Figure S2. Perifosine rescues the defective phenotype caused by Eva1a ... E12.5 were cultured under proliferative conditions with or without perifosine (10μM).
Stem Cell Reports, Volume 6
Supplemental Information
EVA1A/TMEM166 Regulates Embryonic Neurogenesis by Autophagy Mengtao Li, Guang Lu, Jia Hu, Xue Shen, Jiabao Ju, Yuanxu Gao, Liujing Qu, Yan Xia, Yingyu Chen, and Yun Bai
Supplemental Figures and Legends
Figure S1. EVA1A expression in the developing mouse brain. (A) Western blot analysis of the levels of EVA1A, LC3B and β-TUBULIN III in the mouse brain at the indicated time points. ACTB was used as a loading control. (B) Quantifications of the indicated protein levels relative to ACTB are shown. The average value in E10.5 whole brain tissue was normalized as 1. Data are means ± SD of the results from three independent experiments. (C, D and E) Representative confocal microscopy images of the expression of EVA1A, LC3B and β-TUBULIN III in mouse brain cortex from E10.5 to P2. Scale bar = 100 μm.
Figure S2. Perifosine rescues the defective phenotype caused by Eva1a depletion through the suppression of the PI3CA-AKT pathway and the activation of autophagy. (A and C) NSCs obtained from Eva1a+/+ and Eva1a-/- at E12.5 were cultured under proliferative conditions with or without perifosine (10μM) for 3 h, after which the cells were lysed and subjected to immunoblot analysis using the indicated antibodies. (B and D) Densitometric analysis of the immunoblots. ACTB was assessed as a loading control. The average value in the Eva1a+/+ group was normalized as 1. Data are means ± SD of the results from three independent experiments. *P < 0.05, **P < 0.01; One-way ANOVA with Tukey’s post hoc test. (E and F) NSCs obtained from Eva1a+/+ and Eva1a-/- at E12.5 were cultured under
proliferative conditions in 96 well non-coated plates with 500 or 1 000 cells per well with or without perifosine (10μM). The number and the size (diameter) of secondary neurospheres were evaluated. The average value in the Eva1a+/+ group was normalized as 100%. Data are means ± SD of the results from three independent experiments. *P < 0.05, **P < 0.01; One-way ANOVA with Tukey’s post hoc test. (G-H) NSCs obtained from Eva1a+/+ and Eva1a-/- at E12.5 were cultured under differentiation conditions with or without perifosine (10 μM). Then the cells were subjected to immunostaining against β-TUBULIN III and viewed under a fluorescence microscope. Five randomly selected areas from each slide were examined for the percentage of β-TUBULIN III+-differentiated neurons in total cells and β-TUBULIN III+ cells with neurites under fluorescence microscope. All experiments were performed in triplicate. Data are means ± SD. *P < 0.05; One-way ANOVA with Tukey’s post hoc test.
Figure S3. MP treatment recovers impaired autophagic flux in Eva1a-/- NSCs. (A) NSCs from Eva1a-/- at E12.5 were grown as neurospheres in the presence or absence of MP (10 mM), and treated with chloroquine (CQ, 25 μM) for 2h. Cells were then harvested for the TEM analysis. Scale bars: 5 μm (left), 1 μm (right). (B) Quantification of autophagic structures per cell treated as in (A). Data are means ± SD of at least 20 cells scored (*p < 0.05; t-test).
Figure S4. mTOR-dependent autophagy modulator rescues the defective neurogenesis caused by Eva1a knockout. (A and C) NSCs from Eva1a+/+ and Eva1a-/- at E12.5 were grown as neurospheres, and then plated under differentiation conditions for 6 days with or without rapamycin (100 nM) or LiCl (10 mM) treatment for 4 h.
Treated NSCs were lysed and subjected to immunoblot analysis using the
indicated antibodies. (B and D) Histogram showing the quantification of the ratio of the relative proteins/ACTB. The average value in the Eva1a+/+ group (lane1) without rapamycin was normalized as 1. Data are means ± SD of the results from three independent experiments. *P < 0.05, ns, not significance; One-way ANOVA with Tukey’s post hoc test. (E and F) The treatment of NSCs was same as in (A-B), after which the cells were subjected to immunostaining against β-TUBULIN III and viewed
under a fluorescence microscope. Five randomly selected areas from each slide were examined for the percentage of β-TUBULIN III+-differentiated neurons in total cells and β-TUBULIN III+ cells with neurites under fluorescence microscope. All experiments were performed in triplicate. Data are means ± SD. *P < 0.05, ns, not significance; One-way ANOVA with Tukey’s post hoc test. (G and H) The treatment of NSCs was same as in (C-D), after which the cells were subjected to immunostaining against β-TUBULIN III and viewed under a fluorescence microscope. Five randomly selected areas from each slide were examined for the percentage of β-TUBULIN III+-differentiated neurons in total cells and β-TUBULIN III+ cells with neurites under fluorescence microscope. All experiments were performed in triplicate. Data are means ± SD. *P < 0.05, ns, not significance; One-way ANOVA with Tukey’s post hoc test.
Supplemental Table 1. Sequence of the primers used in genome PCR
Genes
Eva1a-GT(5)
Eva1a-GT(3)
Cre
Primer sequence Forward
5’ ATCTGTTAGGGACAAGGGTA 3’
Reverse
5’ CAAAGGAGAATGGCAAATGG 3’
Forward
5’ TCTGAGGCGGAAAGAACCAG 3’
Reverse
5’ CAGCCCAGGAAATAGGATGA 3’
Forward
5’ GCCTGCATTACCGGTCGATGC 3’
Reverse
5’ CAGGGTGTTATAAGCAATCCC 3’
Supplemental Table 2. Sequence of the primers used in RT-PCR
Genes
Eva1a
Nestin
β-tubulin III
Atg5
Atg6
Atg7
Atg8
Atg12
Atg16
Gapdh
Primer sequence Forward
5’ GCCGCTCTGTACTTTGTC 3’
Reverse
5’ TCTCCCTGATGATTCGTT 3’
Forward
5’ GGAGGGCAGAGAAGACAGTG 3’
Reverse
5’ TGACATCCTGGACCTTGACA 3’
Forward
5’ TTTCGTCTCTAGCCGCGTG 3’
Reverse
5’ ACCACGCTGAAGGTGTTCAT 3’
Forward
5’ AAGGATGCGGTTGAGGCT 3’
Reverse
5’ GGCGACTGCGGAAGGACA 3’
Forward
5’ ATAAGATGGGTCTGAAGTT 3’
Reverse
5’ GGTTTTGATGGAATAGGAG 3’
Forward
5’ CCGGTGGCTTCCTACTGTTA 3’
Reverse
5’ AAGGCAGCGTTGATGACC 3’
Forward
5’ TTCGCCGACCGCTGTAAG 3’
Reverse
5’ TGCGTGGGGTTGAGTTGC 3’
Forward
5’ ATGTCGGAAGATTCAGAGGT 3’
Reverse
5’ TCATCCCCATGCCTGGGATT 3’
Forward
5’ ATGTCGTCGGGCCTGCGC 3’
Reverse
5’ CAGACCAAAGATATTAGTGA 3’
Forward
5’ TGAAGGTCGGAGTCAACGGATTTGGT 3’
Reverse
5’ CATGTGGGCCATGAGGTCCACCAC 3’
Supplemental Experimental Procedures
Antibodies and reagents
The following antibodies were used: Rabbit anti-mouse p62/SQSTM1 antibody (MBL International, PM045, Japan), anti-UBIQUITIN (CST, 3936, Danvers, MA, US), anti-LC3B (CST, 2775, Danvers, MA, US), anti-β-TUBULIN III (TuJ1, Covance, MRB-435P, Princeton, New Jersey, US), anti-SOX2 (Abcam, ab133337, Cambridge, MA,
US),
anti-NESTIN
(Sigma-Aldrich,
anti-β-ACTIN/ACTB (Proteintech, 60008–1-Ig,
C9848,
St.
Louis,
MO,
US),
Campbell Park, Chicago, USA), anti-
mTOR (CST, 7C10, Danvers, MA, US), anti phospho-mTOR (Ser2448)（CST, 2971S, Danvers, MA, US), anti RPS6KB1(CST, 4907, Danvers, MA, US), anti phosphoRPS6KB1 (Thr389) (CST, 9234s, Danvers, MA, US), anti PIK3CA p85 antibody (CST, 4292, Danvers, MA, US), anti phospho-PIK3CA p85 (Tyr458) (CST, 4228, Danvers, MA, US), anti AKT antibody (CST, 9272s, Danvers, MA, US), anti phospho-AKT (Ser473) (CST, 4060s, Danvers, MA, US), anti EIF4EBP1 antibody (CST, 9644, Danvers, MA, US), anti phospho-EIF4EBP1 (Thr37/46) (CST, 2855, Danvers, MA, US), anti phospho-TSC2 (Thr1462) (CST, 7249, Danvers, MA, US) and anti-EVA1A (Novus, NB110-74787, Littleton, US). Secondary antibodies included Alexa Fluor 488-labeled goat anti-mouse and rabbit IgG(H+L) (ZSGB-BIO, ZF-0512 and ZF-0511, Beijing, China), Alexa Fluor 594-labeled goat anti-mouse and rabbit IgG(H+L) (ZSGB-BIO, ZF-0513 and ZF-0516, Beijing, China), DyLight™ 680-conjugated anti-mouse and rabbit IgG (H&L) (Rockland,
610-144-002 and 611-144-002,
Limerick, PA), DyLight™ 800-conjugated anti-mouse and rabbit IgG (H&L)( Rockland, 610-145-002 and 611-145-002, Limerick, PA). Other reagents used in this study were: methylpyruvate
(Santa
Cruz,
sc-250384,
Dallas,
Texas,
US),
chloroquine
(Sigma-Aldrich; c6628), perifosine (Selleck; S1037), rapamycin (Selleck; S1039), LiCl (Sigma-Aldrich; V900067), Hoechst 33342 (Life, H1399, Waltham, MA US).
Transmission electron microscopy (TEM)
Treated NSCs were initially fixed in 0.1 M sodium phosphate buffer containing 3% glutaraldehyde (pH 7.4) and then fixed in 0.1 M sodium phosphate buffer containing 1% OsO4 (pH 7.2) for 2 h at 4°C. The cells were dehydrated in a graded series of ethanol. Cells were embedded in Ultracut (LEICA ULTRACUT R) and sliced into 60-nm sections. Ultrathin sections were stained with uranyl acetate and lead citrate, and observed under a JEM-1230 transmission electron microscope.
