Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: RASSF10 is Epigenetically Inactivated and Suppresses Cell Proliferation and Induces Cell Apoptosis by Activating the p53 Signalling Pathway in Papillary Thyroid Carcinoma Cancer.

doi: 10.1159/000464386

Figure Lengend Snippet: Fig. 5. RASSF10 and p53 regulated PTC cell apoptosis. Apoptosis was determined using flow cytometry analysis of Annexin V/PI double-stained K1 cells. *P < 0.05 versus control group, #P < 0.05 versus RASSF10 group.

Article Snippet: Expression vector construction and transfection The open reading frame (ORF) of the RASSF10 gene was generated by RT-PCR, and p53 siRNA and shRASSF10 were purchased from OriGene Technologies (Rockville, USA).

Techniques: Flow Cytometry, Staining, Control

Journal: Molecular Cancer Therapeutics

Article Title: Interleukin-24 overcomes temozolomide resistance and enhances cell death by down-regulation of O6-methylguanine-DNA methyltransferase in human melanoma cells

doi: 10.1158/1535-7163.mct-08-0516

Figure Lengend Snippet: Figure 2. Reversal of temozolomide resistance by Ad-IL-24 occurs via down-regulation of MGMT. A, Ad-IL-24 enhanced temozolomide-induced killing and overcame temozolomide resistance in melanoma cells. Temozolomide-resistant cell lines (MeWo, SK-MEL-28, and WM1341) and temozolomide-sensitive cell line (A375 and WM35) were treated with either PBS control (C), 200 Amol/L temozolomide (T), 2,000 vp/cell Ad-IL- 24 (M), 2,000 vp/cell Ad-luc control (L), temozolomide + Ad-luc (TL), or temozolomide + Ad-IL-24 (TM) for 72 h. Results of cell counting by trypan blue exclusion assay. Mean F SD of three independent experi- ments. Killing activity of temozolomide + Ad-IL-24 was significantly greater than that of temozolomide in all melanoma cells (P < 0.01). Combinatorial synergy (+) or without this effect () was statistically analyzed. B, Ad-IL-24 down-regulates MGMT. MeWo and SK-MEL-28 cells were treated with 3,000 vp/cell Ad-luc or varying doses (1,000- 3,000 vp/cell) Ad-IL-24 for 72 h. Western blot analysis of MGMT was done as in Fig. 1A. C, Ad-IL-24 activates p53 pathway. Temozolomide- resistant (MeWo) cells were treated with either PBS control, 200 Amol/L temozolomide, 2,000 vp/cell Ad-IL-24, 2,000 vp/cell Ad-luc control, temozolomide + Ad-luc, or temozolomide + Ad-IL-24 for 48 h and cytosol proteins were analyzed by Western blot for p53, phospho-p53, p21WAF1/CIP1, and MGMT. h-Actin was used a loading control.

Article Snippet: The transfections of MGMT and p53 siRNA were executed according to the Santa Cruz Biotechnology provided siRNA transfection protocol.

Techniques: Control, Cell Counting, Trypan Blue Exclusion Assay, Activity Assay, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: Interleukin-24 overcomes temozolomide resistance and enhances cell death by down-regulation of O6-methylguanine-DNA methyltransferase in human melanoma cells

doi: 10.1158/1535-7163.mct-08-0516

Figure Lengend Snippet: Figure 5. Requirement of p53 accumulation for MGMT expression. A, blocking MGMT abolishes synergistic cell killing by IL-24 + temozolomide combination. MeWo cells were transfected with control siRNA or MGMT siRNA for 72 h. Left, cell lysates were extracted and Western blot analysis was done to examine p53, p21, and MGMT. h-Actin was used a loading control. Middle, A375M transfected with control shRNA- or MGMT-targeted shRNA for 48 h. Cell lysates were extracted and Western blot analysis was done to examine MGMT. A375 and A375M was used as control. Right, MeWo and A375M cells transfected with MGMT shRNA or control shRNA were treated with PBS, 39 ng/mL IL-24, 200 Amol/L temozolomide + Ad-IL-24, or temozolomide + IL-24 as indicated for 96 h. Cell death was plotted against treatment. Mean F SD of two independent experiments using triplicate samples. *, P < 0.01, compared with control siRNA-transfected cells. +, combinatorial synergy; -, without combinatorial synergy. B, blocking p53 with siRNA abolishes combinatorial synergy of cell killing by IL-24 + temozolomide. Left, MeWo cells were transfected with control siRNA or p53 siRNA for 72 h. Cell lysates were extracted and Western blot analysis was done to examine p53, p21, and MGMT. h-Actin was used a loading control. Middle, A375M transfected with control siRNA- or MGMT-targeted siRNA for 48 h. Cell lysates were extracted and Western blot analysis was done to examine p53. A375 and A375M was used as control. Right, MeWo and A375M cells transfected with SignalSilence p53 siRNA or control siRNA were treated with PBS, 39 ng/mL IL-24, 200 Amol/L temozolomide, or temozolomide + IL-24 as indicated for 96 h. Cell death was plotted against treatment. Mean F SD of two independent experiments using triplicate samples. *, P < 0.01, compared with control siRNA-transfected cells. C, blocking p53 function with dominant-negative mutant abolishes synergistic activity on cell killing by IL-24 + temozolomide combination. MeWo cells were transfected with pEGFP- N1, pP53wt, or pP53mut. Cell lysates were extracted after 48 h and Western blot analysis was done to examine p53 and p21. h-Actin was used a loading control. p53 wild-type, p53 mutant, or GFP plasmid transfected MeWo cells were treated with PBS, 39 ng/mL IL-24, 200 Amol/L temozolomide + Ad-IL- 24, or temozolomide IL-24 as indicated for 96 h. Cell death was plotted against treatment. Mean F SD of two independent experiments using triplicate samples. *, P < 0.01, compared with pEGFP-N1-transfected MeWo cells.

Article Snippet: The transfections of MGMT and p53 siRNA were executed according to the Santa Cruz Biotechnology provided siRNA transfection protocol.

Techniques: Expressing, Blocking Assay, Transfection, Control, Western Blot, shRNA, Dominant Negative Mutation, Activity Assay, Mutagenesis, Plasmid Preparation

Journal: Oncotarget

Article Title: Mechanisms of the anti-tumor activity of Methyl 2-(-5-fluoro-2-hydroxyphenyl)-1 H-benzo[d]imidazole-5-carboxylate against breast cancer in vitro and in vivo

doi: 10.18632/oncotarget.16263

Figure Lengend Snippet: ( A ) Illustration of p53 status and its downstream effect in MCF-7 and MDA-MB-231 cells post MBIC treatment: Functional wild-type p53 in MCF-7 cells suppresses survivin and Cdk1 protein levels after MBIC application. Low protein level of Cdk1 causes lack of phosphorylated survivin. Low protein level of survivin and p-survivin causes release and activation of caspases in cytoplasm which leads to apoptosis (at IC 50 dosage of MBIC and higher). In contrast, dysfunctional mutated p53 in MDA-MB-231 cells, is not capable of inhibiting survivin and Cdk1 proteins after MBIC application. Therefore, there are plenty of survivin in the cell to be phosphorylated and there are enough Cdk1 to phosphorylate the survivin in Thr34 region. Thus, phosphorylated survivin binds to caspases and inhibits their activation (at dosage above 40 μM). ( B ) MBIC alters affinity of survivin for caspases in MCF-7 and MDA-MB-231 cell-lines with different variations: MDA-MB-231 and MCF-7 cells were treated with MBIC dose-dependently. Complex formation of p-survivin with caspases-3/7/9 was detected while performing Co-IP assay followed by Western blot analysis. ( C ) Bar graph represents dual quantification of p-survivin and caspases-3/7/9 protein intensities in complex formation which are normalized with β-actin. Data were results of three independent experiments with mean ± SD. All the treatment groups were compared with control.”*” indicates statistically significant at P < 0.05.

Article Snippet: For gene knockdown, Signal Silence ® p53 siRNA II (#6562) and Signal Silence ® survivin siRNA II (#65546) (Cell Signaling Technology (CST), USA) were used.

Techniques: Functional Assay, Activation Assay, Co-Immunoprecipitation Assay, Western Blot, Control

Journal: Oncotarget

Article Title: Mechanisms of the anti-tumor activity of Methyl 2-(-5-fluoro-2-hydroxyphenyl)-1 H-benzo[d]imidazole-5-carboxylate against breast cancer in vitro and in vivo

doi: 10.18632/oncotarget.16263

Figure Lengend Snippet: ( A ) Cells were transfected with different concentrations of p53 and survivin siRNA, mock-transfection control (Mock-TF) or not transfected control (Non-TF). Western blot analysis was carried out to identify the effective concentration of each siRNA. P53 siRNA at concentration of 26.6 nM succeeded to silence p53 in MCF-7 cells. Survivin siRNA at concentration of 100 nM succeeded to silence survivin in MDA-MB-231 and MCF-7 cells. β-actin served as a loading control. ( B ) IC 50 of both cell-lines after silencing p53 and survivin. ( C ) Western blot analysis was carried out to evaluate caspases-3/7/9 activation after survivin siRNA transfection in MDA-MB-231 cells. Cells were divided into six groups: (1) Non-TF and untreated, (2) Non-TF and 80 μM (4-fold > IC 50 ) of MBIC treated, (3) Mock-TF and 10 μM (2-fold < IC 50 ) of MBIC treated, (4) Survivin siRNA transfected and 10 μM of MBIC treated, (5) Mock-TF and 20 μM (= IC 50 ) of MBIC treated and (6) Survivin siRNA transfected and 20 μM of MBIC treated. β-actin served as a loading control. ( D ) Cell cycle analysis was performed to investigate the absence or presence of mitotic slippage in MDA-MB-231 cell-line, in four different survivin siRNA transfected groups (with/without MBIC treatment), one Non-TF-untreated group and one Mock-TF-untreated group. Data were results of three independent experiments with mean ± SD.

Article Snippet: For gene knockdown, Signal Silence ® p53 siRNA II (#6562) and Signal Silence ® survivin siRNA II (#65546) (Cell Signaling Technology (CST), USA) were used.

Techniques: Transfection, Control, Western Blot, Concentration Assay, Activation Assay, Cell Cycle Assay

Journal: International Journal of Molecular Sciences

Article Title: Differences in the Relative Abundance of ProBDNF and Mature BDNF in A549 and H1299 Human Lung Cancer Cell Media

doi: 10.3390/ijms22137059

Figure Lengend Snippet: Treatment of cells with the MMP2/9 inhibitor resulted in a comparable decrease and increase in the levels of mBDNF and proBDNF, respectively, in the media of both A549 and H1299 cells, while the opposite was observed upon treatment with the p53 inhibitor, pifithrin-α, in A549 cell media only. Cells (0.2 × 10 5 ) were grown in 10% FBS-supplemented media for 24 h. The following day, the cell monolayers were incubated in serum-free media for 24 h, then treated as indicated for 72 h with the inhibitors, as described in the Methods Section. The media was collected, and then the same amount of protein (3 µL of 600 µg/mL total protein) of each sample was used to quantitate mBDNF and proBDNF (Methods) in media from A549 ( A ) and H1299 ( B ) cells. Data from five independent assays, each carried out in triplicate, were quantitated, averaged, normalized, and expressed as fold change relative to control media in the absence of added inhibitors using the GraphPad 9.0.2 software. The graphs summarize the results expressed as means ± SD ( n = 5). Asterisks (*) indicate a statistically significant difference from the corresponding cell line control, * p < 0.05, ** p < 0.01, Mann–Whitney test.

Article Snippet: SignalSilence p53 siRNA I (6231), SignalSilence Control siRNA (Unconjugated, 6568), and p53 antibody (9282) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Incubation, Control, Software, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Differences in the Relative Abundance of ProBDNF and Mature BDNF in A549 and H1299 Human Lung Cancer Cell Media

doi: 10.3390/ijms22137059

Figure Lengend Snippet: Higher levels of MMP2 and MMP9 were found in the conditioned media of A549 cells upon treatment with the p53 inhibitor, pifithrin-α. Cells (0.2 × 10 5 ) were grown in 10% FBS-supplemented media for 24 h. The following day, the cell monolayers were incubated in serum-free media for 24 h and then treated as indicated for 72 h with the inhibitors as described in the Methods Section. The media was collected then the same amount of protein (3 µL of 600 µg/mL total protein) of each sample was used to quantitate MMPs (Methods) in media from A549 ( A ) and H1299 ( B ) cells. Data from five independent assays, each carried out in triplicate, were quantitated, averaged, normalized, and expressed as fold change relative to cells not treated with inhibitors (Media) using the GraphPad 9.0.2 software. The graphs summarize the results expressed as means ± SD ( n = 5). Asterisks (*) indicate a statistically significant difference from the samples (Media) without inhibitor treatment for each cell line, Mann–Whitney test. Statistical differences between different groups were analyzed by an ordinary one-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test. ** p < 0.01.

Article Snippet: SignalSilence p53 siRNA I (6231), SignalSilence Control siRNA (Unconjugated, 6568), and p53 antibody (9282) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Incubation, Software, MANN-WHITNEY, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Differences in the Relative Abundance of ProBDNF and Mature BDNF in A549 and H1299 Human Lung Cancer Cell Media

doi: 10.3390/ijms22137059

Figure Lengend Snippet: The activity of either PI3K or AKT and the phospho/Total NFκB ratio are upregulated upon treatment of A549 cells with the p53 inhibitor, pifithrin-α. Cells (0.2 × 10 5 ) were grown in 10% FBS-supplemented media for 24 h. The following day, the cell monolayers were incubated in serum-free media for 24 h and then treated as indicated for 72 h with the inhibitors. The PI3K activity ( A ) was assayed by the total in-cell ELISA Kit, and the AKT ( B ) and NFκB ( C ) activities were measured on the same amount of protein (3 µL of 600 µg/mL total protein) of the cell lysate as described in the Methods Section. Data from five independent assays, each carried out in triplicate, were quantitated, averaged, normalized, and expressed as fold change relative to cells not treated with inhibitors (Media) using the GraphPad 9.0.2 software. The graphs summarize the results expressed as means ± SD ( n = 5). Asterisks (*) indicate a statistically significant difference from the corresponding samples without inhibitor treatment (Media) for each cell line, Mann–Whitney test, while the absence of asterisks indicates no significance. * p < 0.05, ** p < 0.01.

Article Snippet: SignalSilence p53 siRNA I (6231), SignalSilence Control siRNA (Unconjugated, 6568), and p53 antibody (9282) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, Incubation, In-Cell ELISA, Software, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Differences in the Relative Abundance of ProBDNF and Mature BDNF in A549 and H1299 Human Lung Cancer Cell Media

doi: 10.3390/ijms22137059

Figure Lengend Snippet: Treatment of A549 cells with p53 siRNA resulted in increased levels of mBDNF while treatment with MMP9 siRNA decreased those levels in the media of both cell lines and corresponded with increased proBDNF. Cells (0.2 × 10 5 ) were grown in 10% FBS-supplemented media for 24 h. The following day, the cell monolayers were incubated in serum-free media for 24 h and then treated for 72 h with the indicated siRNAs as described in the Methods Section. The same concentration of total protein (15 µL of 600 µg/mL) of the cell lysates ( A ) was used for Western blotting using the indicated antibodies. As a loading control, anti α-tubulin antibodies were used. The same amount of protein (3 µL of 600 µg/mL total protein) of the media was used to quantitate the levels of mBDNF ( B ) and proBDNF ( C ) (Methods). Data from five independent assays, each carried out in triplicate, were quantitated, averaged, normalized, and expressed as fold change relative to cells transfected with control siRNA using the GraphPad 9.0.2 software. The graphs summarize the results expressed as means ± SD ( n = 5). Asterisks (*) indicate a statistically significant difference from the corresponding samples transfected with control siRNA, ** p < 0.01 of each cell line. Absence of asterisks indicates no significance, Mann–Whitney test.

Article Snippet: SignalSilence p53 siRNA I (6231), SignalSilence Control siRNA (Unconjugated, 6568), and p53 antibody (9282) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Incubation, Concentration Assay, Western Blot, Control, Transfection, Software, MANN-WHITNEY

Journal: Scientific Reports

Article Title: LF-MF inhibits iron metabolism and suppresses lung cancer through activation of P53-miR-34a-E2F1/E2F3 pathway

doi: 10.1038/s41598-017-00913-2

Figure Lengend Snippet: Low frequency MF up-regulated the expression of miR-34a by stabilizing p53 protein. ( A ) Q-PCR analyzes the mRNA levels of P53 in LLC cells treated with MF or Sham MF. ( B ) Protein level of P53 in LLC cells treatment with MF or Sham MF was detected using western Blot. ( C ) The mRNA levels of P53 in LLC cells transfected with siRNA-P53 was detected using Q-PCR. ( D ) Protein level of P53 in LLC cells transfected with siRNA-P53 was detected using western Blot. Numbers under each blot are relative intensity of the blot. (E ) Q-PCR analyzes the miR-34a level after treatment of siRNA-TP53 and H2O2. ( F ) P53 expression in tumor tissue of LLC murine model was detected using IHC staining; Scale bars, 100 µm. ( G ) IHC images were calculated using Image Pro Plus software 6.0 and bar graphs. All experiments were repeated three times. Data represent Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: P53 specific siRNAs were purchased from RiboBio (Guangzhou, China).

Techniques: Expressing, Western Blot, Transfection, Immunohistochemistry, Software

Journal: Scientific Reports

Article Title: LF-MF inhibits iron metabolism and suppresses lung cancer through activation of P53-miR-34a-E2F1/E2F3 pathway

doi: 10.1038/s41598-017-00913-2

Figure Lengend Snippet: Low frequency MF stabilized p53 protein via inducing cell iron metabolism dysfunction. A549 and LLC cells were pre-incubated with medium containing FAC (100 uM) for 24 h and were exposure to MF or Sham MF for 6 days. ( A ) Protein level of P53 in LLC cells was detected using western blot. Numbers under each blot are relative intensity of the blot. ( B ) Protein expression of TfR and ferritin in LLC cells were detected using western blot. Numbers under each blot are relative intensity of the blot. ( C , D ) Cell proliferation of A549 cells and LLC cells was detected using CCK-8 assay. ( E , F ) Cell cycle of A549 and LLC cells were detected using flow cytometery. ( G , H ) Mean cell proportion in each phase was shown. ( I , J ) Representative image of SA-β-Gal staining assay was showed and numbers of SA-β-Gal positive cells was calculated. All experiments were repeated three times. Data represent Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: P53 specific siRNAs were purchased from RiboBio (Guangzhou, China).

Techniques: Incubation, Western Blot, Expressing, CCK-8 Assay, Staining

Journal: Theranostics

Article Title: Glucose homeostasis controls N-acetyltransferase 10-mediated ac4C modification of HK2 to drive gastric tumorigenesis

doi: 10.7150/thno.104310

Figure Lengend Snippet: NAT10-mediated ac4C modification of HK2 mRNA maintains its stability. (A) The distribution of ac4C-containing peaks across mRNAs in NAT10-overexpressing (left panel) and NAT10 knockout (right panel) GC cells is displayed in a metagene plot. (B) Representative pie chart of the peak distribution showing the proportions of total ac4C peaks in the indicated regions, including the 5′-untranslated region (5′-UTR), coding sequence (CDS), 3′- untranslated region (3′-UTR), start codon, and stop codon. (C) Consensus motif in AGS WT and NAT10-knockout cells identified by HOMER. (D) Venn diagram showing HK2 as the selected candidate target gene of NAT10. (E) The mRNA level of HK2 in NAT10-overexpressing and NAT10-KO GC cells was measured by qRT-PCR. (F) The protein level of HK2 in NAT10-overexpressing and NAT10-KO GC cells was measured by Western blotting. (G) Sections of xenograft tumors derived from NAT10-overexpressing (upper panel) and NAT10-knockdown (bottom panel) GC cells subcutaneously injected into nude mice were stained with an anti-HK2 antibody for IHC analysis (scale bars = 50 μm). (H) Sections of organoids formed from cells transduced with the NAT10 overexpression or control lentiviral vector were subjected to H&E staining or IHC staining with an anti-HK2 antibody (scale bars = 50 μm). (I) Sections of stomachs from NAT10 WT and cKO mice were stained with an anti-HK2 antibody for IHC analysis (scale bars = 100 μm). (J) Integrative Genomics Viewer (IGV) tracks revealing the ac4Cseq read distribution on HK2 mRNA in NAT10-overexpressing and NAT10-knockout GC cells. (K) acRIP-qPCR analysis was employed to demonstrate the presence of NAT10-mediated HK2 ac4C modifications. ac4C modification of HK2 was increased upon overexpression of NAT10, while it was decreased upon knockout of NAT10. (L) The level of HK2 expression in NAT10-overexpressing, NAT10-knockout and the corresponding control GC cells treated with actinomycin D (2 μg/mL) at the indicated time points was measured by qRT-PCR. (M) Schematic representation of the construction of the luciferase reporter vectors containing the HK2 WT, Mut1, Mut2, or Both Mut sequences (left panel). The relative luciferase activities of the HK2 WT, Mut1, Mut2, and Both Mut luciferase reporters in AGS cells with NAT10 knockout and the corresponding control cells were measured (right panel). H&E, haematoxylin and eosin; IHC, immunohistochemical; GC, gastric cancer; WT, wild type; KO, knockout. The statistical data in this figure are presented as the mean values ± SDs of three independent experiments. Statistical significance was determined by a two-tailed t test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: siRNAs targeting NAT10 or HK2 were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Modification, Knock-Out, Sequencing, Quantitative RT-PCR, Western Blot, Derivative Assay, Knockdown, Injection, Staining, Transduction, Over Expression, Control, Plasmid Preparation, Immunohistochemistry, Expressing, Luciferase, Immunohistochemical staining, Two Tailed Test

Journal: Theranostics

Article Title: Glucose homeostasis controls N-acetyltransferase 10-mediated ac4C modification of HK2 to drive gastric tumorigenesis

doi: 10.7150/thno.104310

Figure Lengend Snippet: NAT10 accelerates malignant progression of GC by upregulating HK2. (A) The HK2 protein level was measured in GC tissues and paired normal gastric mucosal tissues by Western blotting (n = 5). (B) Representative IHC images of the tissue microarray analysed with the anti-HK2 antibody (scale bars = 200 or 100 μm) are shown (left panel). The distribution of the difference in the HK2 immunoreactivity score (IRS) (△IRS = IRST - IRSN) is shown. The IRS for HK2 staining was available for 192 pairs of tissues. (C) Representative images of HK2 IHC staining in tissues from GC patients in the SUVmax-high and SUVmax-low groups (left panel). Scale bars = 50 μm; n = 22; statistical analysis of the difference in HK2 expression between the SUVmax-high group and the SUVmax-low group (right panel). (D) Correlation analysis between the SUVmax and HK2 protein expression level based on the IHC score in GC tissue microarrays with associated PET/CT data. R, Pearson correlation coefficient; n = 22. (E) Kaplan-Meier analysis of OS in GC patients stratified by HK2 expression (n = 192, P = 6.4e-15; log-rank test). (F) Multivariate analyses were performed for the GC cohort. (G) Glucose uptake (left panel) and lactate production (right panel) in HK2-knockdown AGS cells were measured. (H) A colony formation assay was performed in stable HK2-knockdown AGS cells (left panel). Quantitative analysis of the colony formation assay results (right panel). (I) NAT10 expression was positively correlated with HK2 protein expression in GC tissues (linear regression) according to analysis of the IHC score from the TMA data (n = 192). (J) Correlation analysis of NAT10 and HK2 protein expression based on the IHC score in GC tissue microarrays with associated PET/CT data (n = 22). (K) The colony formation ability was evaluated in NAT10-overexpressing BGC823 cells with or without stable HK2 knockdown or the corresponding controls. Representative images (left panel) and quantitative results (right panel) are shown. (L) Lactate production was measured in NAT10-overexpressing BGC823 cells with or without stable HK2 knockdown or the corresponding controls. CI, confidence interval; GC, gastric cancer; HR, hazard ratio; IHC, immunohistochemical; OS, overall survival; TNM, tumor, node, metastasis. The statistical data in this figure are presented as the mean value ± SD of three independent experiments. Statistical significance was determined by a two-tailed t test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: siRNAs targeting NAT10 or HK2 were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Western Blot, Microarray, Staining, Immunohistochemistry, Expressing, Positron Emission Tomography-Computed Tomography, Knockdown, Colony Assay, Immunohistochemical staining, Two Tailed Test

Journal: Theranostics

Article Title: Glucose homeostasis controls N-acetyltransferase 10-mediated ac4C modification of HK2 to drive gastric tumorigenesis

doi: 10.7150/thno.104310

Figure Lengend Snippet: Targeting the NAT10-HK2 axis in GC cells has clinical value. (A) Different GC cell lines were treated with 10 μM remodelin for 24 h. Then, the protein level of NAT10 was measured by Western blot analysis (upper panel). mRNA isolated from GC cells were subjected to dot blot analysis with an anti-ac4C antibody (bottom panel). MB staining served as a loading control. (B) Glucose uptake (left panel) and lactate production (right panel) were measured in MKN45 cells after treatment with 10 μM Remodelin for 24 h. (C) Remodelin inhibited subcutaneous tumor growth in nude mice (n = 6, left panel). The tumor volume was monitored every other day, and tumor growth curves were generated (middle panel). The tumors were extracted and weighed after 25 days (right panel). (D) Sections of tumors were stained with anti-Ki-67, anti-HK2, and anti-NAT10 antibodies for IHC analysis (scale bars = 50 μm). (E) Representative images of GC organoids treated with 10 μM Remodelin for the indicated durations (scale bars = 100 μm, left panel) and quantification of organoid diameters (right panel). (F) Sections of organoids treated with Remodelin were subjected to H&E staining or stained with anti-Ki-67, anti-HK2, and anti-NAT10 antibodies for IHC analysis (scale bars = 50 μm). (G) The combination of NAT10 and HK2 was evaluated as a new two-gene risk signature, and multivariate analyses were performed for the GC cohort. (H) GC patients were divided into three subgroups according to the median expression level of each protein: high expression of both NAT10 and HK2, low expression of both NAT10 and HK2, and other expression patterns (NAT10 high and HK2 low OR NAT10 low and HK2 high). Kaplan-Meier analysis of survival in the three subgroups of GC patients. (I) Time-dependent receiver operating characteristic (ROC) curve analysis of the NAT10 risk score, the HK2 risk score, and the combined NAT10/HK2 score in the GC cohort. AUC, area under the curve; CI, confidence interval; GC, gastric cancer; HR, hazard ratio; IHC, immunohistochemical; OS, overall survival; TNM, tumor, node, metastasis. (J) Graphical illustration of the mechanism by which NAT10 modulates glycolysis, promoting GC growth. The statistical data in this figure are presented as the mean value ± SD of three independent experiments. Statistical significance was determined by a two-tailed t test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: siRNAs targeting NAT10 or HK2 were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Western Blot, Isolation, Dot Blot, Staining, Control, Generated, Expressing, Immunohistochemical staining, Two Tailed Test

Journal: Frontiers in Physiology

Article Title: The Oncogenic Role of Tribbles 1 in Hepatocellular Carcinoma Is Mediated by a Feedback Loop Involving microRNA-23a and p53

doi: 10.3389/fphys.2017.00789

Figure Lengend Snippet: TRIB1 is overexpressed in human HCC tissues and cell lines. (A) qRT-PCR analysis of TRIB1 and p53 mRNA expression in HCC tumors and adjacent non-tumor tissues. (B) We have made a pooled dot plot to verify the correlation again in HCC patients. (C) Immunohistochemical analysis of TRIB1 in HCC (left panel) and adjacent liver tissues (right panel) (original magnification, × 200). (D) Western blot analysis of TRIB1 protein expression in the normal liver cell line LO2 and the HCC cell lines SMMC-7721, HepG2, and Huh 7. β-actin was used as an internal control. (E) qRT-PCR analysis of TRIB1 and p53 mRNA expression in HCC and normal liver cells. The data were normalized to the expression levels in LO2 cells. Bar graphs (mean ± SEM) and representative images are shown. * p < 0.05, ** p < 0.01 compared with the LO2 group ( n = 3).

Article Snippet: Expression vectors encoding the TRIB1 or p53 genes, and small interfering RNA (siRNA) against TRIB1 or p53 were purchased from Shanghai GenePharma Co. Ltd. (Shanghai, China).

Techniques: Quantitative RT-PCR, Expressing, Immunohistochemical staining, Western Blot, Control

Journal: Frontiers in Physiology

Article Title: The Oncogenic Role of Tribbles 1 in Hepatocellular Carcinoma Is Mediated by a Feedback Loop Involving microRNA-23a and p53

doi: 10.3389/fphys.2017.00789

Figure Lengend Snippet: TRIB1 downregulates p53 and promotes HCC cell growth. (A–D) The mRNA and protein expression levels of TRIB1 and p53 in HepG2 (A,C) and Huh7 (B,D) cells treated with or without siTRIB1 or negative control (siNC) or TRIB1 plasmid were determined by qRT-PCR and western blotting. * p < 0.05 or ** p < 0.01, compared with the control group. Immunocytochemistry were used to detect the p53 and TRIB1 expression in (E) HepG2 cells and (F) Huh7 (Y220C) cells. Cell growth of HepG2 (G) and Huh7 (H) cells treated as described above was determined using the MTT assay at the indicated time points. * p < 0.05 or ** p < 0.01, compared with the control group ( n = 3).

Article Snippet: Expression vectors encoding the TRIB1 or p53 genes, and small interfering RNA (siRNA) against TRIB1 or p53 were purchased from Shanghai GenePharma Co. Ltd. (Shanghai, China).

Techniques: Expressing, Negative Control, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Control, Immunocytochemistry, MTT Assay

Journal: Frontiers in Physiology

Article Title: The Oncogenic Role of Tribbles 1 in Hepatocellular Carcinoma Is Mediated by a Feedback Loop Involving microRNA-23a and p53

doi: 10.3389/fphys.2017.00789

Figure Lengend Snippet: miR-23a regulates the TRIB1/p53 axis in HCC cells. (A–C) The expression levels of p53, TRIB1, and miR-23a were examined in HCC cells transfected with or without a p53 overexpression plasmid by qRT-PCR. (D) TRIB1 is a predicted target of miR-23a by TargetScan. (E) HepG2 cells were co-transfected with miR-23a and wild-type (wt) or mutant (mut) pGL3-TRIB1 constructs and subjected to luciferase reporter assays. (F,G) The expression levels of TRIB1 and p53 in HepG2 cells transfected with or without miR-NC or miR-23a were determined by qRT-PCR and western blotting. (H) Schematic showing the miR-23a/TRIB1/p53 feed-forward loop ( n = 3). * p < 0.05, ** p < 0.01 compared with the control group.

Article Snippet: Expression vectors encoding the TRIB1 or p53 genes, and small interfering RNA (siRNA) against TRIB1 or p53 were purchased from Shanghai GenePharma Co. Ltd. (Shanghai, China).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Mutagenesis, Construct, Luciferase, Western Blot, Control

Journal: Frontiers in Physiology

Article Title: The Oncogenic Role of Tribbles 1 in Hepatocellular Carcinoma Is Mediated by a Feedback Loop Involving microRNA-23a and p53

doi: 10.3389/fphys.2017.00789

Figure Lengend Snippet: TRIB1 stimulates β-catenin signaling in a p53-dependent manner. (A) The expression of β-catenin, c-Myc, and MMP7 was detected by western blotting in HepG2 and Huh-7 cells transfected with siNC or siTRIB1 or Vector or TRIB1. β-actin was used as a loading control. * p < 0.05, ** p < 0.01 compared with the siNC group ( n = 3). (B) Effect of p53 and TRIB1 silencing on β-catenin expression in HepG2 and Huh-7 cells was detected by western blotting. * p < 0.05, ** p < 0.01 compared with the siNC[siP53(−)] group ( n = 3).

Article Snippet: Expression vectors encoding the TRIB1 or p53 genes, and small interfering RNA (siRNA) against TRIB1 or p53 were purchased from Shanghai GenePharma Co. Ltd. (Shanghai, China).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Frontiers in Physiology

Article Title: The Oncogenic Role of Tribbles 1 in Hepatocellular Carcinoma Is Mediated by a Feedback Loop Involving microRNA-23a and p53

doi: 10.3389/fphys.2017.00789

Figure Lengend Snippet: TRIB1 knockdown inhibits in vivo tumor formation from HepG2 cells in nude mice. Stable TRIB1 knockdown and control HepG2 cells were implanted subcutaneously in the left flank of nude mice. (A) After 35 days, mice were sacrificed and tumors were excised. (B) Effect of TRIB1 knockdown on tumor volume in a nude mice model. (C) The TUNEL assay (×400) was performed to determine the apoptotic indices. (D) Western blot analysis of the expression of p53, β-catenin, and c-myc in tumor tissues from control and shTRIB1 mice. β-actin was used as an internal control. Bar graphs (mean ± SEM) and representative images are shown. * p < 0.05, ** p < 0.01 compared with the control group ( n = 3).

Article Snippet: Expression vectors encoding the TRIB1 or p53 genes, and small interfering RNA (siRNA) against TRIB1 or p53 were purchased from Shanghai GenePharma Co. Ltd. (Shanghai, China).

Techniques: Knockdown, In Vivo, Control, TUNEL Assay, Western Blot, Expressing