Journal: Clinical and Translational Medicine

Article Title: Smad3‐mediated lncRNA HSALR1 enhances the non‐classic signalling pathway of TGF‐β1 in human bronchial fibroblasts by binding to HSP90AB1

doi: 10.1002/ctm2.1292

Figure Lengend Snippet: HSALR1 was regulated by Smad3 and was seem to affect PI3K/Akt pathway and cell proliferation through RNA‐seq. (A) Schematic of the binding motif of transcription factor Smad3, and three transcription factor binding sites (162–169, 696–703 and 1544–1553 bp) of Smad3 predicted on lnc9 promoter using JASPAR ( http://jaspar.genereg.net/ ). The red arrow indicates the EBF1 transcription factor. (B) qRT‐PCR analysis of lnc9 expression after treatment with TGF‐β or SIS3 + TGF‐β ( n = 3 biological replicates, Student's t ‐test). (C) ChIP‐PCR analysis of Smad3 occupancy on HSALR1 promoter in unstimulated HBF cells and TGF‐β‐stimulated HBF cells. IgG was used as the negative control ( n = 3 biological replicates, Student's t ‐test). (D) Schematic representation of the two mutation sequences of potential Smad3‐binding sites on HSALR1 promoter. (E) Luciferase activity in the HSALR1 promoter after transfection with a reporter containing wild‐type or mutant HSALR1 promoter in unstimulated HBF cells and TGF‐β‐stimulated HBF cells with TGF‐β ( n = 3 biological replicates, Student's t ‐test). (F) qRT‐PCR analysis of HSALR1 expression after transfection with Smad3 siRNA in HBF cells ( n = 3 biological replicates, one‐way ANOVA). (G) Schematic representation of the cell samples divided into four groups (si‐NC + NC, si‐ HSALR1 + NC, si‐NC + TGF‐β and si‐ HSALR1 + TGF‐β) for RNA‐seq ( n = 3 biological replicates). (H) The volcano plot of the DEGs between si‐NC + NC and si‐ HSALR1 + NC/si‐NC + TGF‐β and si‐ HSALR1 + TGF‐β ( n = 3 biological replicates). (I) Results of GSEA analysis of RNA sequencing by using Hallmark pathway database. (J) The intersection of the Venn diagram showing the upregulated and downregulated overlapping genes between both comparison DEGs in si‐NC + NC and si‐lnc9 + NC/si‐NC + TGF‐β and si‐lnc9 + TGF‐β; and enrichment analysis of DEGs in overlapping genes. (K) qRT‐PCR analysis of proliferation‐associated genes ( CDC6, CDC45, CCNE2, E2F8, CLSPN , etc.) and cytokine‐associated genes ( IL1R1, IL6R, FGFR4, IGTB8, ANGPT1, PIK3R3, LAMA4 , etc.) after HSALR1 knockdown using siRNA or HSALR1 overexpression using overexpression lentiviral vector in HBF cells ( n = 4 biological replicates, Student's t ‐test). Data information: Error bars represent means ± SD. * p < .05, ** p < .01 and *** p < .05.

Article Snippet: After blocking, membranes were incubated with antibodies against human SMAD3 (C67H9, CST, USA), HSP90AB1 (5087S, CST, USA), AKT (4691S, CST, USA), p‐AKT (ab81283, Abcam, UK), p‐p65 (ab76302, Abcam, UK), p65 (ab32536, Abcam, UK), MAPK (Erk1/2) (9107S, CST, USA), p‐MAPK (Erk1/2) (4370S, CST, USA), NLRP3 (ab263899, Abcam, UK), CDC37 (10218‐1‐AP, Proteintech, China) and GAPDH (10494‐1‐AP, Proteintech, China) overnight at 4°C.

Techniques: RNA Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, Negative Control, Mutagenesis, Luciferase, Activity Assay, Transfection, Comparison, Knockdown, Over Expression, Plasmid Preparation

Journal: Clinical and Translational Medicine

Article Title: Smad3‐mediated lncRNA HSALR1 enhances the non‐classic signalling pathway of TGF‐β1 in human bronchial fibroblasts by binding to HSP90AB1

doi: 10.1002/ctm2.1292

Figure Lengend Snippet: HSALR1 promotes the activation of TGF‐mediated non‐classical pathways. Western blot assays of p‐Akt and p‐MAPK with GAPDH as the control after HBFs stimulation with TGF‐β [(A) 2 ng/mL and (B) 4 ng/mL] at various time points (0, 1, 6, 12 and 24 h) ( n = 3 biological replicates, one‐way ANOVA). (C,D) Western blot and qRT‐PCR showing the expression of HSP90AB1 after knockdown of Smad3 by siSmad3‐1 and siSmad3‐2 ( n = 3 biological replicates, Student's t ‐test). (E) Western blot assay results showing the expression of HSP90AB1 and activation level of the Akt signalling pathway after overexpression of HSALR1 and stimulation 12 h by TGF‐β (2 ng/mL) ( n = 4 biological replicates, Student's t ‐test). (F) Western blot indicating the expression of NLRP3 and the activation level of the MAPK signalling pathway after HSALR1 overexpression and stimulation 1 h by TGF‐β (2 ng/mL) ( n = 4 biological replicates, Student's t ‐test). (G) Western blot showing the expression of HSP90AB1 and the activation level of the Akt signalling pathway after HSALR1 knockdown and stimulation 12 h by TGF‐β (2 ng/mL) ( n = 4 biological replicates, Student's t ‐test). (H) Western blot showing the expression level of NLRP3 and the activation level of the MAPK signalling pathway after HSALR1 knockdown and stimulation 1 h by TGF‐β (2 ng/mL) ( n = 4 biological replicates, Student's t ‐test). Data information: Error bars represent means ± SD. * p < .05, ** p < .01 and *** p < .05.

Article Snippet: After blocking, membranes were incubated with antibodies against human SMAD3 (C67H9, CST, USA), HSP90AB1 (5087S, CST, USA), AKT (4691S, CST, USA), p‐AKT (ab81283, Abcam, UK), p‐p65 (ab76302, Abcam, UK), p65 (ab32536, Abcam, UK), MAPK (Erk1/2) (9107S, CST, USA), p‐MAPK (Erk1/2) (4370S, CST, USA), NLRP3 (ab263899, Abcam, UK), CDC37 (10218‐1‐AP, Proteintech, China) and GAPDH (10494‐1‐AP, Proteintech, China) overnight at 4°C.

Techniques: Activation Assay, Western Blot, Control, Quantitative RT-PCR, Expressing, Knockdown, Over Expression

Journal: Clinical and Translational Medicine

Article Title: Smad3‐mediated lncRNA HSALR1 enhances the non‐classic signalling pathway of TGF‐β1 in human bronchial fibroblasts by binding to HSP90AB1

doi: 10.1002/ctm2.1292

Figure Lengend Snippet: Smad3‐mediated HSALR1 promotes HBFs proliferation via Akt pathway by binding to HSP90AB1. (A) Western blot showing the expression of HSP90AB1, the activation level of the Akt and MAPK signalling pathway in HBFs after HSALR1 overexpression and Samd3 knockdown, with GAPDH as the control ( n = 5 biological replicates, one‐way ANOVA). (B) Western blot showing the expression of HSP90AB1, the activation level of the Akt and MAPK signalling pathway in HBFs after HSALR1 overexpression, and Smdd3 knockdown with GAPDH as the control ( n = 5 biological replicates, one‐way ANOVA). (C) Edu assay, (D) cell count assay and CCK‐8 of HBF cells after HSALR1 overexpression and Samd3 or HSP90AB knockdown ( n = 4 biological replicates, one‐way ANOVA). qRT‐PCR analysis of above proliferation‐associated genes and cytokine‐associated genes after Smad3 knockdown using two siRNA (si‐Smad3‐1 and si‐Smad3‐2) in HBF cells ( n = 4 biological replicates, Student's t ‐test). (D) qRT‐PCR analysis of above proliferation‐associated genes and cytokine‐associated genes after HSP90AB1 knockdown using siRNA (si‐HSP90AB1‐1 and si‐HSP90AB1‐2) in HBF cells ( n = 4 biological replicates, Student's t ‐test). (E) Cell cycle Flow cytometry and PI staining assessing the HBF cells after HSALR1 overexpression and Samd3 or HSP90AB knockdown ( n = 4 biological replicates, one‐way ANOVA). (F) qRT‐PCR analysis of proliferation‐associated genes ( CDC6 , CDC45 , CCNE2 , E2F8 , CLSPN , etc.) and cytokine‐associated genes ( IL1R1 , IL6R , FGFR4 , IGTB8 , ANGPT1 , PIK3R3 , LAMA4 , etc.) after HSALR1 overexpression and Samd3 or HSP90AB1 knockdown in HBFs ( n = 4 biological replicates, one‐way ANOVA). Data information: Error bars represent means ± SD. * p < .05, ** p < .01 and *** p < .05.

Article Snippet: After blocking, membranes were incubated with antibodies against human SMAD3 (C67H9, CST, USA), HSP90AB1 (5087S, CST, USA), AKT (4691S, CST, USA), p‐AKT (ab81283, Abcam, UK), p‐p65 (ab76302, Abcam, UK), p65 (ab32536, Abcam, UK), MAPK (Erk1/2) (9107S, CST, USA), p‐MAPK (Erk1/2) (4370S, CST, USA), NLRP3 (ab263899, Abcam, UK), CDC37 (10218‐1‐AP, Proteintech, China) and GAPDH (10494‐1‐AP, Proteintech, China) overnight at 4°C.

Techniques: Binding Assay, Western Blot, Expressing, Activation Assay, Over Expression, Knockdown, Control, EdU Assay, Cell Counting, CCK-8 Assay, Quantitative RT-PCR, Flow Cytometry, Staining