Experiments
Ethanol consumption (g/kg/day)
during stabilization period with
10% ethanol in drinking water
Total fluid consumed
(g/kg/day)
Number of mice
Immunohistochemistry
9.42 ± 0.57
140.50 ± 13.62
Water n=6
Ethanol n=9
RT-qPCR
9.17 ± 0.62
140.00 ± 17.84
Water n=6
Ethanol n=9
Protein analysis
(Western Blot/ELISA)
10.60 ± 0.53
135.30 ± 10.25
Water n=6
Ethanol n=9
ChIP
10.48 ± 0.45
146.30 ± 14.15
Water n=6
Ethanol n=9
Neurogenesis
2 weeks: 10.24 ± 0.99
4 weeks: 9.25 ± 0.33
149.50 ± 9.80
144.30 ± 8.79
Water 2 weeks n=6
Ethanol 2 weeks n=9
Water 4 weeks n=6
Ethanol 4 weeks n=9
ANA-12
Ethanol vehicle-treated: 9.54 ± 0.88
Ethanol ANA12-treated: 8.87 ± 0.77
139.50 ± 17.70
134.00 ± 13.53
Water vehicle-treated n=6
Ethanol vehicle-treated n=9
Water ANA12-treated n=6
Ethanol ANA12-treated n=9
Table S1: Ethanol intake and total fluid consumed in the different groups of mice with free access
to 10% ethanol that had been included in the various experimental series.
mRNA expression analysis
Genes
5’ primer
3’ primer
Gdnf
TGGGTTTGGGCTATGAAACC
AACATGCCTGGCCTACTTTG
Fgf-1
GCGGTCCTCGGACTCACTA
AGCCAATGGTCAAGGGAAC
Fgf-2
TATGAAGGAAGATGGACGGC
TCGTTTCAGTGCCACATACC
Trkb
TGGACCACGCCAACTGACATT
GAATGTCTCGCCAACTTGAG
Bcl-2
ATGACTGAGTACCTGAACCG
ATGCTGGGGCCATATAGTTC
Bdnf I
GTGTGACCTGAGCAGTGGGCAAAGGA
-
Bdnf II
GGAAGTGGAAGAAACCGTCTAGAGCA
-
Bdnf III
GCTTTCTATCATCCCTCCCCGAGAGT
-
Bdnf IV
CTCTGCCTAGATCAAATGGAGCTTC
-
Bdnf V
CTCTGTGTAGTTTCATTGTGTGTTC
-
Bdnf VI
GCTGGCTGTCGCACGGTTCCCATT
-
Bdnf VII
CCTGAAAGGGTCTGCGGAACTCCA
-
Bdnf VIII
GTGTGTGTCTCTGCGCCTCAGTGGA
-
Bdnf IX
TGCAGGGGCATAGACAAAAG
TGAATCGCCAGCCAATTCTC
BDNF Rev
-
GAAGTGTACAAGTCCGCGTCCTTA
Hprt
GATGATGAACCAGGTTATGAC
GTCCTTTTCACCAGCAAGCTTG
β-actin
CCACCATGTACCCAGGCATT
CGGACTCATCGTACTCCTGC
Table S2: Primer sequences used for quantitative real-time PCR analysis of mRNAs with
Absolute SYBR Green technology.
mRNA expression analysis
Genes
References
Mecp2
Mm01193537_g1
Mbd1
Mm01262472_g1
Ncor
Mm00448681_m1
Hdac1
Mm02745760_g1
Hdac2
Mm00515108_m1
Hdac3
Mm00515916_m1
Hdac4
Mm01299565_m1
Hdac5
Mm00515941_g1
Hdac9
Mm00458454_m1
Mash1
Mm03058063_m1
Neurod1
Mm01280117_m1
Neurog1
Mm00440466_s1
Neurog2
Mm00437603_g1
Id2
Mm01293217_g1
Id3
Mm00492575_m1
Gapdh
Mm99999915_g1
Table S3: References of primers used for quantitative real-time PCR analysis of mRNAs with
Taqman technology.
ChIP analyses
Gene promoters
5’ primer
3’ primer
Bdnf PII
GGGGTGGAGAGGGTAAGAGT
GTTTTGTCACGTGTGGATGG
Bdnf PIII
TATCCCTGCCTCCCTCTCTT
CGGGGAGGGATGATAGAAAG
Bdnf PVI
AAATGGAGAGAAGCCAGTGC
TCTTCGGTTGAGCTTCGATT
Bdnf PVIII
TGACCCACATGGATTGTGAC
TGGAGAAAACTGGTGGTTCC
Gapdh
CTCCCAGGAAGACCCTGCTT
GGAACAGGGAGGAGCAGAGA
Table S4: Primer sequences used for ChIP analyses.
Gene
Mecp2
Water
1.005 ± 0.040 (6)
Ethanol
1.278 ± 0.051 (8)
Percent over
Water
+27.16
Mbd1
1.005 ± 0.038 (6)
1.076 ± 0.055 (8)
+7.06
ns
Ncor
1.005 ± 0.049 (6)
0.788 ± 0.050 (8)
-21.59
**
Hdac1
1.003 ± 0.027 (6)
0.798 ± 0.059 (9)
-20.44
**
Hdac2
1.000 ± 0.026 (6)
0.822 ± 0.029 (9)
-17.80
*
Hdac3
1.005 ± 0.048 (6)
0.954 ± 0.042 (9)
-5.07
ns
Hdac4
1.003 ± 0.040 (6)
1.169 ± 0.027 (8)
+16.55
ns
Hdac5
1.008 ± 0.029 (6)
0.715 ± 0.030 (8)
-29.07
****
Hdac9
1.000 ± 0.024 (6)
1.025 ± 0.041 (8)
+2.50
ns
P value
***
Table S5: Effects of chronic ethanol intake on hippocampal levels of mRNAs encoding
transcriptional repressors.
mRNA levels were measured by real-time RT-qPCR in the hippocampus of “water” and
“ethanol” mice. Results [mean ± S.E.M. of (n) mice] are expressed as arbitrary units after
normalization to the endogenous reference genes encoding Gapdh or Hprt and β-actin. * p
<0.05, ** p <0.01, *** p <0.001, **** p <0.0001, significantly different in “ethanol”
versus “water” mice, ns: not significant, one way ANOVA followed by Bonferroni’s
multiple comparison test.
Gene
Water
Ethanol
Percent over
Water
Mash1
0.941 ± 0.069 (6)
0.750 ± 0.041 (7)
-20.30
ns
Neurod1
1.003 ± 0.034 (6)
1.151 ± 0.045 (8)
+14.76
ns
Neurog1
1.012 ± 0.075 (6)
1.238 ± 0.062 (8)
+22.33
ns
Neurog2
1.020 ± 0.088 (6)
1.340 ± 0.094 (8)
+31.37
**
Id2
1.017 ± 0.083 (6)
1.171 ± 0.034 (7)
+15.14
ns
Id3
1.054 ± 0.102 (6)
0.771 ± 0.064 (7)
-26.85
*
P value
Table S6: Effects of chronic ethanol intake on hippocampal levels of mRNAs encoding
transcription factors of the bHLH family.
mRNA levels were measured by real-time RT-qPCR in the hippocampus of “water” and
“ethanol” mice. Results [mean ± S.E.M. of (n) mice] are expressed as arbitrary units after
normalization to the endogenous reference genes encoding Gapdh or Hprt and β-actin.
* p <0.05, ** p <0.01 significantly different in “ethanol” versus “water” mice, ns: not
significant, one way ANOVA followed by Bonferroni’s multiple comparison test.