dnmt inhibitor rg108  (FUJIFILM)

Journal: Journal of cellular biochemistry

Article Title: SP1 regulates KLF4 via SP1 binding motif governed by DNA methylation during odontoblastic differentiation of human dental pulp cells

doi: 10.1002/jcb.28730

Figure Lengend Snippet: Inhibition of DNA methyltransferase enhances the efficiency of odontoblastic differentiation. A, After treatment of RG108, protein level of DNMT1 and KLF4 were measured with Western blot. B, RG108 treated and untreated cells were cultured in odontoblastic differentiation medium or normal growth medium for 14 days. Mineralization was determined by alizarin red staining. On induction day of 0, 1, 5, 7, 11, and 14, RNA and protein were extracted from both RG108 treated and untreated cells. The protein level, C, of KLF4, DSP, and mRNA level, D, of Klf4, Dmp1, and Dspp were determined by Western blot and real-time RT-PCR respectively. All data were based on three independent experiments: *P < 0.05. **P < 0.01. RT-PCR, real-time reverse transcription-polymerase chain reaction

Article Snippet: RG108 treatment RG108, a novel DNA methyltransferase (DNMTs) inhibitor, was purchased from Selleck Chemicals (Houston, TX).

Techniques: Inhibition, Western Blot, Cell Culture, Staining, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: Bronchial epithelial DNA methyltransferase 3b dampens pulmonary immune responses during Pseudomonas aeruginosa infection

doi: 10.1371/journal.ppat.1009491

Figure Lengend Snippet: BEAS-2B cells were pretreated with the DNMT inhibitor RG108 at 10 μM/ml (open bars) or vehicle (black bars) for 12 hours and then stimulated with heat killed PAK (MOI = 50) or medium control for 2, 6 or 12 hours. CXCL1 , CXCL8 and CCL20 mRNA expression was measured by RT-qPCR (A); DNMT3B mRNA levels in BEAS-2B cells incubated with heat killed PAK or medium control for 12 hours (B); Dnmt3b deficient (knock out, DNMT3B KO; open bars) and control (Ctr; black bars) BEAS-2B cells were stimulated with heat killed PAK (+) or medium control (-) for 12 hours. CXCL1 , CXCL8 and CCL20 mRNA levels were measured by RT-qPCR (C) and corresponding protein levels in the supernatant by ELISA (D). Data are presented as means ± SEM (n = 4) and representative of two to three independent experiments and for two DNMT3B KO and control clones. *p < 0.05, **p < 0.01, *** p < 0.001.

Article Snippet: For some experiments, cells were pretreated with the Dnmt inhibitor RG108 at 10 μM/ml [ ] (Sigma, Zwijndrecht, Nederland) or vehicle DMSO for 12 hours, and then stimulated with heat killed bacteria at MOI = 50.

Techniques: Expressing, Quantitative RT-PCR, Incubation, Knock-Out, Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: eLife

Article Title: Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia

doi: 10.7554/eLife.18299

Figure Lengend Snippet: ( A ) Experimental protocol. The times of occurrence of the pretests, training, posttests, and drug/vehicle injections are shown relative to the end of the last training session. Either RG108 or vehicle was injected into the hemocoel at the time indicated by the red arrow. ( B ) The mean duration of the SWR measured at 24 h and 48 h for the Veh-Control-3XTrained ( n = 7), Veh-5XTrained ( n = 7), RG-5XTrained ( n = 8), and RG-5XTrained-3XTrained ( n = 7) groups. A repeated-measures ANOVA indicated that there was a significant group x time interaction (F [6,50] = 73.6, p < 0.0001). Subsequent planned comparisons showed that the overall differences among the four groups for the 24-h and 48-h posttests were highly significant (24 h, F [3,25] = 197.9, p < 0.0001; and 48 h, F [3,25] = 82.8, p < 0.0001). As revealed by SNK posthoc tests, the SWR exhibited sensitization at 24 h in the Veh-5XTrained group (mean duration = 54.0 ± 3.4 s) compared with that in the Veh-Control-3XTrained group (mean duration = 1.3 ± 0.3 s, p < 0.001). The differences in duration of the SWR at 24 h among the Veh-Control-3XTrained, RG-5XTrained (3.6 ± 1.3 s), and RG-5XTrained-3XTrained (1.9 ± 0.6 s) groups were not significant. Sensitization of the SWR was maintained in the Veh-5XTrained group (mean duration of the reflex = 55.3 ± 3.6 s) at 48 h, as shown by the comparison with the Veh-Control-3XTrained group (mean duration of the reflex = 1.6 ± 0.6 s, p < 0.001). There were no significant differences among the Veh-Control-3XTrained, RG-5XTrained (mean duration of the SWR = 3.1 ± 1.2 s), and RG-5XTrained-3XTrained (mean duration of the SWR = 5.7 ± 4.4 s) groups at 48 h, indicating that the three additional bouts of tail shocks given to the latter group after the 24-h posttest failed to induce LTS. Asterisks, comparisons of the Veh-5XTrained group with the Veh-Control-3XTrained group, the RG-5XTrained group, and the RG-5XTrained-3XTrained group. DOI: http://dx.doi.org/10.7554/eLife.18299.006

Article Snippet: The DNA methyltransferase (DNMT) inhibitors RG108 (Sigma, St Louis, MO) and 5-azadeoxycytidine (5-aza; EMD Millipore, Billerica, MA) were dissolved in DMSO to a concentration of 25 mM to make a stock solution.

Techniques: Injection, Control, Comparison

Journal: eLife

Article Title: Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia

doi: 10.7554/eLife.18299

Figure Lengend Snippet: ( A ) Experimental protocol. The times at which the pretests, training, posttests, and drug/vehicle injections occurred are shown relative to the end of the last training session. The time of the intrahemocoelic injection of either RG108 or vehicle is indicated by the red arrow. After the 24-h posttest, animals in the Control-Veh-3XTrained and 5XTrained-RG-3XTrained groups received 3X sensitization training. ( B ) The mean duration of the SWR measured at 24 h and 48 h for the Control-Veh-3XTrained ( n = 8), 5XTrained-Veh ( n = 8), 5XTrained-RG ( n = 7), and 5XTrained-RG-3XTrained ( n = 6) groups. A repeated-measures ANOVA showed that the group x time interaction was significant (F [6,50] = 64.7, p < 0.0001). The overall differences among the four groups for the 24-h and 48-h posttests were highly significant, as indicated by a one-way ANOVA (24 h, F [3,25] = 82.6, p < 0.0001; and 48 h, F [3,25] = 69.2, p < 0.0001). SNK posthoc tests revealed significantly greater sensitization in the 5XTrained-Veh group at 24 h (mean duration of the SWR = 53.1 ± 5.1 s) than in the Control-Veh-3XTrained group (mean duration of the SWR = 2.1 ± 0.9 s, p < 0.001). The differences among the 5XTrained-RG (mean duration of the SWR = 1.7 ± 0.7 s), 5XTrained-RG-3XTrained (mean duration of the SWR = 2.2 ± 0.7 s), and Control-Veh-3XTrained groups at 24 h were not significant. Sensitization persisted in the 5XTrained-Veh group at 48 h (mean duration of the SWR = 48.3 ± 5.0 s) compared with the Control-Veh-3XTrained group (mean duration of the SWR = 1.6 ± 0.3 s, p < 0.001). The failure of the 3X training to induce sensitization in the 5XTrained-RG-3XTrained group was shown by the lack of significant differences between this group (mean duration of the SWR = 2.8 ± 1.2 s) and the Control-Veh-3XTrained group at 48 h. There was also no significant difference between the mean duration of the reflex in the 5XTrained-RG-3XTrained group and that in the 5XTrained-RG (2.3 ± 1.0 s) group at 48 h. Asterisks, comparisons of the 5XTrained-Veh group with the Control-Veh-3XTrained group, the 5XTrained-RG group, and the 5XTrained-RG-3XTrained group. DOI: http://dx.doi.org/10.7554/eLife.18299.007

Article Snippet: The DNA methyltransferase (DNMT) inhibitors RG108 (Sigma, St Louis, MO) and 5-azadeoxycytidine (5-aza; EMD Millipore, Billerica, MA) were dissolved in DMSO to a concentration of 25 mM to make a stock solution.

Techniques: Injection, Control

Journal: eLife

Article Title: Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia

doi: 10.7554/eLife.18299

Figure Lengend Snippet: ( A ) Experimental protocol. The occurrences of the pretests, training, posttests, and drug/vehicle injections are shown relative to the end of the last training session. Either RG108 or vehicle was injected into the animals at the time indicated by the red arrow. After the 48-h posttest, animals in the Control-Veh-3XTrained and 5XTrained-RG-3XTrained groups received 3 bouts of sensitization training. ( B ) RG108 treatment at 24 h after training abolished LTS. There were four experimental groups: Control-Veh-3XTrained group ( n = 6), 5XTrained-Veh group ( n = 6), 5XTrained-RG group ( n = 6), and 5XTrained-RG-3XTrained group ( n = 6). A repeated-measures ANOVA disclosed a significant group x time interaction (F [9,60] = 22.9, p < 0.0001). Subsequent planned comparisons showed that the overall differences among the four groups for the 24-, 48- and 72-h posttests were highly significant (24 h, F [3,20] = 13.8, p < 0.0001; 48 h, F [3,20] = 28.6, p < 0.0001; and 72 h, F [3,20] = 27.9, p < 0.0001). Animals in all three groups trained with five bouts of tail shocks exhibited significant sensitization at 24 h, as indicated by SNK posthoc tests. Thus, the mean SWR was longer in the 5XTrained-Veh (45.7 ± 6.9 s), 5XTrained-RG (42.2 ± 6.7 s), and 5XTrained-RG-3XTrained (47.5 ± 6.5 s) groups than that in the Control-Veh-3XTrained group (2.0 ± 0.7 s; p < 0.001 for each comparison). However, although the 5XTrained-Veh group exhibited significant sensitization on both the 48-h (mean SWR = 43.7 ± 7.6 s) and 72-h (mean SWR = 41.0 ± 7.3 s) posttests, sensitization was absent in both groups of RG108-treated animals after 24 h. Posthoc tests revealed no significant differences for any of the comparisons between the Control-Veh-3XTrained group and the 5XTrained-RG group, or the 5XTrained-RG-3XTrained group, on the posttests after 24 h. Therefore, inhibiting DNMT with RG108 24 h after training erased established LTS. There was no evidence of spontaneous recovery of sensitization over the 48-h period after RG108 injection; furthermore, three additional bouts of training failed to reinstate LTS. Asterisks, comparisons of the 5XTrained-Veh, 5XTrained-RG, and 5XTrained-RG-3XTrained groups with the Control-Veh-3XTrained group at 24 h; and comparison of the 5XTrained-Veh group with the Control-Veh-3XTrained group at 48 h and 72 h. Plus signs , comparisons of the 5XTrained-Veh group with the 5XTrained-RG and 5XTrained-RG-3XTrained groups at 48 h and 72 h. DOI: http://dx.doi.org/10.7554/eLife.18299.008

Article Snippet: The DNA methyltransferase (DNMT) inhibitors RG108 (Sigma, St Louis, MO) and 5-azadeoxycytidine (5-aza; EMD Millipore, Billerica, MA) were dissolved in DMSO to a concentration of 25 mM to make a stock solution.

Techniques: Injection, Control, Comparison

Journal: eLife

Article Title: Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia

doi: 10.7554/eLife.18299

Figure Lengend Snippet: ( A ) Experimental protocol. The times at which the pretests, training, posttests, and drug/vehicle injections occurred are shown relative to the end of the last training session. The intrahemocoelic injection of either drug or vehicle is indicated by the red arrow. The animals did not receive a 24 h test prior to the drug/vehicle injection. After the 48-h posttest, animals in the Control-Veh-3XTrained and 5XTrained-5aza-3XTrained groups received 3 bouts of sensitization training (3X training). ( B ) 5-aza injection abolished established LTS in the absence of a posttest at 24 h. Four experimental groups were included: Control-Veh-3XTrained group ( n = 7), 5XTrained-Veh group ( n = 7), 5XTrained-5aza group ( n = 7), and 5XTrained-5aza-3XTrained group ( n = 8). A repeated-measures ANOVA indicated that there was a significant group x time interaction (F [6,50] = 105.8, p < 0.0001). Subsequent planned one-way ANOVAs showed that the overall differences among the four groups at both 48 h and 72 h were highly significant (48 h, F [3,25] = 385.4, p < 0.0001;and 72 h, F [3,25] = 183.3, p < 0.0001). SNK posthoc tests revealed that the SWR in the 5XTrained-Veh group was significantly sensitized at both 48 h (mean = 56.1 ± 2.9 s) and 72 h (mean = 52.4 ± 3.7 s) compared with that in the Control-Veh-3XTrained group (p < 0.001 for each comparison). Furthermore, the mean duration of the SWR in the 5XTrained-Veh group was significantly longer than that in the 5XTrained-5aza (1.1 ± 0.1 s at 48 h, p < 0.001; and 1.6 ± 0.6 s at 72 h, p < 0.001) and 5XTrained-5aza-3XTrained (1.1 ± 0.1 s at 48 h, p < 0.001; and 1.9 ± 0.9 s at 72 h, p < 0.001) groups. The Control-Veh-3XTrained, 5XTrained-5aza, and 5XTrained-5aza-3XTrained groups did not differ significantly at either 48 h or 72 h. Thus, the erasure of established LTS by inhibition of DNMT did not require elicitation of the SWR immediately preceding the drug injection. Asterisks, comparisons of the 5XTrained-Veh group with the Control-Veh-3XTrained, 5XTrained-5aza, and 5XTrained-5aza-3XTrained groups at 48 h and 72 h. DOI: http://dx.doi.org/10.7554/eLife.18299.010

Article Snippet: The DNA methyltransferase (DNMT) inhibitors RG108 (Sigma, St Louis, MO) and 5-azadeoxycytidine (5-aza; EMD Millipore, Billerica, MA) were dissolved in DMSO to a concentration of 25 mM to make a stock solution.

Techniques: Injection, Control, Comparison, Inhibition

Journal: eLife

Article Title: Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia

doi: 10.7554/eLife.18299

Figure Lengend Snippet: ( A ) Experimental protocol. The occurrences of the pretests, training, posttests, and drug/vehicle injections are shown relative to the end of the last training session. The red arrow indicates the time of the intrahemocoelic injection of RG108 or vehicle. After the day six posttest, animals in some groups received partial sensitization training (3 bouts of tail shocks). ( B ) RG108 injection at day five after training (LATE treatment) erased LTS. There were four experimental groups: Control-Veh LATE -3XTrained group ( n = 6), 5XTrained-Veh LATE group ( n = 5), 5XTrained-RG LATE group ( n = 6), and 5XTrained-RG LATE -3XTrained group ( n = 6). A repeated-measures ANOVA indicated that there was a significant group x time interaction (F [9,57] = 66.3, p < 0.0001). Subsequent planned comparisons showed that the overall differences among the four groups on days 5, 6 and 7 were highly significant (day 5, F [3,19] = 43.7, p < 0.0001; day 6, F [3,19] = 105.1, p < 0.0001; and day 7, F [3,19] = 252.5, p < 0.0001). There was significant sensitization at day five prior to RG108/vehicle injection in the 5XTrained-Veh LATE (mean duration of the SWR = 55.0 ± 5.0 s), 5XTrained-RG LATE (mean duration of the SWR = 56.7 ± 2.1 s), and 5XTrained-RG LATE -3XTrained (mean duration of the SWR = 49.3 ± 6.1 s) groups compared with the Control-Veh LATE -3XTrained group (mean duration of the SWR = 1.7 ± 0.7 s) (p < 0.001 for each comparison). The 5XTrained-Veh LATE group also exhibited robust sensitization on day 6 (mean duration of the SWR = 52.4 ± 5.5 s) and 7 (mean duration of the SWR = 53.0 ± 3.3 s) compared with the Control-Veh LATE -3XTrained group. Sensitization was absent in the 5XTrained-RG LATE group on day 6 and 7; thus, there was no spontaneous recovery of LTS during the 48-h period after the application of RG108. Three bouts of training shortly after the day six posttest did not restore LTS in the 5XTrained-RG LATE -3XTrained group the next day. In particular, the mean duration of the SWR in the 5XTrained-RG LATE -3XTrained group (2.5 ± 0.8 s) on day 7 was not significantly different from that in the Control-Veh LATE -3XTrained group, and was significantly shorter than that in the 5XTrained-Veh LATE group (p < 0.001). Asterisks, comparisons of the 5XTrained-Veh LATE , 5XTrained-RG LATE , and 5XTrained-RG LATE -3XTrained groups with the Control-Veh LATE -3XTrained group on day 5; and comparison of the 5XTrained-Veh LATE group with the Control-Veh LATE -3XTrained group on days 6 and 7. Plus signs , comparisons of the 5XTrained-Veh LATE group with the 5XTrained-RG LATE and 5XTrained-RG LATE -3XTrained groups on days 6 and 7. DOI: http://dx.doi.org/10.7554/eLife.18299.011

Article Snippet: The DNA methyltransferase (DNMT) inhibitors RG108 (Sigma, St Louis, MO) and 5-azadeoxycytidine (5-aza; EMD Millipore, Billerica, MA) were dissolved in DMSO to a concentration of 25 mM to make a stock solution.

Techniques: Injection, Control, Comparison

Journal: eLife

Article Title: Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia

doi: 10.7554/eLife.18299

Figure Lengend Snippet: ( A ) Experimental protocol. The times at which the pretests, training, posttests, and drug/vehicle injections occurred are shown relative to the end of the last training session. The time of the intrahemocoelic injection of either RG108 or vehicle is indicated by the red arrow. After the 48-h posttest, animals in the 5XTrained-RG-5XTrained group received a second round of full sensitization training (five bouts of electrical tail shocks). ( B ) Sensitization retraining produced LTS in animals following erasure of LTM by RG108. There were four experimental groups: Control-Veh group ( n = 6), 5XTrained-Veh group ( n = 6), 5XTrained-RG group ( n = 4), and 5XTrained-RG-5XTrained group ( n = 6). A repeated-measures ANOVA indicated that the group x time interaction was significant (F [9,54] = 61.4, p < 0.0001). Subsequent one-way ANOVAs indicated that the overall differences among the four groups at 24 h, 48 h and 72 h were highly significant (24 h, F [3,18] = 33.3, p < 0.0001; 48 h, F [3,18] = 70.7, p < 0.0001; and 72 h, F [3,18] = 54.9, p < 0.0001). SNK posthoc tests performed on the 24-h data revealed that the initial training produced significant sensitization in the 5XTrained-Veh group (mean duration of the SWR = 50.8 ± 6.1 s), 5XTrained-RG group (mean duration of the SWR = 57.8 ± 2.3 s), and 5XTrained-RG-5XTrained group (mean duration of the SWR = 54.2 ± 5.8 s) compared with Control-Veh group (mean duration of the SWR = 1.2 ± 0.2 s; p < 0.001 for each comparison). The SWR of 5XTrained-Veh group also exhibited sensitization at 48 h (mean duration = 49.2 ± 5.2 s) and 72 h (mean duration = 45.2 ± 5.6 s) compared with the Control-Veh group (48 h, mean duration = 1.2 ± 0.2 s, p < 0.001; and 72 h, mean duration = 1.2 ± 0.2 s, p < 0.001). Sensitization memory was significantly disrupted at 48 h in both the 5XTrained-RG (mean SWR = 1.3 ± 0.3 s) and 5XTrained-RG-5XTrained (mean SWR = 2.2 ± 0.7 s) groups by the RG108 injection immediately after the 24-h posttest (p > 0.05 for the comparisons with the Control-Veh group). Retraining after the 48-h posttest reestablished full LTM. The mean duration of the SWR in the 5XTrained-RG-5XTrained group at 72 h (55.8 ± 4.2 s) was significantly greater than that for the Control-Veh group (mean duration = 1.2 ± 0.2 s, p < 0.001), as well as for the 5XTrained-RG group at 72 h (2.5 ± 1.0 s, p < 0.001). Asterisks, comparisons of the 5XTrained-Veh, 5XTrained-RG, and 5XTrained-RG-5XTrained groups with the Control-Veh group at 24 h; comparisons of the 5XTrained-Veh group with the Control-Veh, 5XTrained-RG, and 5XTrained-RG-5XTrained groups at 48 h; and comparison of the 5XTrained-Veh group with the Control-Veh and 5XTrained-RG groups at 72 h. Plus signs , comparison of the 5XTrained-RG-5XTrained group with the 5XTrained-RG group at 72 h. DOI: http://dx.doi.org/10.7554/eLife.18299.012

Article Snippet: The DNA methyltransferase (DNMT) inhibitors RG108 (Sigma, St Louis, MO) and 5-azadeoxycytidine (5-aza; EMD Millipore, Billerica, MA) were dissolved in DMSO to a concentration of 25 mM to make a stock solution.

Techniques: Injection, Produced, Control, Comparison