Journal:

Article Title: Loss of Oncogenic H-ras-Induced Cell Cycle Arrest and p38 Mitogen-Activated Protein Kinase Activation by Disruption of Gadd45a

doi: 10.1128/MCB.23.11.3859-3871.2003

Figure Lengend Snippet: Disruption of Gadd45a abolishes p38 activation after H-ras overexpression. (A) wt and Gadd45a−/− MEF were infected with H-ras-expressing retrovirus, and activities of ERK, JNK, and p38 were analyzed, with MBP, GST-Jun, and GST-ATF2 as substrates, respectively. puro, puromycin. (B) At the same time that the protein extracts were obtained, the levels of p16/Ink4a, p53, and p21/Waf1 were determined. (C) MEF were infected with either puromycin- or H-ras-expressing retroviruses, and 5 days after selection with puromycin, mRNA was purified and the levels of p53-inducible genes (those encoding p21/Waf1, XPC, and ATF2) were analyzed by using a quantitative filter hybridization procedure (29). The relative induction ratio was obtained after dividing the relative level of mRNA after infection with H-ras retrovirus by the level of mRNA after infection with puromycin vector alone. (D) The levels of Gadd45a and p19/ARF mRNA in wt and Gadd45a−/− MEF on day 5 after retroviral infection were measured by Northern blotting. GAPD was included as a loading control.

Article Snippet: Analysis of protein levels was carried out by using polyclonal antibodies (Abs) against p16/ink4a (M-156; Santa Cruz) and p53 (AB7; Oncogene), monoclonal Abs against p21/Waf1 (AB4; Oncogene) and actin (AB1; Oncogene), anti-Flag monoclonal Ab M2 (Sigma) or anti-Flag monoclonal Ab M2 conjugated with horseradish peroxidase (Sigma), anti-HA polyclonal Ab (Covance Research Products), anti-Myc-tag monoclonal Ab 9E10 (Santa Cruz), anti-Gadd45a polyclonal Ab H-165 (Santa Cruz), anti-p38 polyclonal Ab C-20 (Santa Cruz), anti-JNK polyclonal Ab C-17 (Santa Cruz), and anti-ERK2 polyclonal Ab C-14 (Santa Cruz).

Techniques: Activation Assay, Over Expression, Infection, Expressing, Selection, Purification, Hybridization, Plasmid Preparation, Northern Blot

Journal:

Article Title: Loss of Oncogenic H-ras-Induced Cell Cycle Arrest and p38 Mitogen-Activated Protein Kinase Activation by Disruption of Gadd45a

doi: 10.1128/MCB.23.11.3859-3871.2003

Figure Lengend Snippet: Gadd45a and p38 are required for p53 activation after H-ras overexpression. (A) wt and Gadd45a−/− MEF were incubated in the presence of a MEK1 (50 μM PD98059) or p38 (10 μM SB202190) inhibitor, and protein extracts were obtained on day 5 after selection (see Materials and Methods). The levels of p16/Ink4a, p21/Waf1, and p53 proteins were analyzed. DMSO, dimethyl sulfoxide; puro, puromycin. (B) wt and Gadd45a−/− MEF were cotransfected with p53RE-CAT reporter plasmid and expression vectors containing either puromycin or H-ras. Some cells were additionally transfected with a dominant-negative p38α vector (p38DN). Four days later, cells were treated with either a MEK1 (PD90859) or p38 (SB202190) inhibitor, and CAT assays were carried out 12 h later. (C) wt and Gadd45a−/− MEF were cotransfected with p53RE-CAT reporter plasmid and expression vectors containing either puromycin or MKK6(E). Four days later, CAT activity was analyzed, and representative results are shown. Relative induction, as measured by increased CAT activity, was consistently twofold or greater in wt MEF compared to that in Gadd45a−/− MEF.

Article Snippet: Analysis of protein levels was carried out by using polyclonal antibodies (Abs) against p16/ink4a (M-156; Santa Cruz) and p53 (AB7; Oncogene), monoclonal Abs against p21/Waf1 (AB4; Oncogene) and actin (AB1; Oncogene), anti-Flag monoclonal Ab M2 (Sigma) or anti-Flag monoclonal Ab M2 conjugated with horseradish peroxidase (Sigma), anti-HA polyclonal Ab (Covance Research Products), anti-Myc-tag monoclonal Ab 9E10 (Santa Cruz), anti-Gadd45a polyclonal Ab H-165 (Santa Cruz), anti-p38 polyclonal Ab C-20 (Santa Cruz), anti-JNK polyclonal Ab C-17 (Santa Cruz), and anti-ERK2 polyclonal Ab C-14 (Santa Cruz).

Techniques: Activation Assay, Over Expression, Incubation, Selection, Plasmid Preparation, Expressing, Transfection, Dominant Negative Mutation, Activity Assay

Journal: bioRxiv

Article Title: Oral 4’-fluorouridine completely suppresses epidemic Oropouche virus replication in mice and confers protection against virus-induced disease

doi: 10.1101/2025.09.22.677733

Figure Lengend Snippet: (a) 4’FIU inhibits the replication of OROV in three cell lines. Dose–response curves of the antiviral activity of 4’-FIU determined by MTS-based CPE reduction assay. Cytotoxicity was determined by MTS cell viability assay in mock-infected cells. Data points are the mean ± SD of n = 4 biologically independent experiments. (b) OROV viral load reduction in human A549 and JEG-3 cells based on the quantification of viral RNA in the supernatant by RT-qPCR 3 dpi. Data points are mean ± SD of n=3 biologically independent experiments.

Article Snippet: We explored whether (i) a panel of molecules with reported antiviral activity against multiple RNA viruses [favipiravir, GS-441524 (the parent nucleoside of remdesivir), obeldesivir (an oral prodrug form of GS-441524), ribavirin, molnupiravir, galidesivir, sofosbuvir and 4′-Fluorouridine (4′FlU)], as well as (ii) baloxavir [an influenza endonuclease inhibitor with modest antiviral activity against Bunyamwera virus ], can inhibit the in vitro replication of an epidemic clinical isolate (OROV-IRCCS; obtained from a patient who returned from Cuba to Italy in July 2024) and a pre-epidemic reference OROV strain (isolated in the 1960’s; OROV-TR9760) in VeroE6, A549 and JEG3 cells ( ).

Techniques: Activity Assay, Viability Assay, Infection, Quantitative RT-PCR

Journal: BMC Medicine

Article Title: NUPR1 contributes to radiation resistance by maintaining ROS homeostasis via AhR/CYP signal axis in hepatocellular carcinoma

doi: 10.1186/s12916-022-02554-3

Figure Lengend Snippet: NUPR1 acts as a radioresistant oncogene in HCC in vitro . a Western blot was used to determine the protein expression of NUPR1 in a panel of HCC cell lines (MHCC-97H, MHCC-97L, Hep1, Hep3B, Huh7, and QGY-7701). Bar graphs presented the quantification of NUPR1 levels in HCC cells (below). b The protein and mRNA levels of NUPR1 in MHCC-97H/MHCC-97L cells with NUPR1 overexpression and QGY-7701/Hep3B with NUPR1 knockdown were verified by western blot and qRT-PCR. c Colony formation assays were employed in MHCC-97H/MHCC-97L cells with NUPR1 overexpression and QGY-7701/Hep3B cells with NUPR1 knockdown after an increased dose of IR treatment (0, 2, 4, 6 Gy). Survival curves were represented. d Bar graphs show the quantification of cell death in NUPR1 overexpression or knockdown cells by staining with 7-AAD after IR treatment (8 Gy). e DNA double-strand breaks of NUPR1-overexpressing MHCC-97H and NUPR1-knockdown QGY-7701 cells were detected by comet assays at 24 h after exposure to IR (8 Gy) (left, representative images, scale bar: 50 μm; right, bar graphs indicating the average tail moment per cell). f Western blot analysis was used to determine the protein levels of γH2AX in cells with a different NUPR1 expression status at the indicated time points after IR (8 Gy). Data are the mean of biological triplicates and are shown as the mean ± SD. P values: * P < 0.05; ** P < 0.01; *** P < 0.001 and ns, not significant by two-tailed Student’s t- test

Article Snippet: Antibodies against NUPR1 (15056-1-AP, 1:500, Proteintech), AhR (GTX22770, 1:200, GeneTex), CYP1A1 (13241-1-AP, 1: 200, Proteintech), Ki67 (#9449, 1:800, Cell Signaling), PCNA (A12427, 1:200, ABclonal), γH2AX (#9718, 1:400, Cell Signaling), MDA (ab24066, 1:100, Abcam) were incubated with the sections overnight at 4°C, respectively.

Techniques: In Vitro, Western Blot, Expressing, Over Expression, Knockdown, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: BMC Medicine

Article Title: NUPR1 contributes to radiation resistance by maintaining ROS homeostasis via AhR/CYP signal axis in hepatocellular carcinoma

doi: 10.1186/s12916-022-02554-3

Figure Lengend Snippet: NUPR1 enhances the radiation resistance of HCC cells in vivo . a , b Subcutaneous tumor formation in nude mice was established with NUPR1-overexpressing or control MHCC-97H cells ( n = 5/group). Tumor sizes were measured every 4 days using calipers (left, representative tumor samples; middle, growth curves of subcutaneous tumors; right, the statistical graph of tumor volumes). c Representative images of each group were photographed at the end of the experiment. d Growth curves of subcutaneous tumors (left) and tumor growth rates of mice treated with IR, ZZW-115, or IR plus ZZW-115 were represented (right). e IHC images of Ki67 and γH2AX expression in xenograft tumors derived from MHCC-97H cells with NUPR1 overexpression or empty vector were represented (left, scale bar: 50 μm). The positive stain (in percentages) was analyzed (right). f IHC images of PCNA and γH2AX expression in xenograft tumors derived from Hepa1-6 cells with different treatments were shown (left, scale bar: 50 μm). The positive stain (in percentages) was analyzed (right). Data are the mean of biological triplicates and are shown as the mean ± SD. P values: * P < 0.05; ** P < 0.01; *** P < 0.001 by two-tailed Student’s t -test, or by two-way ANOVA

Article Snippet: Antibodies against NUPR1 (15056-1-AP, 1:500, Proteintech), AhR (GTX22770, 1:200, GeneTex), CYP1A1 (13241-1-AP, 1: 200, Proteintech), Ki67 (#9449, 1:800, Cell Signaling), PCNA (A12427, 1:200, ABclonal), γH2AX (#9718, 1:400, Cell Signaling), MDA (ab24066, 1:100, Abcam) were incubated with the sections overnight at 4°C, respectively.

Techniques: In Vivo, Control, Expressing, Derivative Assay, Over Expression, Plasmid Preparation, Staining, Two Tailed Test

Journal: BMC Medicine

Article Title: NUPR1 contributes to radiation resistance by maintaining ROS homeostasis via AhR/CYP signal axis in hepatocellular carcinoma

doi: 10.1186/s12916-022-02554-3

Figure Lengend Snippet: NUPR1 inhibits ROS generation and oxidative stress via CYPs in HCC cells. a KEGG enrichment analysis of differentially expressed genes between NUPR1-overexpressing and control MHCC-97H cells showed that the metabolic pathway mediated by cytochrome P450 was downregulated in LV-NUPR1 cells. b The mRNA levels of genes included in CYP superfamily in MHCC-97H/MHCC-97L cells transfected with LV-NUPR1 or LV-NC were analyzed by qRT-PCR. Individual RNA values were normalized to β-actin values. c Western blot analysis was used to detect the protein expression of CYP1A1, CYP1B1, and CYP3A4 in NUPR1 overexpressing or knockdown cell lines treated with or without IR (8 Gy). d, e ROS levels and NADPH/NADP + ratio in stable NUPR1 overexpressing or knockdown cell lines were measured after exposure to 8 Gy of IR. f Bar graphs show the relative levels of apoptotic cells after IR treatment (8 Gy) by staining with annexin V and DAPI in indicated cells. g Western blot was used to detect the protein levels of total/cleaved caspase-3 and total/cleaved PARP in different NUPR1 expressing cell lines treated with or without IR (8 Gy). Data are the mean of biological triplicates and are shown as the mean ± SD. P values: * P < 0.05; ** P < 0.01; *** P < 0.001 and ns, not significant by Student’s t- test

Article Snippet: Antibodies against NUPR1 (15056-1-AP, 1:500, Proteintech), AhR (GTX22770, 1:200, GeneTex), CYP1A1 (13241-1-AP, 1: 200, Proteintech), Ki67 (#9449, 1:800, Cell Signaling), PCNA (A12427, 1:200, ABclonal), γH2AX (#9718, 1:400, Cell Signaling), MDA (ab24066, 1:100, Abcam) were incubated with the sections overnight at 4°C, respectively.

Techniques: Control, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Staining

Journal: BMC Medicine

Article Title: NUPR1 contributes to radiation resistance by maintaining ROS homeostasis via AhR/CYP signal axis in hepatocellular carcinoma

doi: 10.1186/s12916-022-02554-3

Figure Lengend Snippet: ROS generated by CYPs is indispensable for IR-induced cytotoxicity in cells with NUPR1 inactivation. a , b Representative images of colony formation were displayed in NUPR1-overexpressing MHCC-97H/MHCC-97L (3000 cells) and NUPR1-knockdown QGY-7701/Hep3B (500 cells and 3000 cells, respectively) pretreated with 5 mM NAC followed by exposure to 6 Gy of IR (left). The survival data were normalized to those of unirradiated control cells (right). c Quantification of cell death was employed in LV-NC/LV-NUPR1 or sh-NC/sh-NUPR1 cell lines pretreated with or without 5 mM NAC followed by exposure to 8 Gy of IR. d Western blot analysis was utilized to determine the expression levels of total/cleaved caspase-3 and total/cleaved PARP in the indicated cells pretreated with or without NAC upon IR (8 Gy). e Relative ROS levels were measured in NUPR1 overexpressing or knockdown cell lines pretreated with or without 20 μM alizarin followed by 8 Gy of IR. f Colony formation assays were applied in stably transfected NUPR1 overexpression or knockdown cells after IR (6 Gy) or combination with 20 μM alizarin treatment. Data are the mean of biological triplicates and are shown as the mean ± SD. P values: * P < 0.05; ** P < 0.01; *** P < 0.001 and ns, not significant by Student’s t- test

Article Snippet: Antibodies against NUPR1 (15056-1-AP, 1:500, Proteintech), AhR (GTX22770, 1:200, GeneTex), CYP1A1 (13241-1-AP, 1: 200, Proteintech), Ki67 (#9449, 1:800, Cell Signaling), PCNA (A12427, 1:200, ABclonal), γH2AX (#9718, 1:400, Cell Signaling), MDA (ab24066, 1:100, Abcam) were incubated with the sections overnight at 4°C, respectively.

Techniques: Generated, Knockdown, Control, Western Blot, Expressing, Stable Transfection, Transfection, Over Expression

Journal: BMC Medicine

Article Title: NUPR1 contributes to radiation resistance by maintaining ROS homeostasis via AhR/CYP signal axis in hepatocellular carcinoma

doi: 10.1186/s12916-022-02554-3

Figure Lengend Snippet: NUPR1 binds to AhR and promotes degradation of AhR in the autophagy-lysosome pathway. a Western blot was used to examine the protein levels of AhR, ARNT, and HSP90 in NUPR1-overexpressing MHCC-97H/MHCC-97L cells and NUPR1-knockdown QGY-7701/Hep3B cells with or without IR (8 Gy). b Immunofluorescence staining was performed to determine the location of AhR (green) in the indicated cell lines. The nuclei were counterstained with DAPI (blue). Scale bar: 10 μm. c Western blot was used to detect AhR levels in indicated cell lysates collected at different timing (0, 3, 6, 9 h) after 20 μg/mL CHX treatment. d Western blot of AhR protein was performed in cell lysates from different NUPR1 expression status cell lines with or without 20 μM CQ treatment. e Representative immunofluorescence images show the distribution of AhR (red), LAMP1 (green), and DAPI (blue) in LV-NC and LV-NUPR1 MHCC-97L cells. Scale bar: 10 μm. f The levels of LC3-II and p62 in cells with a different NUPR1 expression were determined by western blot. g The anti-Flag and anti-IgG products after incubating with lysates from MHCC-97H/MHCC-97L ectopically expressed Flag-tagged NUPR1 were used to detect Flag-NUPR1 and AhR protein by western blot. h Western blot analysis of NUPR1 and AhR in anti-AhR and anti-IgG products was performed. Cell lysates were immunoprecipitated with antibodies against AhR or IgG. i Representative immunofluorescence images of QGY-7701/MHCC-97H cells show the staining of NUPR1 (red), AhR (green), and nuclei counterstained with DAPI (blue) (left). Scale bar: 10 μm. Pearson correlation of the signal intensity of indicated molecules in different subcellular locations was quantified by ImageJ software (N/nucleus, C/cytoplasm, right)

Article Snippet: Antibodies against NUPR1 (15056-1-AP, 1:500, Proteintech), AhR (GTX22770, 1:200, GeneTex), CYP1A1 (13241-1-AP, 1: 200, Proteintech), Ki67 (#9449, 1:800, Cell Signaling), PCNA (A12427, 1:200, ABclonal), γH2AX (#9718, 1:400, Cell Signaling), MDA (ab24066, 1:100, Abcam) were incubated with the sections overnight at 4°C, respectively.

Techniques: Western Blot, Knockdown, Immunofluorescence, Staining, Expressing, Immunoprecipitation, Software

Journal: BMC Medicine

Article Title: NUPR1 contributes to radiation resistance by maintaining ROS homeostasis via AhR/CYP signal axis in hepatocellular carcinoma

doi: 10.1186/s12916-022-02554-3

Figure Lengend Snippet: AhR/CYP signal axis is required for NUPR1-mediated radioresistance in HCC. a Western blot analysis was utilized to examine the expression levels of AhR, CYP1A1, and CYP1B1 in different NUPR1 expressing cell lines transfected with AhR-overexpressing vector or control vector. b Relative ROS levels in different NUPR1 expressing cells with AhR overexpression or negative control were measured after exposure to 8 Gy of IR. c Quantification of cell death in indicated cells with AhR overexpression or negative control was measured after IR (8 Gy). d Cells with different NUPR1 expressions were pretreated with 5 μM FICZ or CH223191 for 24 h, followed by being exposed to 8 Gy of IR. The cells were collected to measure relative ROS levels. e, f Colony formation assays were performed in stably transfected NUPR1 overexpression or knockdown cell lines pretreated with FICZ or CH223191, followed by exposure to 6 Gy of IR. Data are the mean of biological triplicates and are shown as the mean ± SD. P values: * P < 0.05; ** P < 0.01; *** P < 0.001 and ns, not significant by two-tailed Student’s t- test

Article Snippet: Antibodies against NUPR1 (15056-1-AP, 1:500, Proteintech), AhR (GTX22770, 1:200, GeneTex), CYP1A1 (13241-1-AP, 1: 200, Proteintech), Ki67 (#9449, 1:800, Cell Signaling), PCNA (A12427, 1:200, ABclonal), γH2AX (#9718, 1:400, Cell Signaling), MDA (ab24066, 1:100, Abcam) were incubated with the sections overnight at 4°C, respectively.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Over Expression, Negative Control, Stable Transfection, Knockdown, Two Tailed Test

Journal: BMC Medicine

Article Title: NUPR1 contributes to radiation resistance by maintaining ROS homeostasis via AhR/CYP signal axis in hepatocellular carcinoma

doi: 10.1186/s12916-022-02554-3

Figure Lengend Snippet: NUPR1 is upregulated in HCC tissues and predicts radiotherapeutic resistance of HCC. a NUPR1 mRNA expression in unpaired HCC tissues and non-tumor liver tissues from TCGA and GSE14520 datasets were shown (left and middle). NUPR1 mRNA expression in 50 paired HCC tissues and the adjacent matched noncancerous tissues was displayed on the right. b Representative IHC images show the NUPR1 expression in HCC ( n = 50) and corresponding adjacent liver tissues ( n = 50, left). Scale bar: 200 μm in 4 × magnification and 50 μm in 20 × magnification. Quantification of IHC score was shown in boxplot (right). c Gene set enrichment analysis (GSEA) with relatively low-expressed versus high-expressed NUPR1 from TCGA LIHC and GSE14520 datasets showed that the oxygen species pathway was positively correlated with NUPR1 expression. d The mRNA level of NUPR1 was negatively correlated with the CYP1B1 and CYP3A4 expression in the GSE15654 dataset by using Pearson correlation analysis. e Kaplan-Meier survival analysis was used to assess the correlation between NUPR1 expression or combined with AhR, CYP1B1 level, and the overall survival of HCC patients in TCGA LIHC dataset ( n = 366) and GES15654 dataset ( n = 216). f NUPR1 and AhR expression in 13 HCC tissues from the patients who accepted RT treatment were analyzed by IHC. Representative IHC images (left) and quantified IHC score (right) are shown

Article Snippet: Antibodies against NUPR1 (15056-1-AP, 1:500, Proteintech), AhR (GTX22770, 1:200, GeneTex), CYP1A1 (13241-1-AP, 1: 200, Proteintech), Ki67 (#9449, 1:800, Cell Signaling), PCNA (A12427, 1:200, ABclonal), γH2AX (#9718, 1:400, Cell Signaling), MDA (ab24066, 1:100, Abcam) were incubated with the sections overnight at 4°C, respectively.

Techniques: Expressing

Journal: BMC Medicine

Article Title: NUPR1 contributes to radiation resistance by maintaining ROS homeostasis via AhR/CYP signal axis in hepatocellular carcinoma

doi: 10.1186/s12916-022-02554-3

Figure Lengend Snippet: A schematic model of the NUPR1/AhR/CYP signal axis showing it promotes radiation resistance of HCC

Article Snippet: Antibodies against NUPR1 (15056-1-AP, 1:500, Proteintech), AhR (GTX22770, 1:200, GeneTex), CYP1A1 (13241-1-AP, 1: 200, Proteintech), Ki67 (#9449, 1:800, Cell Signaling), PCNA (A12427, 1:200, ABclonal), γH2AX (#9718, 1:400, Cell Signaling), MDA (ab24066, 1:100, Abcam) were incubated with the sections overnight at 4°C, respectively.

Techniques: