Fig 2: Balance of RTK expression determines tumorsphere p-ERK response to ligand and pathway inhibitors. Phospho-ERK activation was analyzed as a function of EGFR and PDGFRA expression and plotted as in Fig. 3. (a) Ligand effect on p-ERK. (i – v) EGF and PDGFB stimulate p-ERK only in cells harboring high levels of both RTKs (cell-bins in dashed squares) and activation patterns are similar for both ligands. (b) Mono- and (c) combination RTK inhibitor treatment effect on p-ERK: Treatment of TS600 with either (i) gefitinib or (ii) imatinib or (c) dual inhibition in the presence of ligand demonstrates a similar pattern of mixed response among cell populations defined by RTK expression, with greater inhibition seen in populations with higher EGFR and PDGFRA co-expression.

Fig 3: Temporal and spatial expression of Igf1 and Igf1r during neonatal lung development in mice.(A, B) Temporal expression of Igf1 (A) and Igf1r (B) from embryonic day 18 (E18) to postnatal day 30 (PN30), quantified by RT-PCR and normalized to Gapdh. RNA used was collected from the whole lung, and data was presented as box plots with each stage represented by at least five lungs. (C, D) Spatial localization of mRNA for Igf1 (C) and Igf1r (D) in PN7 lungs as detected by RNAscope and their overlapped expression with Pdgfra. Outlined area on the left is magnified on the right. Scale bars: 20 um under the whole view and 10 um under the magnified view.

Fig 4: Functional pathways analysis reveals networks of molecular interaction and a potential mechanism of subgroup TNC overexpression in pediatric gliomaPathways analysis was performed to evaluate the biological functions and interactions of differentially expressed genes between TNC cDNA and shRNA cell lines (Log2Fold Change > 2 or < − 2, adjusted p-value < 0.05). a Organismal Development and Small Molecule Biochemistry is indicated as the top network of molecular interaction (22 focus molecules, score 45), with TNC over expression, miRNA30a downregulation, PDGF and NOTCH signaling activation, and interaction with extracellular matrix proteins. These results suggest that the observed biological effects of elevated TNC expression in vitro may be due to these pathway activations. b VEGFRA and STAT3 activation, potentially affected by miRNA30 expression level, are identified as potential upstream regulators of TNC overexpression in a subgroup of DIPGs lacking PDGFRA amplification or mutation. c EZH2 is implicated as a top activated upstream regulator in TNC cDNA (left) relative to TNC shRNA (right) cell lines (activation z-score 1.639, p-value of overlap 0.004), with a direct downstream effect on TNC expression

Fig 5: Anisomycin attenuates the molecular sponge effect in the lncRNA-Meg3/miR-421/PDGFRA axis. (A) Heatmap showing that the expression of lncRNA-Meg3 in the anisomycin-treated group was significantly lower than that of the control group. (B) Expression levels in clinical tumour samples and (C) in xenograft tumours suggest an inverse trend in expression for lncRNA-Meg3 and miR-421. (D) Expression levels of miR-421 in HuOCSCs following anisomycin treatment. **P<0.01 vs. non-treated (n=4). (E) Schematic of the complementary sites of mature miR-421 and the 3'UTR of lncRNA-Meg3 and PDGFRA mRNA. (F) Schematic of the structure of luciferase reporter plasmids. (G) The molecular sponge effect of lncRNA-Meg3/miR-421/PDGFRA. (H-J) Results of the luciferase reporter assays. **P<0.01 vs. blank plasmid (n=3). (K) RIP results revealed that the binding between Meg3 and Ago2 decreased significantly following treatment of HuOCSCs with anisomycin. lncRNA, long non-coding RNA; Meg3, maternally expressed 3; miR, microRNA; PDGFRA, platelet derived growth factor receptor a; HuOCSCs, human ovarian cancer stem cells; RIP, RNA immunoprecipitation; Ago2, argonaute 2; ORF, open reading frame; UTR, untranslated region; mut, mutant; IgG, immunoglobulin G.