRI31215 (10 μM) as indicated for 6 hours. Cell lysates were analyzed by immunoblotting for phospho- and total MET, EGFR, Gab1, AKT and ERK 1/2. *, p
    Figure Legend Snippet: Inhibition of pro-HGF activation overcomes fibroblast-mediated resistance to MET tyrosine kinase inhibition ( A) EBC-1 cells were treated with JNJ38877605 (25 nM) alone or in the presence of conditioned medium (CM) from WI38 fibroblasts (FIB). CM was also prepared from fibroblasts with silenced HGF (HGF −/− FIB), or from fibroblasts cultured with HGF neutralizing antibody (α-HGF Ab) or SRI31215 (10 μM) as indicated. Cell viability was determined by CellTiter Glo ® 72 h after treatment. (B) H1993 cells were treated with JNJ38877605 (25 nM), fibroblast CM (FIB) and SRI31215 (10 μM) as indicated and cell viability was determined after 72 h. (C) Serum-starved EBC-1 cells were treated with JNJ38877605, recombinant HGF (100 nM), FIB and SRI31215 (10 μM) as indicated for 6 hours. Cell lysates were analyzed by immunoblotting for phospho- and total MET, EGFR, Gab1, AKT and ERK 1/2. *, p

    Techniques Used: Inhibition, Activation Assay, Cell Culture, Recombinant

    9) Product Images from "Mael is essential for cancer cell survival and tumorigenesis through protection of genetic integrity"

    Article Title: Mael is essential for cancer cell survival and tumorigenesis through protection of genetic integrity

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13756

    Mael is essential for Myc/Ras-induced transformation and suppression of oncogenic Ras-induced senescence A, B . p53 −/− MEFs were co-transfected with Mael and Myc/Ras or Ras. Transformed colonies were visualized by crystal violet staining, and the colonies were counted in triplicates and the average number of colonies was calculated. C . MAEL protein expression was compared in MEF and Myc/Ras transformed MEF. D . Effects of mMael depletion on the viability of p53 −/− MEFs and their transformed cells were tested using crystal violet staining. E, F . Mael protein expression and cellular responses to siRNA transfection were compared in non-transformed normal human WI38 cells and WI38 cells transformed with SV40Tag. G . β-galactosidase staining was performed to verify the effects of Mael on cellular senescence induced by infection with H-RasV12- expressing retrovirus in BJ-T cells.
    Figure Legend Snippet: Mael is essential for Myc/Ras-induced transformation and suppression of oncogenic Ras-induced senescence A, B . p53 −/− MEFs were co-transfected with Mael and Myc/Ras or Ras. Transformed colonies were visualized by crystal violet staining, and the colonies were counted in triplicates and the average number of colonies was calculated. C . MAEL protein expression was compared in MEF and Myc/Ras transformed MEF. D . Effects of mMael depletion on the viability of p53 −/− MEFs and their transformed cells were tested using crystal violet staining. E, F . Mael protein expression and cellular responses to siRNA transfection were compared in non-transformed normal human WI38 cells and WI38 cells transformed with SV40Tag. G . β-galactosidase staining was performed to verify the effects of Mael on cellular senescence induced by infection with H-RasV12- expressing retrovirus in BJ-T cells.

    Techniques Used: Transformation Assay, Transfection, Staining, Expressing, Infection

    10) Product Images from "Activated hepatic stellate cells play pivotal roles in hepatocellular carcinoma cell chemoresistance and migration in multicellular tumor spheroids"

    Article Title: Activated hepatic stellate cells play pivotal roles in hepatocellular carcinoma cell chemoresistance and migration in multicellular tumor spheroids

    Journal: Scientific Reports

    doi: 10.1038/srep36750

    HSCs promote chemoresistance and compactness in multicellular tumor spheroids (MCTS). ( A,B ) Drug sensitivity was measured by EthD-1 (dead cell marker) staining in HepG2 spheroids ( A ) and Huh7 spheroids ( B ), which were co-cultured with or without LX2, WI38, and HUVECs and treated with 10 μM sorafenib for 4 days (upper panel). The intensity of EthD-1 was analyzed and values were normalized to control (0.01% DMSO) (lower panel). Data are shown as means ± SD from three independent experiments with duplicates. ( C ) HepG2 spheroids with or without LX2, WI38, and HUVECs at a 1:1 ratio were treated with 10 μM cisplatin for 4 days (upper panel). Intensity of EthD-1 was examined and values were normalized to control (0.01% DMSO) (lower panel). Data are shown as means ± SD from three independent experiments with duplicates. ( D ) HepG2 spheroids alone or with LX2 or WI38 were treated with 10 μM doxorubicin for the indicated number of hr. The penetration of doxorubicin into the spheroids was measured with LSM with various confocal stacks. ( E ) 10 μM of doxorubicin was treated in HepG2 spheroids alone or with LX2 or WI38 for indicated time. Absorbance of doxorubicin, which was penetrated in each spheroid, was examined. Values were normalized to 1 hr. Data are shown as means ± SD from two independent experiment with duplicate. *P
    Figure Legend Snippet: HSCs promote chemoresistance and compactness in multicellular tumor spheroids (MCTS). ( A,B ) Drug sensitivity was measured by EthD-1 (dead cell marker) staining in HepG2 spheroids ( A ) and Huh7 spheroids ( B ), which were co-cultured with or without LX2, WI38, and HUVECs and treated with 10 μM sorafenib for 4 days (upper panel). The intensity of EthD-1 was analyzed and values were normalized to control (0.01% DMSO) (lower panel). Data are shown as means ± SD from three independent experiments with duplicates. ( C ) HepG2 spheroids with or without LX2, WI38, and HUVECs at a 1:1 ratio were treated with 10 μM cisplatin for 4 days (upper panel). Intensity of EthD-1 was examined and values were normalized to control (0.01% DMSO) (lower panel). Data are shown as means ± SD from three independent experiments with duplicates. ( D ) HepG2 spheroids alone or with LX2 or WI38 were treated with 10 μM doxorubicin for the indicated number of hr. The penetration of doxorubicin into the spheroids was measured with LSM with various confocal stacks. ( E ) 10 μM of doxorubicin was treated in HepG2 spheroids alone or with LX2 or WI38 for indicated time. Absorbance of doxorubicin, which was penetrated in each spheroid, was examined. Values were normalized to 1 hr. Data are shown as means ± SD from two independent experiment with duplicate. *P

    Techniques Used: Ethidium Homodimer Assay, Marker, Staining, Cell Culture

    Collagen 1A1 expression is increased by the interaction between HSCs and HCC cells in MCTS. HepG2 and PLC/PRF/5 spheroids were cultured alone or with LX2, WI38, and HUVEC stromal cells at 1:1 ratio for 3 days. After 3 days, mRNA expression in each spheroid was evaluated by ( A ) RT-PCR and ( B ) real-time PCR using collagen and non-collagen-related ECM primers. Values were normalized to GAPDH. Data are shown as means ± SD from two independent experiments with duplicates.
    Figure Legend Snippet: Collagen 1A1 expression is increased by the interaction between HSCs and HCC cells in MCTS. HepG2 and PLC/PRF/5 spheroids were cultured alone or with LX2, WI38, and HUVEC stromal cells at 1:1 ratio for 3 days. After 3 days, mRNA expression in each spheroid was evaluated by ( A ) RT-PCR and ( B ) real-time PCR using collagen and non-collagen-related ECM primers. Values were normalized to GAPDH. Data are shown as means ± SD from two independent experiments with duplicates.

    Techniques Used: Expressing, Planar Chromatography, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Activated HSCs possess a migration capacity as a result of increased MMP9 in HCC-MCTS. ( A ) HepG2 alone and co-culture spheroids with LX2 cells, which is stained by DiO, WI38 cells stained by DiI, and HUVECs stained by DiD were cultured for 3 days. After spheroid formation, each spheroid was transferred to a collagen I coated plate and cultivated for 4 days. The cells were stained with Hoechst33342 after fixation and the images were obtained (left). The distance from surface of spheroid to boundary of stretched cells was measured (right). ( B,C ) HepG2 and PLC/PRF/5 spheroids were cultured alone or with LX2, WI38, and HUVEC stromal cells at a 1:1 ratio for 3 days. After 3 days, mRNA expression in each spheroid was evaluated by ( B ) RT-PCR and ( C ) real-time PCR using MMP and cadherin primers. Values were normalized to GAPDH. ( D ) LX2 cells were transfected with siCont and siMMP9 and its efficacy was examined by real-time PCR. ( E ) HepG2 alone and co-culture spheroids with LX2-siCont or LX2-siMMP9, which were stained by DiO, were cultured for 3 days. After 3 days, each spheroid was transferred to a collagen I coated plate and cultivated for 4 days. The cells were fixed and stained with Hoechst33342. The images were obtained using the same method (left). The distance from surface of spheroid to boundary of stretched cells was measured (right). Data represent the mean values ± SD from three independent experiments. **P
    Figure Legend Snippet: Activated HSCs possess a migration capacity as a result of increased MMP9 in HCC-MCTS. ( A ) HepG2 alone and co-culture spheroids with LX2 cells, which is stained by DiO, WI38 cells stained by DiI, and HUVECs stained by DiD were cultured for 3 days. After spheroid formation, each spheroid was transferred to a collagen I coated plate and cultivated for 4 days. The cells were stained with Hoechst33342 after fixation and the images were obtained (left). The distance from surface of spheroid to boundary of stretched cells was measured (right). ( B,C ) HepG2 and PLC/PRF/5 spheroids were cultured alone or with LX2, WI38, and HUVEC stromal cells at a 1:1 ratio for 3 days. After 3 days, mRNA expression in each spheroid was evaluated by ( B ) RT-PCR and ( C ) real-time PCR using MMP and cadherin primers. Values were normalized to GAPDH. ( D ) LX2 cells were transfected with siCont and siMMP9 and its efficacy was examined by real-time PCR. ( E ) HepG2 alone and co-culture spheroids with LX2-siCont or LX2-siMMP9, which were stained by DiO, were cultured for 3 days. After 3 days, each spheroid was transferred to a collagen I coated plate and cultivated for 4 days. The cells were fixed and stained with Hoechst33342. The images were obtained using the same method (left). The distance from surface of spheroid to boundary of stretched cells was measured (right). Data represent the mean values ± SD from three independent experiments. **P

    Techniques Used: Migration, Co-Culture Assay, Staining, Cell Culture, Planar Chromatography, Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Transfection

    Human hepatic stellate cells (HSCs; LX2) enhance the compactness of loosely aggregated spheroids from HCC cell lines and primary HCC cells. ( A ) HepG2 and PLC/PRF/5 cells, which formed loosely aggregated spheroids alone, were co-cultured with stromal cells (LX2, WI38, and HUVECs) at a 1:1 ratio for 3 days (upper panel). To calculate the volume of spheroids, their long and short diameter were examined (lower panel). ( B ) 118965, which is a primary HCC cell line that formed loosely aggregated spheroids alone, were co-cultured with LX2, WI38, and HUVEC cells at a 1:1 ratio for 3 days (upper panel). Volume of spheroids were analyzed (lower panel). All bright field images of spheroids were obtained using the Operetta ® High Content Screening System with a 10× objective. Data are shown as means ± SD from three independent experiments with triplicate. **P
    Figure Legend Snippet: Human hepatic stellate cells (HSCs; LX2) enhance the compactness of loosely aggregated spheroids from HCC cell lines and primary HCC cells. ( A ) HepG2 and PLC/PRF/5 cells, which formed loosely aggregated spheroids alone, were co-cultured with stromal cells (LX2, WI38, and HUVECs) at a 1:1 ratio for 3 days (upper panel). To calculate the volume of spheroids, their long and short diameter were examined (lower panel). ( B ) 118965, which is a primary HCC cell line that formed loosely aggregated spheroids alone, were co-cultured with LX2, WI38, and HUVEC cells at a 1:1 ratio for 3 days (upper panel). Volume of spheroids were analyzed (lower panel). All bright field images of spheroids were obtained using the Operetta ® High Content Screening System with a 10× objective. Data are shown as means ± SD from three independent experiments with triplicate. **P

    Techniques Used: Planar Chromatography, Cell Culture, High Content Screening

    11) Product Images from "Induction of IL-25 secretion from tumour-associated fibroblasts suppresses mammary tumour metastasis"

    Article Title: Induction of IL-25 secretion from tumour-associated fibroblasts suppresses mammary tumour metastasis

    Journal: Nature Communications

    doi: 10.1038/ncomms11311

    IL-25 secreted by Q2-3-treated fibroblasts suppresses the growth of mammary tumour cells. ( a ) Western blot analysis of the IL-25 secretion activity of mouse (3T3) and human (WI38) fibroblasts in response to Q2-3 treatment. Different fibroblast-conditioned media (CM), including 3T3-CM and WI38-CM, were collected from the 3D cultures and were stained with coomassie blue, revealing that the total protein level in tested CM was normalized. Aliquots of 3T3-CM and WI38-CM were immunodepleted for IL-25. Rabbit IgG (isotype control) was used as a negative control. Amounts of IL-25 (relative staining intensity) were further normalized with the value detected for Q2-3-treated 3T3-CM (blue) or Q2-3-treated WI38-CM samples (purple). ( b ) Reduction in cytotoxicity of 3T3-CM on 4T1 cells after immunodepletion of IL-25. The control (fresh) medium, 3T3-CM, Q2-3-treated 3T3-CM, Q2-3-treated 3T3-CM with added IL-25 protein, Q2-3-treated 3T3-CM with the immunodepletion of IL-25 and Q2-3-treated 3T3-CM with control IgG-mediated immunodepletion, were compared for their suppressive effect on growth of 4T1 cells. ( c ) Reduction in cytotoxicity of WI38-CM on MDA-MB-231 after immunodepletion of IL-25. Similarly, WI38-CM with added IL-25 protein, WI38-CM with the immunodepletion of IL-25, or Q2-3-treated WI38-CM with control IgG-mediated immunodepletion, were compared for their effect on growth of MDA-MB-231 cells. The growth activity of 4T1 cells or MDA-MB-231 cells was determined using MTT assay at 5 days post cultivation, and was normalized to the control group (with fresh medium only). Data are reported as mean±s.d. ( n =3). * P
    Figure Legend Snippet: IL-25 secreted by Q2-3-treated fibroblasts suppresses the growth of mammary tumour cells. ( a ) Western blot analysis of the IL-25 secretion activity of mouse (3T3) and human (WI38) fibroblasts in response to Q2-3 treatment. Different fibroblast-conditioned media (CM), including 3T3-CM and WI38-CM, were collected from the 3D cultures and were stained with coomassie blue, revealing that the total protein level in tested CM was normalized. Aliquots of 3T3-CM and WI38-CM were immunodepleted for IL-25. Rabbit IgG (isotype control) was used as a negative control. Amounts of IL-25 (relative staining intensity) were further normalized with the value detected for Q2-3-treated 3T3-CM (blue) or Q2-3-treated WI38-CM samples (purple). ( b ) Reduction in cytotoxicity of 3T3-CM on 4T1 cells after immunodepletion of IL-25. The control (fresh) medium, 3T3-CM, Q2-3-treated 3T3-CM, Q2-3-treated 3T3-CM with added IL-25 protein, Q2-3-treated 3T3-CM with the immunodepletion of IL-25 and Q2-3-treated 3T3-CM with control IgG-mediated immunodepletion, were compared for their suppressive effect on growth of 4T1 cells. ( c ) Reduction in cytotoxicity of WI38-CM on MDA-MB-231 after immunodepletion of IL-25. Similarly, WI38-CM with added IL-25 protein, WI38-CM with the immunodepletion of IL-25, or Q2-3-treated WI38-CM with control IgG-mediated immunodepletion, were compared for their effect on growth of MDA-MB-231 cells. The growth activity of 4T1 cells or MDA-MB-231 cells was determined using MTT assay at 5 days post cultivation, and was normalized to the control group (with fresh medium only). Data are reported as mean±s.d. ( n =3). * P

    Techniques Used: Western Blot, Activity Assay, Staining, Negative Control, Multiple Displacement Amplification, MTT Assay

    Dihydrobenzofuran lignan (Q2-3) exhibits a specific cytotoxic effect on breast tumour cells. ( a ) Chemical reaction formula for synthesis of Q2-3 compound (methyl ( E )-3-[2-(3,4-dihydroxyphenyl)-7-hydroxy-3-methoxycarbonyl-2,3-dihydro-1-benzofuran-5yl]prop-2-enoate) from two caffeic acid methyl ester molecules. ( b , c ) MTT assays for cytotoxic effect of Q2-3 on SKBR3 (ER − Her + human breast cancer cell line), MDA-MB-231 (ER − Her − human breast cancer cell line), M10 (human normal epithelial cell line) and WI38 (human lung fibroblast cell line). 2 × 10 4 cells were seeded and compared for their growth activity after treatment with Q2-3 for 24 or 72 h. The beginning cytotoxic dosage of Q2-3 was detected at 0.3 μM, with > 50% kill rate on SKBR3 and MDA-MB-231 cells. Data are reported as mean±s.d. ( n =4). ** P
    Figure Legend Snippet: Dihydrobenzofuran lignan (Q2-3) exhibits a specific cytotoxic effect on breast tumour cells. ( a ) Chemical reaction formula for synthesis of Q2-3 compound (methyl ( E )-3-[2-(3,4-dihydroxyphenyl)-7-hydroxy-3-methoxycarbonyl-2,3-dihydro-1-benzofuran-5yl]prop-2-enoate) from two caffeic acid methyl ester molecules. ( b , c ) MTT assays for cytotoxic effect of Q2-3 on SKBR3 (ER − Her + human breast cancer cell line), MDA-MB-231 (ER − Her − human breast cancer cell line), M10 (human normal epithelial cell line) and WI38 (human lung fibroblast cell line). 2 × 10 4 cells were seeded and compared for their growth activity after treatment with Q2-3 for 24 or 72 h. The beginning cytotoxic dosage of Q2-3 was detected at 0.3 μM, with > 50% kill rate on SKBR3 and MDA-MB-231 cells. Data are reported as mean±s.d. ( n =4). ** P

    Techniques Used: MTT Assay, Multiple Displacement Amplification, Activity Assay

    12) Product Images from "NKG2D ligands mediate immunosurveillance of senescent cells"

    Article Title: NKG2D ligands mediate immunosurveillance of senescent cells

    Journal: Aging (Albany NY)

    doi:

    Senescent cells upregulate MICA and ULBP2 on the cell surface ( A ) ImageStream analysis demonstrates higher expression levels of MICA and ULBP2 on the cell surface membrane of DIS IMR-90 fibroblasts (Senescent) compared to control (Growing) cells. ( B ) Quantification of total intensity and total membrane intensity indicate higher levels of MICA and ULBP2 in DIS IMR-90 cells compared to growing (control) cells. ( C ) Representative immunofluorescence staining of MICA and ULBP2 performed on non-permeabilized cells demonstrates higher expression levels of these proteins on the cell surface membrane in DIS WI38 and BJ cells compared to growing (control) cells. Scale bar: 50μm. MICA and ULBP2 are shown in red. DAPI is shown in blue. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P
    Figure Legend Snippet: Senescent cells upregulate MICA and ULBP2 on the cell surface ( A ) ImageStream analysis demonstrates higher expression levels of MICA and ULBP2 on the cell surface membrane of DIS IMR-90 fibroblasts (Senescent) compared to control (Growing) cells. ( B ) Quantification of total intensity and total membrane intensity indicate higher levels of MICA and ULBP2 in DIS IMR-90 cells compared to growing (control) cells. ( C ) Representative immunofluorescence staining of MICA and ULBP2 performed on non-permeabilized cells demonstrates higher expression levels of these proteins on the cell surface membrane in DIS WI38 and BJ cells compared to growing (control) cells. Scale bar: 50μm. MICA and ULBP2 are shown in red. DAPI is shown in blue. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P

    Techniques Used: Expressing, Immunofluorescence, Staining, Two Tailed Test

    MICA and ULBP2 expression is stably upregulated in senescent, but not quiescent cells Growing IMR-90, WI38 and BJ cells were treated with Etoposide (100μM) and the level of MICA and ULBP2 expression were assessed by RT-PCR. The expression of MICA ( A ) and ULBP2 ( B ) was elevated 24 hours following treatment and was maintained at higher levels throughout the indicated time points. Growing cells at day 0 served as control. ( C ) Growing cells were treated with a low concentration of Etoposide (10μM) to induce transient growth arrest and the level of MICA and ULBP2 expression were assessed by RT-PCR. ( D ) IMR-90 cells were grown to confluence and maintained for a further 7 days until cells became quiescent. Cell-cycle arrest induced by quiescence was not sufficient to elevate MICA and ULBP2 expression as assessed by RT-PCR and compared to growing and senescent cells. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P
    Figure Legend Snippet: MICA and ULBP2 expression is stably upregulated in senescent, but not quiescent cells Growing IMR-90, WI38 and BJ cells were treated with Etoposide (100μM) and the level of MICA and ULBP2 expression were assessed by RT-PCR. The expression of MICA ( A ) and ULBP2 ( B ) was elevated 24 hours following treatment and was maintained at higher levels throughout the indicated time points. Growing cells at day 0 served as control. ( C ) Growing cells were treated with a low concentration of Etoposide (10μM) to induce transient growth arrest and the level of MICA and ULBP2 expression were assessed by RT-PCR. ( D ) IMR-90 cells were grown to confluence and maintained for a further 7 days until cells became quiescent. Cell-cycle arrest induced by quiescence was not sufficient to elevate MICA and ULBP2 expression as assessed by RT-PCR and compared to growing and senescent cells. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P

    Techniques Used: Expressing, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Two Tailed Test

    Senescent cells upregulate NKG2D ligands RT-PCR analysis demonstrates a consistent up-regulation of MICA, ULBP1, and ULBP2 in DNA damage-induced senescent (DIS) ( A ), replicative senescent (RS) ( B ), and H-RAS v12 -mediated oncogene-induced senescent (OIS) ( C ) IMR-90 fibroblasts compared to growing (control) cells. Similarly, DIS WI38 ( D ) and BJ ( E ) fibroblasts and DIS hepatic stellate cells (HSCs) ( F ) also show an up-regulation of NKG2D ligands. The graphs represent the mean and the S.E.M of at least triplicate measurements from at least four independent experiments. Two-tailed t-test *P
    Figure Legend Snippet: Senescent cells upregulate NKG2D ligands RT-PCR analysis demonstrates a consistent up-regulation of MICA, ULBP1, and ULBP2 in DNA damage-induced senescent (DIS) ( A ), replicative senescent (RS) ( B ), and H-RAS v12 -mediated oncogene-induced senescent (OIS) ( C ) IMR-90 fibroblasts compared to growing (control) cells. Similarly, DIS WI38 ( D ) and BJ ( E ) fibroblasts and DIS hepatic stellate cells (HSCs) ( F ) also show an up-regulation of NKG2D ligands. The graphs represent the mean and the S.E.M of at least triplicate measurements from at least four independent experiments. Two-tailed t-test *P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    13) Product Images from "NKG2D ligands mediate immunosurveillance of senescent cells"

    Article Title: NKG2D ligands mediate immunosurveillance of senescent cells

    Journal: Aging (Albany NY)

    doi:

    Senescent cells upregulate MICA and ULBP2 on the cell surface ( A ) ImageStream analysis demonstrates higher expression levels of MICA and ULBP2 on the cell surface membrane of DIS IMR-90 fibroblasts (Senescent) compared to control (Growing) cells. ( B ) Quantification of total intensity and total membrane intensity indicate higher levels of MICA and ULBP2 in DIS IMR-90 cells compared to growing (control) cells. ( C ) Representative immunofluorescence staining of MICA and ULBP2 performed on non-permeabilized cells demonstrates higher expression levels of these proteins on the cell surface membrane in DIS WI38 and BJ cells compared to growing (control) cells. Scale bar: 50μm. MICA and ULBP2 are shown in red. DAPI is shown in blue. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P
    Figure Legend Snippet: Senescent cells upregulate MICA and ULBP2 on the cell surface ( A ) ImageStream analysis demonstrates higher expression levels of MICA and ULBP2 on the cell surface membrane of DIS IMR-90 fibroblasts (Senescent) compared to control (Growing) cells. ( B ) Quantification of total intensity and total membrane intensity indicate higher levels of MICA and ULBP2 in DIS IMR-90 cells compared to growing (control) cells. ( C ) Representative immunofluorescence staining of MICA and ULBP2 performed on non-permeabilized cells demonstrates higher expression levels of these proteins on the cell surface membrane in DIS WI38 and BJ cells compared to growing (control) cells. Scale bar: 50μm. MICA and ULBP2 are shown in red. DAPI is shown in blue. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P

    Techniques Used: Expressing, Immunofluorescence, Staining, Two Tailed Test

    MICA and ULBP2 expression is stably upregulated in senescent, but not quiescent cells Growing IMR-90, WI38 and BJ cells were treated with Etoposide (100μM) and the level of MICA and ULBP2 expression were assessed by RT-PCR. The expression of MICA ( A ) and ULBP2 ( B ) was elevated 24 hours following treatment and was maintained at higher levels throughout the indicated time points. Growing cells at day 0 served as control. ( C ) Growing cells were treated with a low concentration of Etoposide (10μM) to induce transient growth arrest and the level of MICA and ULBP2 expression were assessed by RT-PCR. ( D ) IMR-90 cells were grown to confluence and maintained for a further 7 days until cells became quiescent. Cell-cycle arrest induced by quiescence was not sufficient to elevate MICA and ULBP2 expression as assessed by RT-PCR and compared to growing and senescent cells. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P
    Figure Legend Snippet: MICA and ULBP2 expression is stably upregulated in senescent, but not quiescent cells Growing IMR-90, WI38 and BJ cells were treated with Etoposide (100μM) and the level of MICA and ULBP2 expression were assessed by RT-PCR. The expression of MICA ( A ) and ULBP2 ( B ) was elevated 24 hours following treatment and was maintained at higher levels throughout the indicated time points. Growing cells at day 0 served as control. ( C ) Growing cells were treated with a low concentration of Etoposide (10μM) to induce transient growth arrest and the level of MICA and ULBP2 expression were assessed by RT-PCR. ( D ) IMR-90 cells were grown to confluence and maintained for a further 7 days until cells became quiescent. Cell-cycle arrest induced by quiescence was not sufficient to elevate MICA and ULBP2 expression as assessed by RT-PCR and compared to growing and senescent cells. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P

    Techniques Used: Expressing, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Two Tailed Test

    Senescent cells upregulate NKG2D ligands RT-PCR analysis demonstrates a consistent up-regulation of MICA, ULBP1, and ULBP2 in DNA damage-induced senescent (DIS) ( A ), replicative senescent (RS) ( B ), and H-RAS v12 -mediated oncogene-induced senescent (OIS) ( C ) IMR-90 fibroblasts compared to growing (control) cells. Similarly, DIS WI38 ( D ) and BJ ( E ) fibroblasts and DIS hepatic stellate cells (HSCs) ( F ) also show an up-regulation of NKG2D ligands. The graphs represent the mean and the S.E.M of at least triplicate measurements from at least four independent experiments. Two-tailed t-test *P
    Figure Legend Snippet: Senescent cells upregulate NKG2D ligands RT-PCR analysis demonstrates a consistent up-regulation of MICA, ULBP1, and ULBP2 in DNA damage-induced senescent (DIS) ( A ), replicative senescent (RS) ( B ), and H-RAS v12 -mediated oncogene-induced senescent (OIS) ( C ) IMR-90 fibroblasts compared to growing (control) cells. Similarly, DIS WI38 ( D ) and BJ ( E ) fibroblasts and DIS hepatic stellate cells (HSCs) ( F ) also show an up-regulation of NKG2D ligands. The graphs represent the mean and the S.E.M of at least triplicate measurements from at least four independent experiments. Two-tailed t-test *P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    14) Product Images from "A Viral microRNA Cluster Regulates the Expression of PTEN, p27 and of a bcl-2 Homolog"

    Article Title: A Viral microRNA Cluster Regulates the Expression of PTEN, p27 and of a bcl-2 Homolog

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005405

    The reduced BHRF1 expression in B-cells infected with Δ123 leads to a moderately increased apoptosis. Primary B-cells were infected with wild type, Δ123, ΔBHRF1 or Δ123ΔBHRF1 and monitored over time by immunofluorescence staining of active caspase 3 (a), by TUNEL assay (b), and by phospho-histone H3 staining (PH3) (c). Two out of five blood samples are shown. BrdU incorporation on day 14 was determined in B-cells transformed with wild type, Δ123, ΔBHRF1 and Δ123ΔBHRF1 (d). The results obtained with two samples are shown. Transformation assays were performed by infecting primary B-cells with 0.01 infectious virus per cell. 500 cells per well were seeded on 96-well cluster plates coated with 50Gy irradiated WI38 feeder cells and the number of wells with LCL outgrowth was determined 30 days after infection (e).
    Figure Legend Snippet: The reduced BHRF1 expression in B-cells infected with Δ123 leads to a moderately increased apoptosis. Primary B-cells were infected with wild type, Δ123, ΔBHRF1 or Δ123ΔBHRF1 and monitored over time by immunofluorescence staining of active caspase 3 (a), by TUNEL assay (b), and by phospho-histone H3 staining (PH3) (c). Two out of five blood samples are shown. BrdU incorporation on day 14 was determined in B-cells transformed with wild type, Δ123, ΔBHRF1 and Δ123ΔBHRF1 (d). The results obtained with two samples are shown. Transformation assays were performed by infecting primary B-cells with 0.01 infectious virus per cell. 500 cells per well were seeded on 96-well cluster plates coated with 50Gy irradiated WI38 feeder cells and the number of wells with LCL outgrowth was determined 30 days after infection (e).

    Techniques Used: Expressing, Infection, Immunofluorescence, Staining, TUNEL Assay, BrdU Incorporation Assay, Transformation Assay, Irradiation

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    Genotoxicity induces senescence of <t>human</t> <t>stromal</t> cells which display upregulation of PDK4 and a full spectrum SASP. a. Transcriptome-wide profiling of gene expression changes in primary normal human prostate stromal cell line PSC27 by microarray. Cell lysates were collected for analysis 7 d after treatment. CTRL, control. RAD, radiation. BLEO, bleomycin. HP, hydrogen peroxide. Red highlighted, typical soluble factors of the SASP. Microarray data adapted from Sun et al. with permission from Nature Medicine , copyright 2012, Springer Nature 23 . b. Quantitative RT-PCR to detect PDK4 expression after PSC27 cells were subject to individual treatment as indicated. Cell lysates were collected for measurement 7 d after establishment of stable cell sublines or completion of in vitro treatment. Signals normalized to CTRL. RS, replicative senescence. p16, lentiviral transduction of human tumor suppressor p16 INK4a . RAS, lentiviral transduction of human oncogene HRAS G12V . c. Immunoblot analysis of PDK4 expression in stromal cells as delineated in ( b ). GAPDH, loading control. d. Comparative RT-PCR assay of PDK4 expression after treatment of PSC27 or prostate epithelial cells by agents as indicated. Cell lysates were collected for measurement 7 d after treatment. Signals normalized to CTRL. BPH1, M12, PC3, DU145, LNCaP and VCaP, human epithelial <t>lines</t> of prostate origin. e. Comparative RT-PCR assay of PDK4 expression in human stromal cells 7 d after treatments performed as indicated. <t>WI38,</t> HFL1, HBF1203 and BJ, human stromal lines of different origins. f. A time course RT-PCR assessment of the expression of PDK4 and a set of typical SASP factors (MMP1, WNT16B, SFRP2, SPINK1, MMP3, CXCL8, EREG, ANGPTL4 and AREG) after drug treatment of PSC27 cells in vitro . Numeric numbers indicate the individual days after treatment (indexed at the top line). g. Immunoblot measurement of PDK4 expression at protein level at the individual timepoints as indicated. β-actin, loading control. h. Comparative appraisal of human PDK family expression at transcript level in PSC27 cells after BLEO treatment. Signals normalized to untreated sample per gene. CXCL8, experimental control as a hallmark SASP factor. i. Immunoblot assessment of the expression of PDK4 family members at protein level after BLEO treatment. β-actin, loading control. Data are representative of 3 independent experiments. P values were calculated by Student’s t -test ( b , e , f , h ) and one-way ANOVA ( d ). ^, P > 0.05. *, P
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    Genotoxicity induces senescence of human stromal cells which display upregulation of PDK4 and a full spectrum SASP. a. Transcriptome-wide profiling of gene expression changes in primary normal human prostate stromal cell line PSC27 by microarray. Cell lysates were collected for analysis 7 d after treatment. CTRL, control. RAD, radiation. BLEO, bleomycin. HP, hydrogen peroxide. Red highlighted, typical soluble factors of the SASP. Microarray data adapted from Sun et al. with permission from Nature Medicine , copyright 2012, Springer Nature 23 . b. Quantitative RT-PCR to detect PDK4 expression after PSC27 cells were subject to individual treatment as indicated. Cell lysates were collected for measurement 7 d after establishment of stable cell sublines or completion of in vitro treatment. Signals normalized to CTRL. RS, replicative senescence. p16, lentiviral transduction of human tumor suppressor p16 INK4a . RAS, lentiviral transduction of human oncogene HRAS G12V . c. Immunoblot analysis of PDK4 expression in stromal cells as delineated in ( b ). GAPDH, loading control. d. Comparative RT-PCR assay of PDK4 expression after treatment of PSC27 or prostate epithelial cells by agents as indicated. Cell lysates were collected for measurement 7 d after treatment. Signals normalized to CTRL. BPH1, M12, PC3, DU145, LNCaP and VCaP, human epithelial lines of prostate origin. e. Comparative RT-PCR assay of PDK4 expression in human stromal cells 7 d after treatments performed as indicated. WI38, HFL1, HBF1203 and BJ, human stromal lines of different origins. f. A time course RT-PCR assessment of the expression of PDK4 and a set of typical SASP factors (MMP1, WNT16B, SFRP2, SPINK1, MMP3, CXCL8, EREG, ANGPTL4 and AREG) after drug treatment of PSC27 cells in vitro . Numeric numbers indicate the individual days after treatment (indexed at the top line). g. Immunoblot measurement of PDK4 expression at protein level at the individual timepoints as indicated. β-actin, loading control. h. Comparative appraisal of human PDK family expression at transcript level in PSC27 cells after BLEO treatment. Signals normalized to untreated sample per gene. CXCL8, experimental control as a hallmark SASP factor. i. Immunoblot assessment of the expression of PDK4 family members at protein level after BLEO treatment. β-actin, loading control. Data are representative of 3 independent experiments. P values were calculated by Student’s t -test ( b , e , f , h ) and one-way ANOVA ( d ). ^, P > 0.05. *, P

    Journal: bioRxiv

    Article Title: Senescent cells develop PDK4-dependent hypercatabolism and form an acidic microenvironment to drive cancer resistance

    doi: 10.1101/2022.08.29.505761

    Figure Lengend Snippet: Genotoxicity induces senescence of human stromal cells which display upregulation of PDK4 and a full spectrum SASP. a. Transcriptome-wide profiling of gene expression changes in primary normal human prostate stromal cell line PSC27 by microarray. Cell lysates were collected for analysis 7 d after treatment. CTRL, control. RAD, radiation. BLEO, bleomycin. HP, hydrogen peroxide. Red highlighted, typical soluble factors of the SASP. Microarray data adapted from Sun et al. with permission from Nature Medicine , copyright 2012, Springer Nature 23 . b. Quantitative RT-PCR to detect PDK4 expression after PSC27 cells were subject to individual treatment as indicated. Cell lysates were collected for measurement 7 d after establishment of stable cell sublines or completion of in vitro treatment. Signals normalized to CTRL. RS, replicative senescence. p16, lentiviral transduction of human tumor suppressor p16 INK4a . RAS, lentiviral transduction of human oncogene HRAS G12V . c. Immunoblot analysis of PDK4 expression in stromal cells as delineated in ( b ). GAPDH, loading control. d. Comparative RT-PCR assay of PDK4 expression after treatment of PSC27 or prostate epithelial cells by agents as indicated. Cell lysates were collected for measurement 7 d after treatment. Signals normalized to CTRL. BPH1, M12, PC3, DU145, LNCaP and VCaP, human epithelial lines of prostate origin. e. Comparative RT-PCR assay of PDK4 expression in human stromal cells 7 d after treatments performed as indicated. WI38, HFL1, HBF1203 and BJ, human stromal lines of different origins. f. A time course RT-PCR assessment of the expression of PDK4 and a set of typical SASP factors (MMP1, WNT16B, SFRP2, SPINK1, MMP3, CXCL8, EREG, ANGPTL4 and AREG) after drug treatment of PSC27 cells in vitro . Numeric numbers indicate the individual days after treatment (indexed at the top line). g. Immunoblot measurement of PDK4 expression at protein level at the individual timepoints as indicated. β-actin, loading control. h. Comparative appraisal of human PDK family expression at transcript level in PSC27 cells after BLEO treatment. Signals normalized to untreated sample per gene. CXCL8, experimental control as a hallmark SASP factor. i. Immunoblot assessment of the expression of PDK4 family members at protein level after BLEO treatment. β-actin, loading control. Data are representative of 3 independent experiments. P values were calculated by Student’s t -test ( b , e , f , h ) and one-way ANOVA ( d ). ^, P > 0.05. *, P

    Article Snippet: Human fetal lung stromal lines WI38 and HFL1, and foreskin stromal line BJ were from ATCC and cultured with F-12K medium supplemented with 10% FBS.

    Techniques: Expressing, Microarray, Quantitative RT-PCR, Stable Transfection, In Vitro, Transduction, Reverse Transcription Polymerase Chain Reaction

    Expression profiles of human WI38 cells after knockdown of the HIC1 gene. (A) Down-regulation of HIC1 mRNA in WI38 cells at 24, 48 and 72 h after transfection with HIC1-specific siRNAs from Ambion (Amb HIC1 siRNA) or Dharmacon (Dhar HIC1 siRNA); transfection with non-coding siRNA was used as a control. Graphs show the results of RT-qPCR analysis of WI38 cells transfected with the indicated siRNAs; the obtained quantification cycle (Cq) values were normalized to the UBB mRNA levels; the HIC1 expression level in cells treated with non-silencing control siRNA was arbitrarily set to 1. Error bars indicate standard deviations (SDs); * p-value

    Journal: bioRxiv

    Article Title: Tumor suppressor Hypermethylated in Cancer 1 represses expression of cell cycle regulator E2F7 in human primary cells

    doi: 10.1101/2022.07.25.501405

    Figure Lengend Snippet: Expression profiles of human WI38 cells after knockdown of the HIC1 gene. (A) Down-regulation of HIC1 mRNA in WI38 cells at 24, 48 and 72 h after transfection with HIC1-specific siRNAs from Ambion (Amb HIC1 siRNA) or Dharmacon (Dhar HIC1 siRNA); transfection with non-coding siRNA was used as a control. Graphs show the results of RT-qPCR analysis of WI38 cells transfected with the indicated siRNAs; the obtained quantification cycle (Cq) values were normalized to the UBB mRNA levels; the HIC1 expression level in cells treated with non-silencing control siRNA was arbitrarily set to 1. Error bars indicate standard deviations (SDs); * p-value

    Article Snippet: Cell culture, CRISPR/Cas9 editing of the HIC1 locus, and chromosome analysis Human WI38, HFF-1, and HEK293 cells were purchased from ATCC (cat. no. CCL-75, SCRL-1041, CRL-1573, respectively).

    Techniques: Expressing, Transfection, Quantitative RT-PCR