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1 doi: /nature09667 A critical role for IGF-II in memory consolidation and enhancement Dillon Y. Chen, Sarah A. Stern, Ana Garcia-Osta, Bernadette Saunier-Rebori, Gabriella Pollonini, Dhananjay Bambah-Mukku, Robert D. Blitzer & Cristina M. Alberini Nature 469, (2011) In Supplementary Fig. 2 of this Article, the same western blot representative was inadvertently used for rows 1 and 3 of the left panel. This has now been corrected.
2 doi: /nature % of 0h- 150 C/EBPβ 100 0h 9h 20h 72h Trained (+) No Shock (-) Supplementary Figure 1: Hippocampal C/EBPβ increases after IA training Taubenfeld et al.(ref. 20) showed that, compare to rats that were exposed to the IA box without footshock (No Shock), hippocampal expression of C/EBPβ significantly increased between 6-9h after training and returns to baseline by 48h after training. 1
3 RESEARCH SUPPLEMENTARY INFORMATION Anti-IGF-II rigf-ii rigf-i 25kD 15kD 10kD 15kD 10kD Recombinant Hippocampal kD 15kD 10kD 50kD 37kD Actin Supplementary Figure 2: Anti-IGF-II antibody specifically recognizes IGF-II but not IGF-I. Western blot analyses with anti-igf-ii antibody of recombinant IGF-II and hippocampal extracts non-competed or competed with either recombinant IGF-II or IGF-I. Colloidal gold stained membranes and actin were used as loading controls. 2
4 RESEARCH Mean Latency (s) *** Acq. 24h Test Unpaired protocol Trained Supplementary Figure 3: Unpaired protocol does not lead to long-term memory retention. Unpaired protocol, which dissociates context and footshock by one hour does not elicit long-term IA memory retention. Data are expressed as mean latency ± s.e.m. Two-way ANOVA comparing behavioral protocols and latency revealed a significant interaction F(1,20)=102.2, P<0.0001, behavioral protocol F(1,20)=107.2, P< and latency F(1,20)=107.0, P< Bonferroni post hoc test confirmed that unpaired protocol does not elicit long-term memory retention, where as training leads to significantly memory latency 24h later (***P<0.001). Unpaired: Acq. (10.19± 4.02s); 24h test: (15.63±3.76s), Trained: Acq. (15.82±4.82s); 24h test (493.84±46.16s). n=6/group. 3
5 RESEARCH SUPPLEMENTARY INFORMATION 300bp 200bp MW IGF-II Blank 100bp Supplementary Figure 4: C/EBPβ binds to IGF-II promoter region of exon 1. PCR amplication fragment following C/EBPβ ChIP assay with primers designed to flank the IGF-II promoter region upstream of exon 1 (bp of sequence X ) which contains a putative C/EBPβ consensus sequence. Blank corresponds to PCR reaction carried out without the immunoprecipitated DNA. The sequence of the fragment was confirmed by DNA sequencing. MW= molecular weight in base-pairs (bp). 4
6 RESEARCH Training 0h Test 8h 24h Mean Latency (s) SC-ODN IGF-II-ODN 0h Supplementary Figure 5: Hippocampal single injections of IGF-II-ODN do not impair long-term memory Injections of IGF-II-ODN either immediately after (0h) or 8h after training do not impair memory retention tested at 24h after training. Data are expressed as mean latency ± s.e.m. 0h: SC-ODN (408.2±77.9s); IGF-II-ODN (381.7±103.4s). 8h: SC-ODN (294.9±108.3s); IGF-II-ODN (208.9±118.6s) (n=8/group). 8h 5
7 RESEARCH SUPPLEMENTARY INFORMATION Training Euthanasia 0h 8h 16h IGF-II IGF-I 18s Fold Change of SC-ODN IGF-II IGF-I *** 0.4 SC-ODN IGF-II-ODN Supplementary Figure 6: Double injections of IGF-II-ODN selectively knock down IGF-II, but not IGF-I mrna. Representative example and real-time quantitative PCR of IGF-II and IGF-I show that double Injections of IGF-II-ODN into the hippocampus immediately after (0h) and 8h after training knock down endogenous levels of IGF-II, but not IGF-I mrna measured at 16h after training. Data are expressed as mean Fold change of SC-ODN± s.e.m (Student s t-test ***P=0.0006). IGF-II: (SC-ODN: 1.0±0.1; IGF-II-ODN: 0.3±0.04). IGF-I: (SC-ODN: 1.0±0.09; IGF-II-ODN:0.98±0.07). n=4/group. 6
8 RESEARCH Training Final Test Locomotor Test # of crossing of arbtirary midlines h 24h Veh IGF-II IGF-I Supplementary Figure 7: IGF-II administration does not affect spontaneous locomotor activity. Twenty-four h after the final retention test (experiments described in Figs 3a and 4a), rats injected with either vehicle, IGF-II or IGF-I were placed in a novel box and their activity, which was measured by the number of crossings of arbitrary lines that divide the box into quadrants, in 540 sec. Veh (n=8): 40.8± 7.0; IGF-II (n=8): 44.6± 4.1; IGF-I (n=8): 39.6± 2.3. Data are expressed as mean number of crossing ± s.e.m. 7
9 RESEARCH SUPPLEMENTARY INFORMATION Training Test 0h 24h Mean latency (s) * ** ** Veh 25ng IGF-II 2.5ng IGF-II 250ng IGF-II Supplementary Figure 8: IGF-II enhances memory retention in a dose-dependent manner. Groups of rats were injected with different doses of IGF-II immediately after training. Memory was tested 24h after training. Veh (n=4): 275.3± 83.5s; 2.5ng (n=5): 552.4± 23.3s; 25ng (n=6) 798.4±57.55s; 250ng (n=6): 758.9± 93.6s. One-way ANOVA F(3,20)=9.37, P= followed by Newman-Keuls post-hoc test *P<0.05, **P<0.01). Data are expressed as mean latency ± s.e.m. 8
10 RESEARCH Training Test1 Test2 5h 1h 5h 0h 24h 48h 900 ** ** * ** Mean latency (s) Test1 Test2 Test1 Test2 5h -1h+5h Veh/SC-ODN IGF-II/SC-ODN IGF-II/β-ODN Supplementary Figure 9: IGF-II-mediated memory enhancement does not require the function of C/EBPβ. Neither single Injections of C/EBPβ ODN 5h after memory retreival (Test 1), nor double injections of C/EBPβ ODN, 1h before and 5h after Test 1, affected the IGF-II mediated memory enhancement. Data are expressed as mean latency ± s.e.m. 5h (Veh/SC-ODN: Test 1: ±75.69s, Test 2: 323.6±74.1s; IGF-II/SC-ODN: Test 1: 289.2±85.3s, Test 2: 821.6±53.9s;IGF-II/β ODN: Test 1: 419.7±121.3s, Test 2: ±97.5s). -1h+5h (Veh/2xSC-ODN: Test 1: 315.5±96.4s, Test 2: 329.7±74.2s; IGF-II/2xSC-ODN: Test 1: 427.3±99.3s, Test 2: 743.7±137.7s; IGF-II/2xβ ODN: Test 1: 371.8±107.5s, Test 2: ±90.3s). n=4-6/group. (5h: two-way ANOVA F(2,26)=2.8, P=0.079 for interaction, F(2,26)=5.93, P= for treatment, F(1,26)=11.9, P= for test, Bonferroni post-hoc **P<0.01; -1h+5h: two-way ANOVA F(2,30)=2.53, P= for interaction, F(2,30)=3.72, P= for treatment, F(1,30)=10.27, P= for test, Bonferroni post-hoc *P<0.05,**P<0.01) 9
11 RESEARCH SUPPLEMENTARY INFORMATION T P T P PSD-95 NR-1 Actin Supplementary Figure 10: Biochemical characterization of the synaptoneurosomal fraction. Western blot analyses showed that synaptoneurosomal fraction (P) is enriched for PSD95 and NMDA receptor subunit NR1 compared to total lysate (T), whereas actin is not. 10
12 RESEARCH a PPR a2 before after Freq. (Hz) b1 fepsp amp. (mv) b2-2 0 before after PPR before after Freq. (Hz) fepsp amp. (mv) -2 0 before after Supplementary Figure 11: a, Paired pulse ratio (PPR) and b, spike threshold are not affected by IGF-II or anti-igf-iir antibody. a1, PPRs (n=4) were obtained by evoking two fepsps at 10, 20, 50 and 100 Hz, before the addition of IGF-II (1.34± 0.04, 1.43± 0.02, 1.28± 0.03 and 0.78± 0.04 respectively) and in the presence of IGF-II (1.39± 0.02, 1.47± 0.04, 1.34± 0.02 and 0.84± 0.07 respectively). a2, PPRs (n=5) were obtained as a1, but either before the introduction of anti-igf-iir antibody (1.17± 0.06, 1.39± 0.06, 1.35± 0.09, 0.96± 0.08 at 10, 20, 50 and 100 Hz respectively) or in the presence of the antibody (1.27± 0.04, 1.44± 0.08, 1.23± 0.05 and 0.85± 0.06 at 10, 20, 50 and 100 Hz respectively). In all cases, PPR was unaffected by drug treatment. b, Spike threshold was measured by stimulating input fibers at increasing intensities before and after treatment. Neither IGF-II (b1) (n=7, before: 2.24± 0.16; after 2.12± 0.11 mv) nor anti-igf-iir antibody (b2) (n=5 before 2.07± 0.16; after 2.12± 0.15 mv) produced any changes in spike threshold. 11
13 RESEARCH SUPPLEMENTARY INFORMATION fepsp (% of baseline) * *** whfs no HFS Veh IGF-II Anti-IGF-IIR antibody * Supplementary Figure 12: IGF-II promotes the induction of IGF-IIR dependent stable LTP. Average of the last 10 minutes of data plotted in Fig. 6e showing that IGF-II promotes stable LTP after weak HFS, and anti-igf-iir antibody blocks this effect. Depicted group differences are significant at P<0.001 (***) or P<0.05 (*); no other group differences were statistically significant. 12
14 RESEARCH Table 1: Fold change of IGF-II mrna and protein levels and IGF-I mrna levels Figure 1a Control (h-) Trained (+) 0h ± 11.7% 6h ± 38.3% 90.2 ± 10.2% 9h ± 20.8% ± 59.5% 20h ± 16.7% ± 72.6% 36h ± 25.3% ± 61.0% Figure 1b Fold Change of 20h-/0h- IGF-II IGF-I 20h-/0h ± ± h+/0h ± ± 0.16 Figure 1c % of 0h- 0h ± 7.9% 20h ± 18.8% 20h ± 20.6% 72h ± 24.5% 72h ± 27.6% 96h ± 13.8% 96h ± 14.8% Figure 1d % of SC-ODN Unp SC-ODN Unp ± 14.2% SC-ODN Trained ± 30.8% β-odn Trained ± 17.2% β-odn Unp ± 7.4% 13
15 RESEARCH SUPPLEMENTARY INFORMATION Table 2: Mean latencies of rats after training and treatment Figure 2a n Mean Latency (s) 0h/8h 24h/32h 96h/104h SC-ODN ± ± ± 74.5 IGF-II ODN ± ± ± 33.8 Retrain ± 53.2 Figure 2b Mean Latency (s) SC-ODN/IGF-I ± 47.0 SC-ODN/IGF-II ± 56.8 IGFII-ODN/IGF-I ± 33.7 IGFII-ODN/IGF-I ±
16 RESEARCH Table 3: Mean latencies or percent freezing of rats after training and treatment Figure 3a n Mean Latency (s) Test1 Test2 Veh ± ± IGF-II ± ± IGF-I ± ± Figure 3b Acq. Test Veh ± ± 36.2 IGF-II ± ± Figure 3c Percent freezing Baseline Context Test Tone Test Veh ± ± ± 4.4 IGF-II ± ± ± 4.3 Figure 3d Mean Latency (s) Test Veh ± IGF-II ±
17 RESEARCH SUPPLEMENTARY INFORMATION Table 4: Mean latencies of rats after training and treatment Figure 4a n Mean Latency (s) Test 1 Final Test IGF-I IGF-II ± ± ± ± IGF-I NoR ± 77.3 IGF-II NoR ± 39.3 Figure 4b Test 1 Final Test Veh IGF-II ± ± ± ±
18 RESEARCH Table 5: Mean latencies of rats after training, reactivation and treatment Figure 5a n Mean Latency (s) Veh ± 87.9 IGF-II ± IGF-II/Anti-IGF-II-R ± 86.9s IGF-II/JB ± Anti-IGF2-R ± 136.9s JB ± 91.2 Figure 5b Mean Latency (s) IgG ± 73.2 Anti-IGF-IIR antibody ± 17.4 Figure 5c Mean Latency (s) Test 1 Test 2 Veh ± ± 98.9 IGF-II ± ± 93.4 IGF-II+Aniso ± ± 65.9 Figure 5d Mean Latency (s) Veh/SC-ODN ± 55.0 IGF-II/SC-ODN ± 31.0 IGF-II/Arc-ODN ± Veh/Arc-ODN ±
19 RESEARCH SUPPLEMENTARY INFORMATION Table 6: Fold change of western blot analyses and mean latencies of rats after training, reactivation and treatment Figure 6a n pcreb C/EBPβ Naïve-Veh ± 15.2% ± 6.4% Trained-Veh ± 20.1% ± 10.6% Trained-IGF-II ± 30.0% ± 20.6% Figure 6b GluR1 GluR2 Naïve-Veh ± 21.2% ± 12.0% Trained-Veh ± 30.6% 72.3 ± 4.9% Trained-IGF-II ± 52.2% 83.3 ± 10.8% Trained-IGF-II ± 37.5% 81.4 ± 19.4% anti-igf-iir Figure 6c pgsk3β GSK3β Naïve-Veh ± 13.2% ± 11.1% Trained-Veh ± 3.4% 99.3 ± 10.9% Trained-IGF-II ± 5.7% ± 16.1% Trained-IGF-II ± 9.6% ± 6.9% anti-igf-iir Figure 6d Mean Latency (s) Test 1 Test 2 Veh ± ± 66.7 SB ± ± 67.6 IGF-II ± ± IGF- II+SB ± ±
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Supplemental Figure 1 A S100A4: SFLGKRTDEAAFQKLMSNLDSNRDNEVDFQEYCVFLSCIAMMCNEFFEGFPDK Overlap: SF G DE KLM LD N D VDFQEY VFL I M N FF G PD S100A2: SFVGEKVDEEGLKKLMGSLDENSDQQVDFQEYAVFLALITVMCNDFFQGCPDR
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Supplementary Figure 1
Combination index (CI) Supplementary Figure 1 2. 1.5 1. Ishikawa AN3CA Nou-1 Hec-18.5...2.4.6.8 1. Fraction affected (Fa) Supplementary Figure 1. The synergistic effect of PARP inhibitor and PI3K inhibitor
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Supplementary Figures
Supplementary Figures Supplementary Fig. 1. Galectin-3 is present within tumors. (A) mrna expression levels of Lgals3 (galectin-3) and Lgals8 (galectin-8) in the four classes of cell lines as determined
Supporting Information
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a 2.5 1. AMPAR-fEPSP slope (mv/ms) 2. 1. NMDAR-fEPSP amplitude (mv).8.6.4.5.2. 2 4 6 8. 1 2 3 4 5 Intensity (µa) Intensity (µa) b 2. PPF Ratio (fepsp2/fepsp1) 1..5. 5 1 2 5 Time (ms) Supplementary Figure
Supplementary Figure 1
Supplementary Figure 1 A B mir-141, human cell lines mir-2c, human cell lines mir-141, hepatocytes mir-2c, hepatocytes Relative RNA.1.8.6.4.2 Relative RNA.3.2.1 Relative RNA 1.5 1..5 Relative RNA 2. 1.5
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Nature Neuroscience: doi: /nn Supplementary Figure 1
Supplementary Figure 1 Bidirectional optogenetic modulation of the tonic activity of CEA PKCδ + neurons in vitro. a, Top, Cell-attached voltage recording illustrating the blue light-induced increase in
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Supplementary Figure 1
Supplementary Figure 1 Supplementary Figure 1 SNARE Probes for FRET/2pFLIM Analysis Used in the Present Study. mturquoise (mtq) and Venus (Ven) are in blue and yellow, respectively. The soluble N-ethylmaleimide-sensitive
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Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
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