Fig 2: The two smarcad1 zebrafish genes are expressed in both overlapped and distinct regions during embryonic development. Whole‐mount in situ hybridization of zebrafish embryos at stages 12S (A, E), 20S (B, F), 24 hpf (C, D, G, H), 48 hpf (I–P), and 72 hpf (Q–X). Anterior is to the left in all whole‐mount images, and dorsal is to the top in all transverse sections. Gene names are labeled at the upright corner in the panels of whole‐mount images. (A–D, I–L, and Q–T): gene expression of smarcad1a. (E–H, M–P, and U–X): gene expression of smarcad1b. (A and C) Lateral view of the expression of smarcad1a at 12S, 20S, and 24dpf, respectively. (D) Transverse section through the trunk region of the embryos in panel C. smarcad1a is mainly expressed in the neural tissues, endoderm, and intermediate cell mass at these stages. (E and G) Lateral view of the expression of smarcad1b at 12S, 20S, and 24dpf, respectively. (H) Transverse section through the trunk region of the embryos in panel G. The expression patterns of smarcad1b are very similar to smarcad1a at these early stages. (I, J) Lateral and dorsal view of the expression of smarcad1a at 48 hpf. (K and L) Transverse section through the head and pectoral fin regions. (M and N) Lateral and dorsal view of the expression of smarcad1b at 48 hpf. (O and P) Transverse section through the head and pectoral fin regions. (Q and R) Lateral and dorsal view of the expression of smarcad1a at 72 hpf. (S and T) Transverse section through the head and pectoral fin regions. (U and V) Lateral and dorsal view of the expression of smarcad1b at 72 hpf. (W and X) Transverse section through the head and pectoral fin regions. The dashed lines indicate the positions of sections. The letters below the dashed lines correspond to the panels. de, diencephalon; dm, dermomyotome; end, endoderm; fb, forebrain; hb, hindbrain; icm, intermediate cell mass; ir, iris; le, lens; mand, mandibular cartilage; mb, middle brain; mhb, midbrain‐hindbrain boundary; mz, ciliary marginal zone; n, notochord; nt, neural tube; op, optical cup; pf, pectoral fin; pa, pharyngeal; quad, quadrate cartilage; ret, retina; tb, tail bud

Fig 3: Model of SMARCAD1 tumor suppressor mechanisms. Three mechanisms (DNA damage response, heterochromatin maintaining, and transcription regulation) were proposed based on current knowledge. 33 , 34 , 36 , 38 , 72 , 73 , 80 , 81 The bidirectional arrows indicate protein–protein interactions. The arrows imply cellular consequences. The dashed arrows show a possible result. The involved genes and pathways are solely based on loss‐of‐function studies due to the lack of overexpression reports

Fig 4: Human cells show a defect in double‐strand DNA damage repair. (A, B) Double‐strand DNA damage repair was measured by Western blots with anti‐histone H2AX S139ph (phospho Ser139) in SMARCAD1 knockdown schwannoma cell line HEI‐193 after X‐ray irradiations (10 and 20 Gy, respectively). ULTRA‐3351712 knockdown cell was used for this experiment. Doxycycline (final concentration: 0.1 μg/ml) was added 48 h ahead of the experiments. (C, D) Double‐strand DNA damage repair in SMARCAD1 overexpression MPNST cells (STS26T) by H2AX S139ph. Doxycycline (final concentration: 1 μg/ml) was added 24 h ahead of the experiments. Both cell lines were harvested at 1, 5, and 10 h post‐irradiation along with untreated samples. un, untreated sample. Beta‐actin (ACTB) was used as a loading control. The densitometry ratios of H2A X S139ph over ACTB was listed underneath the blots

Fig 5: Zebrafish have two smarcad1 genes. (A) Extended majority‐rule consensus tree for the Bayesian phylogenetic analysis of SMARCAD1 proteins. Numbers at each node indicate posterior probability (pp) values based on 20 million runs. Branch lengths are proportional to the means of the pp densities for their expected replacements per site. The ME and ML phylogenetic trees (Figures S1 and S2) were generally in agreement with the BP phylogeny: most of the metazoan species have one SMARCAD1 gene, while there are usually two smarcad1 genes in teleost genomes. The two smarcad1s most likely resulted from teleost‐specific whole‐genome duplications, as each formed a distinct clade. The tree was rooted with yeast. (B) Syntenic relationship between human and zebrafish chromosomes. Zebrafish chromosome 8 (Dre8) that contains smarcad1a and three other genes (FAM190A, GRID2, and ATOH1, bottom row) are orthologous to, and in the same order as, genes in the portion of human chromosome 4 (Hsa4) that contains SMARCAD1 (middle row). A portion of Dre10 contains smarcad1b, but not the other three directly linked genes. However, this part of Dre10 is co‐orthologous to the portion of Hsa4 that contains SMARCAD1, as they share other syntenies (top row). Orthologous genes are indicated with colored lines