Additional file 1 of Elevated expression of RAB3B plays important roles in chemoresistance and metastatic potential of hepatoma cells
Creators: name: Tsunedomi, Ryouichi
name: Yoshimura, Kiyoshi
name: Kimura, Yuta
name: Nishiyama, Mitsuo
name: Fujiwara, Nobuyuki
name: Matsukuma, Satoshi
name: Kanekiyo, Shinsuke
name: Matsui, Hiroto
name: Shindo, Yoshitaro
name: Watanabe, Yusaku
name: Tokumitsu, Yukio
name: Yoshida, Shin
name: Iida, Michihisa
name: Suzuki, Nobuaki
name: Takeda, Shigeru
name: Ioka, Tatsuya
name: Hazama, Shoichi
name: Nagano, Hiroaki
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Table S1. Primers and hydrolysis probes used in this study. Supplementary Figure S1. Genome editing of RAB3B. In each panel, upper and lower figures show Sanger sequencing data and the respective amino acid sequence it translates to. a, In the wild-type RAB3Bsequence, a protospaceradjacent motif (PAM) and guide RNA sequences are represented. b, RAB3Bsequence with heterogeneous mutation and resulting truncated amino acid sequence are represented. Supplementary Figure S2. Genome view of edited RAB3B. Genomic DNA from SK-HEP-1 (upper panel) and its derivative, RAB3B-KD cells (lower panel) were subjected to whole exome sequence using the TruSeqRapid Exome Library Prep Kit and NextSeq500 (Illumina). the filtered short reads were mapped to the reference genome (hg19) with BWA (version 0.7.12). The represented image was generated using IGV (version 2.9.4). The RAB3B-KD cells showed reads harboring insertion “T” at chromosome 1: 52,442,589 with 37% (11/30 reads) frequency. Mutation analysis using Strelka(version 0.4.10.2) showed no insertion/deletion variation by the off-target effect of the genome-editing. Supplementary Figure S3. Schematic for identification of genes specific for cancer stem-like cells and HCCs with poor prognosis. On the left, the identification was started with SK-HEP-1 cells in the sphere inducing and control conditions. On the right, the identification started from HCC specimens with/without recurrence after surgery. The represented number of starting genes showed that the number of genes with sum of a fragment-count of all samples was more than half of the sample number. DEGs, differentially expressed genes with &gt; 2-fold change, q 50. Supplementary Figure S4. mRNA levels of the identified genes determined using RNA-seqanalysis. The mRNA levels of the five identified cancer stem-like cell specific upregulated genes in SK-HEP-1 cells (a) and clinical specimens (b) are represented as transcripts per million (TPM). Supplementary Figure S5. Cell cycle analysis. Cell cycle distribution of SK-HEP-1 (aand b), RAB3B-KO (cand d), and KD/pRAB3B (eand f) cells in control (a, c, and e) and sphere inducing (b, d, and f) conditions, respectively. After cultivation, cells were dissociated with Accumax(Innovative Cell Technologies, San Diego, CA, USA). Cell cycle distribution was analyzed by flow cytometry, following propidiumiodide (PI) staining. Cells were fixed with 70% ethanol and then resuspendedin PI/RNase Staining Buffer (BD Biosciences, Franklin Lakes, NJ). The DNA content of cells was analyzed using a MACSQuantanalyzer (MiltenyiBiotec, BergischGladbach, Germany). Each panel shows a representative histogram. Supplementary Figure S6. BCAN9370Liver metastasis ability of RAB3B-KD cells. The metastatic ability of cells was evaluated by splenic injection into NOD-Rag1nullIL2rγnulldouble mutant mice. Representative images in Table 1, mice were injected with 1 x 104 tumor cells. RAB3B-KD is a RAB3B-edited clone derived from SK-HEP-1 cells. Yellow arrows indicate formed tumors. Supplementary Figure S7. Schematic for identification of RAB3B-affected cancer stem-like cell (CSLC) specific genes. With SK-HEP-1 derivative cells; parental SK-HEP-1, RAB3B-KD, and KD/pRAB3B, RAB3Baffected CSLC specific differentially expressed genes (DEGs) were identified using RNA-seqanalysis. On the left, the DEGs in sphere-inducing conditions were identified using the same criteria as mentioned in Supplementary Fig. S3. On the right, RAB3B-affected genes in control conditions were identified as DEGs with &gt; 1.7-fold change, q 30. The circled numbers are the screening criteria as shown in the text. The represented number of starting genes shows that the number of genes with sum of a fragment-count of all samples was more than half of the sample number. Supplementary Figure S8. Full-length blots used in Fig. 2. Supplementary Figure S9. Full-length blots at different exposure time used in Fig. 2.