**2. Materials and Methods**

### *2.1. Generation of Conditional Knockout Mice, Breeding, and Genotyping*

Mice harboring the floxed allele (f) of the *TCTP* gene were generated and genotyped as previously described [15]. Brain neuronal progenitor cell-specific TCTP conditional mutants were obtained by breeding *floxed TCTP* mice with *Nestin-cre* mice (B6.Cg-*Tg (Nes-cre)1Kln*/J, #003771) from Jackson Laboratory [22,23] to produce *NestinCre*/+; *TCTPflox*/+ (heterozygous, het) mice. The *NestinCre*/+; *TCTPflox*/+ mice were crossed with *NestinCre*/+; *TCTPflox*/+ mice or *TCTPf*/*f* alone mice to produce *NestinCre*/+; *TCTPflox*/*flox* homologous conditional mutant mice (TCTP-cKO). *NestinCre*/+; *TCTP*+/+, or *TCTPf*/*f* alone mice were used as a control. Both *floxed TCTP* and *Nestin-Cre* mouse lines were generated in C57BL/6 and 129svj mixed background, and the mice used in this study were back-crossed to C57BL/6 for at least 8 generations. Double-heterozygous littermates (*NestinCre*/+; *TCTPf*/+) were also used as controls for some in vivo experiments. For embryonic time points, the time of plug identification was counted as postnatal day 0.5 (P0.5). Genotyping was performed by PCR using primers P1 (5--TCTAGAAAAGTGGAGGCGGAGC-3-) and P5 (5--GGTGACTACTGTGCTTT CGG TA-3-) for the wild-type (450 base pairs) and floxed (520 base pairs) alleles, and cre-sense (5--TGCCACGACCAAGTGACAGC-3-) and cre-antisense (5--CCTTAGCGCCGTAAAT CAATCG-3- ) for the cre allele (580 base pairs). All animal studies were performed following the recommended procedures approved by the Institutional Animal Care and Use Committee of Tzu Chi University (PPL number: 97043) and conformed to the guidelines of Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes or the National Institutes of Health (NIH) guidelines.

### *2.2. Tissue Processing, Histological Analysis, and Immunohistochemistry*

For histological analysis, whole brains were fixed with 4% paraformaldehyde and embedded in para ffin, sectioned, and stained with hematoxylin and eosin. TCTP expression pattern, BrdU incorporation, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, caspase activation, and Oct4 expression were detected by immunohistochemistry with DAB staining. Immunofluorescence was performed by labeling with anti-TCTP (1:200; Abcam, Cambridge, United Kingdom), anti-Tuj1 (1:100; Millipore, Burlington, MA, USA), anti-active caspase-3 (1:100; Abcam), anti-nestin (1:100; Cell Signaling, Danvers, MA, USA), and anti-MAP (1:100; Invitrogen, Carlsbad, CA, USA) antibodies. The retrieved sections were incubated with the primary antibody overnight at 4 ◦C, washed for 1 h in phosphate-bu ffered saline (PBS), and incubated with the secondary antibody for 1 h, then washed for 1 h in PBS. Sections were followed by DAB staining, and then counterstained with hematoxylin. For the immunofluorescence sections, visualization of staining was achieved using HiLyte Fluor 488- and HiLyte Fluor 555-conjugated secondary antibodies (1:200; AnaSpec, Fremont, CA, USA), then counterstained with 4', 6-diamidino-2-phenylindole (DAPI) for double or triple immunofluorescence staining. Immunofluorescence was visualized and captured using an Olympus BX43 upright fluorescent microscope equipped with a digital camera system (DP-72, Olympus, Tokyo, Japan) or a Zeiss LSM 510 META confocal imaging system (Carl Zeiss, Oberkochen, Germany).

### *2.3. RNA Isolation, cDNA Synthesis, and Quantitative Real-Time Reverse Transcriptase PCR*

To analyze gene expression in the embryonic and postnatal stages of control and TCTP-cKO mice, RNA was isolated from 4 independent biological samples at stages E13.5, E16.5, P0.5, and P56 using TRIzol reagen<sup>t</sup> (Invitrogen #15596018). Total RNA (1 μg) extracted from brain tissue was reverse-transcribed to synthesize cDNA using a ReverTra Aceα- reverse transcription kit (Toyobo #F0937K, Toyobo, Osaka Prefecture, Japan) and oligo (dT) as a primer. Real-time PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems #4368577, Applied Biosystems, Foster City, CA, USA) in an ABI 7300 Real-Time PCR System (Applied Biosystems). The specific primers used for the detection of genes are shown in Table 1. Each reaction was performed in duplicate, and dissociation curves were constructed to ensure that only a single product was amplified. The transcript expression level of the gene of interest was normalized to GAPDH mRNA as the internal control, and the data were expressed as a fold change over the control group.
