Fig 2: A scheme is showing the effect of CR on synaptic microenvironment and plasticity.The growth of thin astrocytic processes triggered by CR increases astrocyte volume fraction in hippocampal neuropil and reduces the escape of K+ and glutamate from the synaptic cleft (spillover). Fewer extrasynaptic N2B-NMDA receptors are activated by glutamate spillover during high-frequency synaptic stimulation. Reduced activation of extrasynaptic N2B-NMDA, in turn, increases the magnitude of LTP. Growth of astroglial perisynaptic leaflets is associated with the downregulation of Cx43 expression and reduced gap-junction coupling of the cells in the network. Fewer channels balance astrocyte input resistance increase produced by larger astrocyte membrane surface.

Fig 3: Analysis of Marker Genes in Multipotent and Neurogenic RPCs(A) Unbiased clustering initially classified cells into six clusters. Cluster 6 contains all neurons. Clusters 1 through 5 collectively contain all RPCs.(B) PCA plot showing the biological meaningful cell subgroups. Although six clusters were initially identified, there were only three biologically meaningful subgroups, each of which express a unique set of marker genes compared with others, which is shown in (C).(C) Heatmap showing the marker genes identified for each of the six clusters. Cluster 6 expresses a unique set of marker genes related to neuronal identity. Clusters 1, 3, and 5 express the same set of marker genes, whereas clusters 2 and 4 express the other set of markers.(D and E) Top: functional enrichment of genes significantly highly expressed in neurogenic RPCs (D) and multipotent RPCs (E). Bottom: heatmaps showing the average expression of representative marker genes in neurogenic RPCs (D) and multipotent RPCs (E).(F) Schematic presentation of RPC lineage tracing. AAVS1-loxp-3Stop-loxp-tdTomato H9 ESC-derived retinal organoids were transduced with hPGK:CreERT2-expressing lentivirus in the combination of low-dose 4-hydroxytamoxifen to achieve sparse labeling of multipotent RPC on day 24 and collected on days 26 and 32, respectively.(G) Coimmunostaining of tdTomato with multipotent RPC markers (JAG1 and GJA1) and neurogenic RPC markers (DCX and ASCL1) at day 26 and day 32, respectively. Scale bars, 25 μm.(H) Quantification of Marker+ cells among tdTomato+ cells (mean ± SD; n ≥ 5 organoids from two differentiations). ∗p < 0.001 (unpaired t test).See also Figure S3.

Fig 4: The results of RT-qPCR (real-time-quantitative polymerase chain reaction) validation of analyzed genes, presented in the form of bar graph. All the fold changes were described in relation to the transcript levels at 24 h of primary culture. LogFC was used to present the data in order to improve the clarity and comparability of upregulation and downregulation results. Connexins 36 (GJA1-gap junction protein alpha), 37 (GJA4-gap junction protein alpha 4), 40 (GJA1-gap junction protein alpha), and 43 (GJA1-gap junction protein alpha 1).

Fig 5: Mechanisms involved in continuous shear stress-enhanced osteogenic differentiation of iPSCs. iPSCs were analyzed after being subjected to shear stress for 48 h. (A) Western blot analysis of cell surface channels Connexin 43 (Cx43) and Trpm7 as well as the phosphorylation of downstream factors including Erk1/2 (phospho-Erk1/2) and p38 (phospho-p38). Original blots are presented in Supplementary Fig. 6. (B) Quantitative analysis of Cx43 and phospho-Erk1/2 expression using ImageJ software. The expression levels were normalized to Gapdh expression. *: P < 0.05, ANOVA with Tukey’s multiple comparison test. Representative data from three independent experiments are shown (mean values ± SD: n = 3). Immunofluorescence analysis was performed to detect the protein expression and localization of (C) Cx43 (green), (D) phospho-Erk1/2 (green), and (F) Trpm7 (green). Normal mouse IgG and normal rabbit IgG were used as a negative control (NC). The cytoskeleton (F-actin; red) and nuclei (blue) were stained using rhodamine-phalloidin and Hoechst, respectively. Scale bars: (C and D) 100 μm, (F) 200 μm. (E) The relative mean fluorescence intensity of Cx43 and phospho-Erk1/2 was quantified by ImageJ software. *: P < 0.05, Student’s t-test. Data represent the mean ± SD (n = 4).