Fig 1.
Effect of microgravity on cell morphology and cell viability of DLD-1 cell cultures.
A DLD-1 cell cultures; Static culture (control) B DLD-1 Microgravity culture at 16 RPM C DLD-1 Microgravity culture at 27RPM Differential staining to detect apoptotic population D DLD-1Static monolayer cultures E Microgravity cultures of DLD1 at 16 RPM F Microgravity cultures of DLD1 at 27 RPM G Cell adhesion and proliferation assay Top panel—static cultures, bottom panel—microgravity cultures shifted to static TCP H Morphological changes in DLD-1; Crystal violet staining of DLD-1 cells in static monolayer culture I Crystal violet staining of DLD-1 cells after transfer of cell aggregates from microgravity to TCP J Colony forming ability assay; Static cultures K Colony forming ability assay; DLD-1 cells after transfer of cell aggregates from microgravity to TCP
Fig 2.
Effect of microgravity on cell viability and cell cycle of DLD-1 and MOLT-4 cell lines.
A Cell viability assay for DLD-1 cells; Viability measured for microgravity cultures (16 RPM and 27 RPM) and static cultures using MTT B Cell cycle analysis for DLD-1 cells; Static C Cell cycle analysis; Microgravity D Cell cycle analysis; Static suspensions on agar underlays E The average sub G0 population in replicates of cell cycle analysis for microgravity, static and static suspension cultures of DLD-1 cells F MOLT-4 cell culture Static and Microgravity cultures of MOLT-4 G Cell viability assay Viability measured for microgravity cultures (16 RPM and 27 RPM) and static cultures using MTT H Cell cycle analysis; Static I Cell cycle analysis; Microgravity cultures of MOLT-4.
Fig 3.
Quantitative PCR analysis for changes in mRNA expression of significant, candidate genes involved in cell proliferation and cancer.
A CDK1—Cell cycle kinase gene, CD117—proto-oncogene, JUNB—transcription factor and immediate early gene, MYC—proto-oncogene expression in DLD-1 and MOLT-4 B Real time PCR analysis in HL-60 CCNE1 and CDK2, CCNB1 and CDK1, Oncogenes: CD117 and MYC, Cancer prognostic markers CD105, CD90 and CD71.
Table 1.
Commonly deregulated genes in DLD-1 and MOLT-4 cells under microgravity.
Fig 4.
Effect of microgravity on gene expression levels in DLD-1 and MOLT-4 cell cultures; Validation of microarray analysis by real time PCR and western blotting.
A Log fold change of CCNB1, ROMO1 and HES1 deregulation in MOLT-4 cells under microgravity as observed by microarray analysis validated by real time PCR B Log fold change of CDK2, HEY1 and STAT3 deregulation in DLD-1 cells under microgravity as observed by microarray analysis validated by real time PCR C Log fold change of commonly up and downregulated genes CCNE1, TFRC (CD71) and CD44 as observed by microarray analysis validated by real time PCR forCCNE1 and TFRC (CD71).
Fig 5.
Dysregulation of stem cell marker CD44 and tumor suppressor microRNA under microgravity.
A Validation of microarray data for CD44 by Western blotting for CD44 protein and beta-actin in static and Microgravity (μG) cultures of DLD1 and MOLT-4 and Densitometric analysis of western blots. B MIR22HG expression under microgravity; Overexpressed MIR22HG, host gene of miR-22 microRNA in DLD-1 cells under microgravity, MOLT-4 cells show no differential expression; Levels of dysregulation of direct targets of miR-22 microRNA CDK6, CCNA2, SP1 and CDKN1A in microarray data; RT-PCR validation of microRNA miR-22 levels and target genes in DLD-1 shows over expressed microRNA miR-22 in DLD-1 cells under microgravity confirming upregulation in microarray data. No significant dysregulation of direct targets CDKN1A (similar to expression levels in microarray data) and CCND1A C GO analysis (by DAVID) of microarray data to depict other dysregulated microRNA host genes in DLD-1 and MOLT-4 cells under microgravity.
Fig 6.
Analysis of microarray data using Gene Functional Classification and Functional Annotation; Dysregulation of genes involved in the Notch signaling system and microRNA processing and regulation.
A Regulation of transcription B RNA mediated gene silencing (PTGS) C Notch signaling pathway D Dysregulated microRNA processors and regulators.
Fig 7.
Functional annotation of microarray data using DAVID.
A Functional annotation of upregulated and downregulated genes of DLD-1 cells under microgravity B Functional annotation of upregulated and downregulated genes of MOLT-4 cells under microgravity.