Fig 1: In vitro and in vivo assay of STK31 expression in cells ectopically expressing STK31. (A) Establishment of STK31-expressing cells. Gastric cancer cell lines MKN74 and AGS were transfected with lentiviral STK31-expression vector. STK31 mRNA and protein expression levels was compared with cells transfected with an empty vector control (pCDH). Tubulin was used as an internal control. (B) Colony formation, (C) proliferation and (D) migration assays were performed. (E and F) Gap closure assay following STK31 overexpression in MKN74 cells at a density of 1×106 cells. Scale bar, 100 µm. (G) Xenograft assay with transfected MKN74 cells. Mice were sacrificed at day 53 and tumor volumes were measured. Scale bar, 1 cm. (H) Tumor growth curves for STK31-expressing and control MKN74 cells in nude mice. P-values were determined using Student's t-test. *P<0.05, **P<0.01, ***P<0.001. STK, serine/threonine kinase.
Fig 2: STK31 expression and bisulfite sequencing analysis of GC cell lines. (A) RT-qPCR analysis of 16 GC cell lines. (B) Analysis of bisulfite sequencing. A total of eight GC cell lines were categorized based on relative STK31 expression (determined by RT-qPCR) as strong (+) or weak/silenced (-). (C) Association between STK31 expression and mean methylation at CpG#23 and #24. Methylation status was based on bisulfite sequencing and pyrosequencing analysis. (D) Restoration of STK31 mRNA levels following treatment with 5-aza and/or TSA. STK31 expression levels were assessed by RT-qPCR and normalized to ß-actin. Data are presented as the mean ± SD of three independent experiments. Pairwise P-values were calculated using Student's t-test and corrected for multiple comparison by Bonferroni method. *P<0.05. STK, serine/threonine kinase; GC, gastric cancer; RT-q, reverse transcription-quantitative; 5-aza, 5-aza-2-deoxycytidine; TSA, trichostatin A.
Fig 3: Methylation profile of the STK31 promoter in cells of a clinical tissue isolated by laser capture microdissection. (A) Gene structure of STK31 on human chromosome 7p15.3. The map was modified from the UCSC Genome Browser (hg19, genome.ucsc.edu). The distance from TSS to transcription end site is ~122.4 kb. Thick black bars denote exons. (B) RRBS methylome profiles in an enlargement of the STK31 promoter region (~2 kb) in paired GM, IM and GC cells by mirroring the UCSC Genome Browser. The height of each vertical line indicates methylation score for individual CpGs. Methylation and non-methylation scores are displayed as purple and blue bars, respectively. The red rectangle highlights differentially methylated region in GM compared with IM or GC. (C) Strategy for analysis. Bisulfite sequencing was performed for Regions 1 (6 CpGs, -386 to -198 nucleotides from TSS) and 2 (39 CpGs, -171 to 249 nucleotides). Pyrosequencing was performed for CpG#23 (+54 nucleotidea from TSS) and #24 (+58 from TSS). Positions of CpG probes CpG#19 (cg05000488, -46 from TSS) and CpG#25 (cg11755819, +67 from TSS) are shown, proximal to the STK31 TSS from 450K HumanMethylation BeadChip. STK, serine/threonine kinase; TSS, transcription start site; RRBS, reduced representation bisulfite sequencing; GM, gastric mucosa; IM, intestinal metaplasia; GC, gastric cancer.
Fig 4: Histone modifications at STK31 promoters in paired normal and GC tissue. Public data for histone modifications in paired tissue, as determined by nano-scale chromatin immunoprecipitation-sequencing (22), were downloaded and processed. H3K4me3 and H3K27ac peak regions from five paired normal and gastric tumor tissue samples were merged and visualized with the UCSC Genome Browser (hg19). Red rectangle highlights gain of promoter activity with increased H3K27ac and H3K4me3 at each promoter in primary GC. STK, serine/threonine kinase; GC, gastric cancer.
Fig 5: In vitro assay of STK31 expression levels in STK31-KD cells. (A) Establishment of STK31-KD cells by expression of shRNA. SNU484 and MKN01 cells were transfected with either of two lentiviral STK31 shRNAs (sh#1, sh#4) or scrambled-sequence shCon and cultured for 2 weeks. KD and control cells were compared by reverse transcription-quantitative PCR and western blotting. Tubulin was used as an internal control. (B) Colony formation assay. Transfected cells were plated on 6-well plates at 1×103 cells per well. After 2 weeks, colonies were stained with crystal violet and counted. (C) Relative viability of STK31-KD cells over 4 days was measured using EZ-Cytox Cell Viability Assay kit and compared with empty vector control (PLKO). (D) Migration assay. Transfected cells were plated on Transwell chambers at 2×104 cells per well. After 18–22 h, Transwell membranes were stained with crystal violet and cells were counted. (E) Cell cycle analysis of STK31-KD MKN1 cells. Following PI staining, cells were assessed by flow cytometry. (F) Western blot analysis of caspase-3 and PARP cleavage in MKN1 cells. Two membranes were used for PARP and Caspase-3. Pairwise P-values were calculated using Student's t-test and corrected for multiple comparison by Bonferroni method (n=2). *P<0.05, **P<0.01, ***P<0.001. STK, serine/threonine kinase; KD, knockdown; sh, short hairpin; Con, control.
Supplier Page from Abcam for Anti-STK31 antibody