Journal: Heliyon

Article Title: Median raphe glutamatergic neuron-mediated enhancement of GABAergic transmission and suppression of long-term potentiation in the hippocampus

doi: 10.1016/j.heliyon.2024.e38192

Figure Lengend Snippet: MRN VGLUT3 neuron-mediated glutamatergic transmission in SR/SLM 5-HT3aR neurons. A) A schematic of AAV injection into the MRN. The left panel shows an image of the MRN from VGLUT3 Cre/5-HT3aR-EGFP mouse which received an AAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA injection into the MRN. Scale 150 μm. B) An image of the hippocampus from MRN VGLUT3 ChR2/5-HT3aR-EGFP mouse. The inset shows EGFP-expressing SR/SLM 5-HT3aR neurons and mCherry-expressing MRN VGLUT3 axons. Scale 300 μm/25 μm (inset). C) The upper panel shows a schematic for recording MRN VGLUT3 neuron-mediated transmission in SR/SLM 5-HT3aR neurons. The bottom panel shows example traces for MRN VGLUT3 neuron-mediated EPSCs in SR/SLM 5-HT3aR neurons without drug application, in the presence of tetrodotoxin (TTX, 1 μM), TTX+4-aminopyridine (4-AP, 100 μM) and TTX+4-AP + DNQX (10 μM). Scale 50 pA/10 ms. The blue line indicates light application. D) EPSC amplitude in SR/SLM 5-HT3aR neurons before the drug application, in the presence of TTX, TTX+4-AP, and TTX+4-AP + DNQX (6 neurons from 3 female mice and 6 neurons from 3 male mice). F (1,11) = 22.01, P = 0.001. Data are presented as mean ± SEM. E) EPSC amplitude in female (17 neurons/5 mice) and male (16 neurons/5 mice) groups. Female and male groups did not show statistically significant difference in EPSC amplitude (P = 0.8). The horizontal line in each group represents the mean and the vertical line represents SEM. F) The delay between the onset of the light stimulation and the onset of EPSCs in female (17 neurons/5 mice) and male (16 neurons/5 mice) groups. Female and male groups did not show a statistically significant difference in the delay between the onset of light stimulation and the onset of EPSCs (P = 0.77). n.s. not statistically significant.

Article Snippet: The slices were then incubated in mCherry polyclonal antibody (Rockland Immunochemicals, Cat. No. 600401P16) or Anti-GFP antibody (Abcam, Cat. No. ab13970, RRID: AB_300798 ) at a dilution of 1:1000 in 0.3 % Triton-X/TBS for 24 h at 4 °C [ , ].

Techniques: Transmission Assay, Injection, Expressing

Journal: International Journal of Cancer

Article Title: Frequent deregulation ofp16 and thep16/G1 cell cycle-regulatory pathway in neuroblastoma

doi: 10.1002/(sici)1097-0215(19990105)80:1<145::aid-ijc26>3.0.co;2-g

Figure Lengend Snippet: FIGURE 1 – Expression and DNA analysis of p16 in neuroblastoma cell lines. (a) p16 expression at the transcript level was assessed by RT-PCR. Lane 1, Saos2 osteosarcoma cell line, which is known to over-express p16 (Li et al., 1994); lane 2, foreskin fibroblasts for a comparison of ‘‘normal’’ p16 expression; lanes 3–9 are neuroblastoma cell lines Be2C/ADR5, NB4, NB14, NB17, NB20, NMB7 and PCL1691, respectively. Only Be2C/ADR5 (lane 3) is deleted for p16. GAPDH is shown as a control. A 100-bp m.w. ladder (GIBCO BRL) is also shown. (b) p16 expression at the protein level was assessed by Western blot. Lanes 1–7 are neuroblastoma cell lines PCL1691, NB17, SMS-KCN, PCL3014, NB20, NB5 and NB14, respectively. b-Actin is shown as a control. (c) Mutation analysis of p16 exon 1 was performed as described previously (Diccianni et al., 1997). Lanes 1–12 are neuroblastoma cell lines SK-N-SH, IMR6, IMR32, Be2C, Be2C/ ADR5 (deleted for p16), NB4, NB5, NB14, NB17, NB20, PCL1643 and PCL2021, respectively. The NB4 cell line was characterized as having a C = A heterozygous substitution 2 bases upstream of the ATG start site.

Article Snippet: Cells were then incubated for 2 hr at RT with rabbit anti-human p16 polyclonal antibody (C-20, Santa Cruz Biotechnology) at 0.5 μg/ml or rabbit immunoglobulin in PBS containing 0.5% non-fat milk, and immunodetection was performed using the Supersensitive Multilink System (BioGenex, San Ramon, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison, Control, Western Blot, Mutagenesis

Journal: International Journal of Cancer

Article Title: Frequent deregulation ofp16 and thep16/G1 cell cycle-regulatory pathway in neuroblastoma

doi: 10.1002/(sici)1097-0215(19990105)80:1<145::aid-ijc26>3.0.co;2-g

Figure Lengend Snippet: FIGURE 2 – Expression and DNA analysis of G1 regulatory genes downstream of p16 in neuroblastoma cell lines. Expression of CDK4 and CDK6 (a); cyclins D1, D2 and D3 (b); and E2F1 and E2F2 (d) was examined at the transcript level by RT-PCR. Lanes 2–9 are neuroblastoma cell lines NB4, NB5, NB14, NB17, NB20, PCL1643, PCL1691 and IMR32, respectively. Lane 1 shows mononuclear cells from a normal individual, and lane 10 is SK-N-MC, a neuroepithelial cell line. GAPDH is shown as a control (c).Amplification of CDK4 was examined by Southern blot using a full-length cDNAprobe generated by PCR, as described in ‘‘Material and Methods.’’Lanes 1–8 are neuroblastoma cell lines PCL3091, PCL3014, PCL1691, IMR6, SK-N-SH, IMR32, NB20 and NB5, respectively. Lane 9 is the CDK4-amplified osteosarcoma cell line SJ-SA1, which is used here as a positive control. The concentration of DNAin lane 8 was inadvertently lower than those of the other samples, accounting for the apparent weak signal from this sample.

Article Snippet: Cells were then incubated for 2 hr at RT with rabbit anti-human p16 polyclonal antibody (C-20, Santa Cruz Biotechnology) at 0.5 μg/ml or rabbit immunoglobulin in PBS containing 0.5% non-fat milk, and immunodetection was performed using the Supersensitive Multilink System (BioGenex, San Ramon, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Southern Blot, Generated, Positive Control, Concentration Assay

Journal: International Journal of Cancer

Article Title: Frequent deregulation ofp16 and thep16/G1 cell cycle-regulatory pathway in neuroblastoma

doi: 10.1002/(sici)1097-0215(19990105)80:1<145::aid-ijc26>3.0.co;2-g

Figure Lengend Snippet: FIGURE 4 – Immuno-histochemical analysis of p16 in neuroblastoma cell lines. Cells were grown on slides, fixed and stained for p16 as described in ‘‘Material and Methods’’. (a) Saos 2 osteosarcoma cell line, known to over-express p16 (Fig. 1a, lane 1; Li et al., 1994); (b) foreskin fibroblasts; (c–f) neuroblastoma cell lines NB4, PCL1691, NMB7 and Be2C/ADR5, respectively. NB4 and PCL1691 are strong p16-expressing cell lines, NMB7 illustrates a pattern in which only an occasional (,3%) p16-immunopositive nucleus is seen and Be2C/ADR5 is a p16-deleted neuroblastoma cell line (Diccianni et al., 1996).

Article Snippet: Cells were then incubated for 2 hr at RT with rabbit anti-human p16 polyclonal antibody (C-20, Santa Cruz Biotechnology) at 0.5 μg/ml or rabbit immunoglobulin in PBS containing 0.5% non-fat milk, and immunodetection was performed using the Supersensitive Multilink System (BioGenex, San Ramon, CA).

Techniques: Staining, Expressing

Journal: International Journal of Cancer

Article Title: Frequent deregulation ofp16 and thep16/G1 cell cycle-regulatory pathway in neuroblastoma

doi: 10.1002/(sici)1097-0215(19990105)80:1<145::aid-ijc26>3.0.co;2-g

Figure Lengend Snippet: FIGURE 5 – RT-PCR analysis of the expression of the G1 regulators p16, CDK4 and cyclin D2 in primary neuroblastoma. Samples 1–6 are shown in the order listed in Table I, except for cylin D2: lane 1 is sample 1176 and lane 2 is sample 2090. The patterns of expression of the G1 regulatory genes investigated here parallelled the expression patterns seen in neuroblastoma cell lines (see text). GAPDH is shown as a control.

Article Snippet: Cells were then incubated for 2 hr at RT with rabbit anti-human p16 polyclonal antibody (C-20, Santa Cruz Biotechnology) at 0.5 μg/ml or rabbit immunoglobulin in PBS containing 0.5% non-fat milk, and immunodetection was performed using the Supersensitive Multilink System (BioGenex, San Ramon, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control