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. 2022 Aug 21;9(29):2201273. doi: 10.1002/advs.202201273

Figure 4.

Figure 4

Transcriptomic analysis of osteoblast enriched spot in frozen tissue sections. A) Mapping of OB‐enriched spots for transcriptomic analysis based on marker gene expression. Top left: Frozen sections of growth plate and adjacent trabecular bone from proximal tibia of a wild type mouse (WT‐1 sample). Top right: Osteoblast‐enriched spots (yellow circles) selected for transcriptomic analysis based on above‐threshold expression of Col1a1, Runx2, Sp7, and Ibsp (similar to Figure 1) as well as below‐threshold expression of Col2a1 (<0.15% UMI) and Col10a1 (<0.03% UMI). Bottom left: Spots with high Ibsp (yellow), Col10a1 (magenta), and Col2a1 (cyan) expression. Bottom right: zoomed area of the left image illustrating Ibsp expression in trabecular area, Col10a1 expression in the hypertrophic zone and adjacent structures, and Col2a1 expression in the rest of the growth plate. B) Expression of ISR genes (top row), endoplasmic reticulum (ER) and mitochondrial HSP70 and HSP90 chaperones (second row), transcription factors regulating cellular response to stress (third row), and genes indicating mitochondrial distress (bottom row). Violin plots are shown for each of the 3 Het (yellow) and 3 WT (green) animals examined by srRNASeq. BIP encoded by Hspa5 is ER‐HSP70, Hspa9 encodes mt‐HSP70, Hsp90b1 and Trap1 encode ER‐HSP90 and mt‐HSP90, respectively. Cyb5r1, Gpt2, and Shmt2 encode mitochondrial proteins. Asparagine synthase gene (Asns) is a known marker of mitochondrial stress response regulated by ATF4.[ 33 ] More than 20% change in gene expression with p < 0.05 (Wilcoxon test) was considered significant. N.S. = not significant, ** p < 0.01, **** p < 0.0001.