Fig 2: IgE promotes collagen expression via miR-486a-5p/Smad1 in CFs. (A-B) Representative immunoblot (upper panel) and quantification (lower panel) of SMAD1 protein expression in IgE-stimulated WT (A) and FcεR1-KO (B) CFs at indicated lengths of time (0, 3, 24 h). (C-D) Representative immunoblot (C) and quantification (D) showing protein levels of α-SMA, COL1A1, and COL3A1 in CFs transfected with small interference RNA (siRNA) against Smad1 or scrambled control. (E-F) Representative immunoblot (E) and quantification (F) showing protein levels of α-SMA, COL1A1, and COL3A1 in CFs transfected with Smad1 ORF clone or scrambled control. (G-H) Representative immunoblot (G) and quantification (H) of SMAD1, COL1A1, and COL3A1 protein expression in CFs transfected with miR-486a-5p mimic and/or IgE. (I-J) Immunoblot (I) and quantification (J) showing protein levels of SMAD1, COL1A1, and COL3A1 in CFs transfected with siRNA against Smad1 and/or miR-486a-5p inhibitor. Data are mean ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s indicates no significant difference in One-way ANOVA with Bonferroni's post hoc test (A-B), Student's t-test (D, F) or Two-way ANOVA with Bonferroni's post hoc test (H, J).

Fig 3: SLC39A5 depletion suppresses the TGF‐β signalling. (A) show the intracellular zinc level of the knockout (KO) cells was reverted after SLC39A5 re‐expression via lentivirus infection. (B,C) show the rescue of expression of the extracellular matrix members (COL1, FN and LN) and Smad proteins (Smad1, pSmad1/5/9, Smad2/3, pSmad2/3 and Smad4) in the knockout (KO) cells after SLC39A5 re‐expression via lentivirus infection. (D–G) show the degradation of cycloheximide (CHX)‐treated Smad1, Smad2/3 and Smad4 at different time points within 8 h. Degradation curves (E–G) were obtained based on statistical analyses of the Western blotting results (D). *p < 0.05, **p < 0.01 and ***p < 0.001

Fig 4: GAS5 interacts with UPF1 to accelerate SMAD7 decay.(A) Western blot analysis of the activation pathway (pSMAD1/5/8, SMAD1) in GAS5 knockdown or overexpression conditions. (B) SMAD7 was regulated by GAS5 knockdown or overexpression by western blot. (C) Relative SMAD7 mRNA levels in GAS5 knockdown or overexpression cells tested by qRT-PCR. (D, E) Western blot analysis and quantification of pSMAD1/5/8/SMAD1 and SMAD7 in the UPF1 knockdown and control groups. (F) RIP assay with anti-UPF1 showed the binding of UPF1 and SMAD7 mRNA. (G) Western blot analysis in the GAS5 knockdown, overexpression and control groups after actinomycin D treatment. NC means the negative control siRNA transfected group. It was also be treated by Actinomycin D but as the control of GAS5 shRNA-1. (H) Relative SMAD7 mRNA expression in the GAS5 knockdown and control groups by actinomycin D treatment tested by qRT-PCR. (I) Relative SMAD7 mRNA expression in the GAS5 overexpression and control groups by actinomycin D treatment tested by qRT-PCR. (J, K) Relative SMAD7 mRNA expression by actinomycin D treatment under the condition of UPF1 siRNA, overexpression of a truncated form of GAS5 or GAS5 full-length. (L) ARS staining and quantification in the GAS5 overexpression and UPF1 siRNA groups. Scale bar, 500 µm. (M) ALP staining and ALP tests in the GAS5 overexpression and UPF1 siRNA groups. Scale bar, 500 µm. Each cellular experimental group was repeated at least three times.

Fig 5: Matured primary astrocytes express BMP receptors and reveal activation of Smad1/5/9 upon treatment with canonical BMPs. (A) RT-qPCR confirms that astrocytes express BMP receptor transcripts. BMP receptor transcripts Bmpr1a, Bmpr1b, Bmpr2, Acvr1, Acvr1b, and Acvr2a are robustly expressed, while Acvr1 exhibits low expression and Acvr1l is not expressed. Values are presented as mean copy numbers per nanogram total RNA input into the cDNA reaction. Dots: individual sample values; Error bars = SEM; n = 4. (B–D) Immunoblots and quantification using antibodies to Smad1 and phosphorylated Smad1/5/9 (pSmad1/5/9) on protein lysates from primary astrocytes after 3 days of BMP treatment. a -tubulin was used as a loading control. *Sig ANOVA LSD post-hoc test; Dots: individual sample values; Error bars = SEM; P < 0.05; n = 3. (C) Smad1 is expressed in all samples and significantly lower in cells treated with BMP2, –4, and –10. (D) pSmad1/5/9 phosphorylation is induced and significantly increased only in samples treated with canonical BMPs –2, –4, and –10.