Journal: Virology

Article Title: Molecular pathways executing the "trophic sentinel" response in HPV-16 E7-expressing normal human diploid fibroblasts upon growth factor deprivation.

doi: 10.1016/j.virol.2003.11.008

Figure Lengend Snippet: Fig. 1. (A) The trophic sentinel response in HPV E7-expressing cells induced by serum starvation is p53 dependent. Induction of apoptosis in the different cell lines as determined by fluorescence microscopic analysis of nuclear abnormalities (left panel) and nucleosomal DNA fragmentation using an ELISA-based quantitative DNA laddering assay (right panel). The values shown were calculated by determining the relative increases of apoptosis and DNA fragmentation, respectively, for each cell population in the absence or presence of fetal bovine serum for 48 h derived from a single experiment. Similar results were obtained in multiple independent experiments. (B) Immunoblot analysis of the steady state levels of p53, p21CIP1, and HPV-16 E7 in the different cell populations in response to DNA damage induced by actinomycin D (actD) treatment. GAPDH expression is shown as a loading control. See Materials and methods for details. (C) Analysis of p53 phosphorylation by immunoblot analysis with various phospho-specific antibodies in control (LXSN) and HPV-16 E7-expressing (E7) IMR-90 cells in the absence (0%) or presence (10%) of fetal bovine serum (FBS). UV-irradiated cos7 and control (LXSN) and HPV-16 E7-expressing (E7) IMR-90 cells were used as positive controls. GAPDH expression is shown to document equal loading. (D) DNA binding capacity of p53 in control (LXSN) and HPV-16 E7-expressing (E7) IMR-90 cells grown in the absence (0%) or presence (10%) of fetal bovine serum (FBS) by EMSA. Wild-type or mutant oligonucleotide competitors or a p53-specific antibody were added to demonstrate the specificity of the shifted band; ns denotes a nonspecific band. Lysates from the human Raji cell line were supplied as a positive control by the manufacturer of the kit (GENEKA).

Article Snippet: The following antibodies were used according to the recommendations of their suppliers: p53 (Ab-6, Calbiochem), p21CIP1 (Ab-1, Oncogene Science), Noxa (Pharmingen), GAPDH (CHEMICON), HPV-16 E7 (mixture of 8C9, 1:100, Zymed and ED17, 1:1000, Santa Cruz), InB (Rabbit polyclonal, Santa Cruz), IGFBP-1, 2,3,4,5,6 (goat polyclonal, Santa Cruz), p50 (Rockland), p65 (Santa Cruz).

Techniques: Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay, DNA Laddering, Derivative Assay, Western Blot, Control, Phospho-proteomics, Irradiation, Binding Assay, Mutagenesis, Positive Control

Journal: Journal of Biological Chemistry

Article Title: Mammary Epithelial Cell Polarity Is Regulated Differentially by p73 Isoforms via Epithelial-to-mesenchymal Transition

doi: 10.1074/jbc.m112.358143

Figure Lengend Snippet: FIGURE 4. p73-KD and TAp73-KD, but not Np73-KD induce EMT pheno- types in MCF10A cells. Western blots were prepared using extracts from MCF10A cells (lane 1), and MCF10A cells with p73-KD (lane 2), with Np73-KD (lane3),orwithTAp73-KD(lane4).MCF10AcellsweregrowninMatrigelfor20 days.TheblotswereprobedwithantibodiesagainstE-cadherin(A),-catenin (B), laminin V (B), Snail (B), Slug (B), Twist (B), p21 (C), PUMA (C), and actin (A–C), respectively. The basal levels of each gene were arbitrarily set at 1.0, and fold change is shown below each lane.

Article Snippet: Antibodies used were purchased from Calbiochem (anti-p73 (ab-1)-ER-13, anti-p73 (ab-2)-ER-15), Millipore (anti-laminin 2), Santa Cruz Biotechnology (anti- - catenin (E-5), anti-Snail-1, anti-Twist, p21, and anti-GAPDH), Cell Signaling (anti-Slug), BDTransduction Laboratories (antiE-cadherin), Prosci (PUMA), Sigma (anti-actin), and Bio-Rad (secondary antibodies against rabbit or mouse IgG conjugated with HRP).

Techniques: Western Blot

Journal: Cancer Medicine

Article Title: The GNL3L ‐ MDM2 Interaction Drives Esophageal Squamous Cell Carcinoma Progression

doi: 10.1002/cam4.71146

Figure Lengend Snippet: GNL3L positively regulated MDM2 expression and interacted with MDM2. (a) After GNL3L knockdown, qRT‐PCR, and western blot (WB) were performed to determine MDM2 expression in TE‐1 cells. (b) Differences in the expression of p53 and p21 in TE‐1 cells after the knockdown of GNL3L. (c) Co‐IP of GNL3L and MDM2 from ESCC cell lines. The input represents the total protein extract used in the IP. The GNL3L protein expression was normalized to that of GAPDH in ESCC cells. (IP, immunoprecipitation; IgG, negative control). (d) qRT‐PCR analysis of MDM2 and GNL3L expression in TE‐1 cells in different groups. (e) Protein expression of MDM2 and GNL3L of TE‐1 in different groups was determined by WB. (f) Cell proliferation was analyzed in different groups using the CCK8 assay for TE‐1 cell lines. (g) Proliferation was analyzed using an EdU assay. (** p < 0.01, ## p < 0.01).

Article Snippet: The primary antibodies were GAPDH polyclonal antibody (1:3000, Affinity, AF7021, 36 kDa), MDM2 polyclonal antibody (1:1000, Affinity, AF0208, 90 kDa), GNL3L antibody (1:1000, Affinity, DF4113, 66 kDa), p53 antibody (1:1000, Affinity, AF0879, 53 kDa), and p21 antibody (1:1000, MedChemExpress, HY‐ P80774 , 18 kDa).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, CCK-8 Assay, EdU Assay

Journal: Cancer Medicine

Article Title: The GNL3L ‐ MDM2 Interaction Drives Esophageal Squamous Cell Carcinoma Progression

doi: 10.1002/cam4.71146

Figure Lengend Snippet: GNL3L knockdown reversed the increased malignant behaviors of ESCC cells induced by MDM2 overexpression. (a) Wound healing assay results in TE‐1 cells from different groups. (b) Invasion and migration of TE‐1 cells were assessed using Transwell assays. (c) Distribution of the cell phases of TE‐1 in the different groups. (d) In the different groups, apoptosis was measured in TE‐1 cells using flow cytometry. (e) Expression of P53 and P21 in TE‐1 cells in different groups. (** p < 0.01, ## p < 0.01).

Article Snippet: The primary antibodies were GAPDH polyclonal antibody (1:3000, Affinity, AF7021, 36 kDa), MDM2 polyclonal antibody (1:1000, Affinity, AF0208, 90 kDa), GNL3L antibody (1:1000, Affinity, DF4113, 66 kDa), p53 antibody (1:1000, Affinity, AF0879, 53 kDa), and p21 antibody (1:1000, MedChemExpress, HY‐ P80774 , 18 kDa).

Techniques: Knockdown, Over Expression, Wound Healing Assay, Migration, Flow Cytometry, Expressing

Journal: Cancer Medicine

Article Title: The GNL3L ‐ MDM2 Interaction Drives Esophageal Squamous Cell Carcinoma Progression

doi: 10.1002/cam4.71146

Figure Lengend Snippet: GNL3L overexpression reversed the decreased malignant behavior of ESCC cells induced by MDM2 knockdown. (a) Protein expression of MDM2 and GNL3L in TE‐1 cells in different groups was determined by western blot (WB). (b) Cell proliferation was analyzed in different groups using an EdU assay. (c) Migration and invasion of TE‐1 cells were assessed using Transwell assays. (d) In the different groups, apoptosis was measured in TE‐1 cells using flow cytometry. (e) Expression of P53 and P21 in TE‐1 cells in different groups. (** p < 0.01, ## p < 0.01).

Article Snippet: The primary antibodies were GAPDH polyclonal antibody (1:3000, Affinity, AF7021, 36 kDa), MDM2 polyclonal antibody (1:1000, Affinity, AF0208, 90 kDa), GNL3L antibody (1:1000, Affinity, DF4113, 66 kDa), p53 antibody (1:1000, Affinity, AF0879, 53 kDa), and p21 antibody (1:1000, MedChemExpress, HY‐ P80774 , 18 kDa).

Techniques: Over Expression, Knockdown, Expressing, Western Blot, EdU Assay, Migration, Flow Cytometry

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Acetylation of Atp5f1c Mediates Cardiomyocyte Senescence via Metabolic Dysfunction in Radiation-Induced Heart Damage

doi: 10.1155/2022/4155565

Figure Lengend Snippet: Ionizing radiation causes cardiomyocyte senescence. (a) Senescence-associated β -galactosidase (SA- β gal) staining of sham-irradiated and irradiated H9C2 cells. (b)–(d) Western blotting and quantification analyses of p21 and p16 protein expression in H9C2 cells in the control group and after 6h, 12h, and 24h of irradiation ( ∗ p < 0.05, ∗∗ p < 0.01). (e) q-PCR analysis of the mRNA levels of inflammation- and fibrosis-associated factors in H9C2 cells of the control, 6h, 12h, and 24h irradiation groups ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). Bar: 100 μ m; h: hour.

Article Snippet: Total proteins from the animal heart apex or H9C2 cells were extracted using RIPA (Solarbio, Beijing, China), and protein concentration and western blotting were performed as previously described [ ] The primary antibodies used in this study were anti-acetyllysine rabbit pAb (1 : 1000; Jingjie PTM BioLabs, Inc., China), anti-Atp5f1 monoclonal antibody (1 : 1000; Proteintech, Wuhan, China), anti-p21 monoclonal antibody (1 : 1000; Boster, Wuhan, China), HA-tagged monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), 6× His-tagged monoclonal antibody (1 : 10000; Proteintech, Wuhan, China), GAPDH monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), and anti- α -tubulin monoclonal antibody (1 : 1000; Boster).

Techniques: Staining, Irradiation, Western Blot, Expressing, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Acetylation of Atp5f1c Mediates Cardiomyocyte Senescence via Metabolic Dysfunction in Radiation-Induced Heart Damage

doi: 10.1155/2022/4155565

Figure Lengend Snippet: Atp5f1c is hyperacetylated at the 55th lysine site, which leads to metabolic disorders and senescence. (a) The expression of anti-His antibody after His-tagged Atp5f1c WT, K55R, and K55Q point mutation plasmids transfection in H9C2 cells. (b) Atp5f1c acetylation level verified by Co-IP after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells. (c, d) Senescence-associated β -galactosidase (SABG) staining and statistical analysis after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; NS: not significant). (e) q-PCR analysis of the mRNA levels of inflammation- and fibrosis-associated factors after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (f)–(i) Western blotting and quantification analyses of p21 and p16 protein expression after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells; relative protein levels were normalized to GAPDH or actin expression ( ∗ p < 0.05). (j, k) ATP synthase activity and ATP production after transfection of Atp5f1c WT, K55R, and K55Q plasmids into H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; NS: not significant). Co-IP: Coimmunoprecipitation; WB: western blotting; WT: wild type.

Article Snippet: Total proteins from the animal heart apex or H9C2 cells were extracted using RIPA (Solarbio, Beijing, China), and protein concentration and western blotting were performed as previously described [ ] The primary antibodies used in this study were anti-acetyllysine rabbit pAb (1 : 1000; Jingjie PTM BioLabs, Inc., China), anti-Atp5f1 monoclonal antibody (1 : 1000; Proteintech, Wuhan, China), anti-p21 monoclonal antibody (1 : 1000; Boster, Wuhan, China), HA-tagged monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), 6× His-tagged monoclonal antibody (1 : 10000; Proteintech, Wuhan, China), GAPDH monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), and anti- α -tubulin monoclonal antibody (1 : 1000; Boster).

Techniques: Expressing, Mutagenesis, Transfection, Co-Immunoprecipitation Assay, Staining, Western Blot, Activity Assay

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 3 CLOCK interference promotes GCs proliferation. A The interference efficiency of CLOCK was measured using RT-qPCR. Data are expressed as mean ± SEM (n = 5), **P < 0.01. B Western blotting reveals the expression levels of CLOCK. C Quantification of the western blot analysis. Data are expressed as mean ± SEM (n = 3), *P < 0.05. D EdU staining was used to detect the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 5), **P < 0.01. E CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 5), *P < 0.05. F RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 5), *P < 0.05, **P < 0.01. G Western blot analysis of proliferation-related gene protein level (CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A). GAPDH as a housekeeping protein. H Quantifying the Western blot analysis of CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), *P < 0.05

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Transfection

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 6 ASB9 interference promotes GCs proliferation. A RT-qPCR detected the interference efficiency of ASB9. Data are expressed as mean ± SEM (n = 6), **P < 0.01. B Western blotting reveals the expression levels of ASB9. C Quantification of the western blot analysis. Data are expressed as mean ± SEM (n = 3), *P < 0.05. D EdU staining was used to detect the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 4), **P < 0.01. E CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 16), ****P < 0.0001. F RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 5), *P < 0.05, **P < 0.01. G Western blot analysis of proliferation-related gene protein level (ASB9, CCNB1, CCNE1, CDK4, and CDKN1A). H Quantifying the western blot analysis of CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), *P < 0.05, **P < 0.01. I RNA expression of ASB9 in GCs. ZT: zone time

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Transfection, RNA Expression