biostudies-arrayexpressFrédéric MartinsMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16618Bone regeneration remains clinically challenging, partly due to limited knowledge of the cellular mechanisms governing bone repair. Osteocytes, which constitute the majority of bone cells, form an dendritic network and are central regulators of skeletal homeostasis. Residing in a mineralized and low-oxygen environment, they are exposed to hypoxic conditions that may shape their function, yet the role of hypoxia in osteocyte behavior and angiogenic signaling during bone regeneration is still unclear. This study investigated the effects of hypoxia on osteocytes and their paracrine interaction with endothelial cells. MLO-Y4 osteocytic cells were cultured under normoxia (21% O₂), moderate hypoxia (5% O₂), or severe hypoxia (1% O₂), and conditioned media were applied to mouse endothelial cells. Transcriptomic analyses were performed following optimization of the co-culture model. Hypoxia promoted osteocyte dendritic network development and induced oxygen-dependent metabolic and transcriptional adaptations. Severe hypoxia induces a stronger hypoxic response and glycolytic shift compared with moderate hypoxia, leading to distinct osteocyte profiles. In contrast, osteocyte-conditioned media did not significantly affect endothelial gene expression, suggesting that hypoxia-independent mechanisms may govern osteocyte-endothelial communication. These findings highlight oxygen availability as a key regulator of osteocyte physiology and underscore the need for further investigation into its role during bone regeneration.biostudies-arrayexpressSequencing - Libraries were sequenced at the PGTB facility with the NextSeq 2000 P3 XLEAP-SBS Reagent Kit (100 Cycles) (Illumina, 20100990) using the NextSeq 2000 sequencing System (Illumina, 20038897). Paired-end reads (R1, 100 bases correspond to the sense strand and R2, 8 bases to UMI) resulted in an average of 29 million paired reads by sample.Sample Collection - The cells on six-well culture plates were washed with sterile PBS 1X and stored dry at -80°C for Total RNA extraction.Nucleic Acid Extraction - Total RNA extraction was performed on six-well culture plates using the QIAGEN RNeasy Micro Kit (QIAGEN, 74004).Library Construction - RNA-sequencing libraries were prepared using the SEQuoia Express Stranded RNA Library Prep Kit (Bio-Rad, 12017297), SEQuoia RiboDepletion Kit (Bio-Rad, 17006487), and SEQuoia Dual Indexed Primers (Bio-Rad, 12011930) according to the manufacturer’s protocol. Briefly, 40ng of total RNA were first fragmented 8min at 98°C. Then, cDNA was synthesized with adapter addition in a continuous synthesis reaction. After twice SPRI cleanup, 11 cycles of PCR were applied to amplify libraries and barcode samples with unique dual indices. An additional SPRI cleanup step allowed to obtaining 200-1000 bp fragments. Six point four nanograms of libraries were ribodepleted and then amplified with 9 cycles of PCR. An additional SPRI cleanup step allowed to obtain 11-40nM of libraries with 378-427 bp of average size.Growth Protocol - MLO-Y4 were seeded in six-well culture plates and cultured for 48h under normoxia (21 % O2), or hypoxic conditions (hypoxia 5% O2 and 1 % O2). Endothelial cells (1x10^4 cells/cm2) were seeded in six-well culture plates and incubated in a 1:1 ratio of endothelial cell medium and osteocyte conditioned medium (21 % O2, 5 % O2, and 1 % O2).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Fastq files were analyzed with Bio-Rad SeqSense Analysis Solution v2.1 with the following parameters: Species: mm10 (GRCm38 Mus musculus); Minimum MapQ Score: 30; Min Base Pairs Per read: 50; 3’ and 5’ Read Quality Cutoff: 30, to produce gene counts files. STAR (2.7.0), picard (2.27.4), FastQC (0.11.9), UMI tools (1.1.4), bedtools (2.31.0) and subread (1.6.4) modules where used for alignment, data quality assessment, UMI parsing, deduplication, read trimming and counting, using the reference genome mm10 (GRCm38 Mus Musculus).Data Transformation - R package DESeq2 was used to normalize and perform differential expression analysis on the quantified reads, identifying differentially expressed genes with FDR-adjusted p-value < 0.1 and preparing the data for functional enrichment analysis.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 2000RNA-seq of total RNAMus musculusDelphine MaurelFrédéric MartinsLéa GelléefalseOxygen-dependent osteocyte transcriptomic adaptations occur without modulation of endothelial gene expression via osteocyte paracrine signalingBone regeneration remains clinically challenging, partly due to limited knowledge of the cellular mechanisms governing bone repair. Osteocytes, which constitute the majority of bone cells, form an dendritic network and are central regulators of skeletal homeostasis. Residing in a mineralized and low-oxygen environment, they are exposed to hypoxic conditions that may shape their function, yet the role of hypoxia in osteocyte behavior and angiogenic signaling during bone regeneration is still unclear. This study investigated the effects of hypoxia on osteocytes and their paracrine interaction with endothelial cells. MLO-Y4 osteocytic cells were cultured under normoxia (21% O₂), moderate hypoxia (5% O₂), or severe hypoxia (1% O₂), and conditioned media were applied to mouse endothelial cells. Transcriptomic analyses were performed following optimization of the co-culture model. Hypoxia promoted osteocyte dendritic network development and induced oxygen-dependent metabolic and transcriptional adaptations. Severe hypoxia induces a stronger hypoxic response and glycolytic shift compared with moderate hypoxia, leading to distinct osteocyte profiles. In contrast, osteocyte-conditioned media did not significantly affect endothelial gene expression, suggesting that hypoxia-independent mechanisms may govern osteocyte-endothelial communication. These findings highlight oxygen availability as a key regulator of osteocyte physiology and underscore the need for further investigation into its role during bone regeneration.2026-09-01T00:00:00Z2026-09-01T01:00:47.039Z2026-02-09T13:41:54.424ZE-MTAB-16618ERP188892EFO_0002944EFO_0004170EFO_0009653EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0004184