Additional file 1 of H3K27ac-activated EGFR-AS1 promotes cell growth in cervical cancer through ACTN4-mediated WNT pathway

Additional file 1: Fig. S1. SiHa and CaSki cells were subjected to different treatments: sh-NC, sh-EGFR-AS1#1 and sh-EGFR-AS1#1 + CHIR99021. (A–C) EdU, flow cytometry and transwell assays were performed to evaluate the proliferation, apoptosis and invasion of the indicated CC cells. **P < 0.01

Image_2_Ca2+-stimulated ADCY1 and ADCY8 regulate distinct aspects of synaptic and cognitive flexibility.TIF

The type 1 and 8 adenylyl cyclase (ADCY1 and ADCY8) exclusively account for Ca2+-stimulated cyclic AMP (cAMP) production and regulate activity-dependent synaptic modification. In this study, we examined distinct forms of synaptic plasticity in the hippocampus of Adcy1−/− and Adcy8−/− mice. We found that, at the Schaffer collateral-CA1 synapses, while the Adcy8−/− mice displayed normal long-term potentiation (LTP) following various induction protocols with high-frequency stimulation (HFS), the Adcy1−/− mice showed protocol-dependent deficits in LTP. We also found that long-term depression (LTD) requires ADCY1 but not ADCY8. Interestingly, both Adcy1−/− and Adcy8−/− mice showed defective synaptic depotentiation (i.e., activity-dependent reversal of LTP); the deficits in Adcy8−/− mice were dependent on the induction protocol. Examination of spatial memory found that ADCY1 is required for the formation of both initial and reversal memory. ADCY8 is only required for reversal memory formation. These data demonstrate that ADCY1 and ADCY8 play distinct roles in regulating synaptic and cognitive flexibility that involves bidirectional modification of synaptic function.</p

Image_1_Ca2+-stimulated ADCY1 and ADCY8 regulate distinct aspects of synaptic and cognitive flexibility.TIF

The type 1 and 8 adenylyl cyclase (ADCY1 and ADCY8) exclusively account for Ca2+-stimulated cyclic AMP (cAMP) production and regulate activity-dependent synaptic modification. In this study, we examined distinct forms of synaptic plasticity in the hippocampus of Adcy1−/− and Adcy8−/− mice. We found that, at the Schaffer collateral-CA1 synapses, while the Adcy8−/− mice displayed normal long-term potentiation (LTP) following various induction protocols with high-frequency stimulation (HFS), the Adcy1−/− mice showed protocol-dependent deficits in LTP. We also found that long-term depression (LTD) requires ADCY1 but not ADCY8. Interestingly, both Adcy1−/− and Adcy8−/− mice showed defective synaptic depotentiation (i.e., activity-dependent reversal of LTP); the deficits in Adcy8−/− mice were dependent on the induction protocol. Examination of spatial memory found that ADCY1 is required for the formation of both initial and reversal memory. ADCY8 is only required for reversal memory formation. These data demonstrate that ADCY1 and ADCY8 play distinct roles in regulating synaptic and cognitive flexibility that involves bidirectional modification of synaptic function.</p

Oncogenic magnesium transporter 1 upregulates programmed death-1-ligand 1 expression and contributes to growth and radioresistance of glioma cells through the ERK/MAPK signaling pathway

Radiotherapy has been established as a major therapeutic modality for glioma, whereas new therapeutic targets are needed to prevent tumor recurrence. This study intends to explore the regulatory role of magnesium transporter 1 (MAGT1) in radiotherapy resistance of glioma through modulating ERK and programmed death-1-ligand 1 (PD-L1). Our bioinformatics analysis identified differentially expressed MAGT1 in glioma, expression of which was subsequently determined in cohort data of TCGA database and microarray dataset as well as glioma cell lines. Artificial modulation of MAGT1, ERK, and PD-L1 expression was performed to examine their effects on glioma cell proliferation and radioresistance, as reflected by MTT and colony formation assays under irradiation. Mouse glioma cells with manipulated MAGT1 and ERK inhibitors were further injected into mice to assess the in vivo tumor formation ability of glioma cells. It was noted that MAGT1 expression was highly expressed in glioma tissues of TCGA data and microarray dataset, which was then validated in glioma cell lines. Ectopic expression of MAGT1 was revealed to promote the proliferation and radioresistance of glioma cells, which was attributed to the MAGT1-mediated activation of the ERK/MAPK signaling pathway. It was illuminated that MAGT1 stimulated PD-L1 expression through the ERK/MAPK pathway and thus facilitated glioma cell growth. Additionally, MAGT1 overexpression accelerated the in vivo tumor formation of glioma cells, while the ERK inhibitor negated its effect. In conclusion, MAGT1 enhances the growth and radioresistance of glioma cells through the ERK/MAPK signaling pathway-mediated upregulation of PD-L1 expression.</p

Transactivation of CYP2B6 5’-flanking reporter constructs by HNF3β.

<p>A computer-based search for the first 2 kb of CYP2B6 upstream resulted in the identification of three potential HNF3β-responsive elements located between -1893bp and -350bp (A). HepG2 cells were transfected with HNF3β expression vector in the presence of CYP2B6 promoter constructs containing sequential deletion fragments (B) or the CYP2B6-2.0k harboring one of the mutated HNF3β binding sites (C). Forty eight hours post-transfection, luciferase activities were determined and expressed relative to the control (pGL3-Basic). Data represent the mean ± SD. (n = 3). (<i>*</i>, <i>p <0</i>.<i>05; **</i>, <i>p<0</i>.<i>01</i>).</p

Effects of HNF3β on hCAR-mediated CYP2B6 activation in HepG2 cells.

<p>HepG2 cells were co-transfected with expression plasmids of hCAR, HNF3β or C/EBPα in the presence (A) or absence (B) of CYP2B6-2kb reporter construct as detailed in the <i>Materials and Methods</i>. Transfected cells were then treated with PB (1mM) and CITCO (1 μM) for 24 h. Dual luciferase activities (A) and CYP2B6 mRNA expression (B) were detected and expressed relative to vehicle control by reporter assay and real-time PCR analysis.</p

Correlation between CYP2B6 and HNF3β expression in HPH.

<p>Total RNA was extracted from HPHs prepared from 35 human liver donors. Expression levels of CYP2B6, hCAR, C/EBPα, HNF4α, HNF3β were measured using real-time RT-PCR assays as detailed in the <i>Materials and Methods</i>. Relative gene expression levels from all donors were normalized against a randomly selected single donor. Leaner regression between CYP2B6 and one of these hepatic transcriptional factors was analyzed individually using Pearson’s Correlation Coefficient (JMP 7.0; SAS, NC).</p

Recruitment of HNF3β to enhancers identified upstream of CYP2B6 promoter.

<p>As detailed in the <i>Materials and Methods</i>, ChIP assays were used to analyze binding of HNF3β to the HNF3β-a (distal), HNF3β-c (proximal) containing, and the -1.6/-1.4kb regions in cultured HPH (A). After precipitation with HNF3β antibody, de-crosslinked DNA fragments were amplified by PCR. Amplification of the promoter of SULT1E1 was used as negative control as reported previously. In separate experiments, BIACORE SPR affinity assays (B) and (C) were carried out to measure the comparative binding kinetics of CYP2B6 enhancers on HNF3β as described under <i>Materials and Methods</i>. Sensorgrams of the interaction generated by the instrument were analyzed by the software BIAeval 3.2.</p

Effects of HNF3β on the expression and activity of CYP2B6 in HepG2 cells.

<p>HepG2 cells were infected with Negative Control Adenovirus (Ad-NC) or various amounts of adenovirus expressing HNF3β (Ad-HNF3β) for 48 h. Expression of HNF3β mRNA (A), CYP2B6 mRNA (B), and their proteins (C) were measured using real-time PCR and Western blotting assays, respectively. CYP2B6 enzymatic activity (D) was detected using P450-Glo^™ CYP2B6 Assay kit (Promega). In separate experiments, HepG2 cells were infected with pGreen Negtive Control Lentivirus (pGreen-NC) or HNF3β-RNAi lentivirus (HNF3β-shRNA) for 96 h before measuring the mRNA expression of HNF3β (E) and CYP2B6 (F) by real-time PCR. Results are expressed as the mean ± S.D. (n = 3). (<i>*P <0</i>.<i>05</i>, <i>**P <0</i>.<i>01</i>).</p