hmga1 150 expression plasmid  (Addgene inc)

Journal: Molecular Cancer

Article Title: Integrated single-cell and spatial transcriptomic profiling decodes lineage plasticity and immune microenvironment remodeling in prostate cancer progression

doi: 10.1186/s12943-026-02617-6

Figure Lengend Snippet: Functional characterization of FOSL1 as a key regulator in double-negative prostate cancer subtype. A Transcription factor activity in malignant epithelial subtypes. Heatmap displaying the top transcription factors with the highest inferred activity (based on SCENIC analysis) in each of the four malignant epithelial subtypes. Color scale (blue to red) represents low to high regulon activity. B Spatial activity of FOSL1 across subtypes. UMAP of malignant epithelial cells colored by the SCENIC-derived regulon activity score of FOSL1, showing its specific enrichment in Subtype 4. Gradient from white to blue indicates low to high activity. C Effect of FOSL1 knockdown on cell proliferation (CCK-8 assay). Growth curves of PC-3 cells transfected with control siRNA or three independent siRNAs targeting FOSL1. Data are presented as mean ± SD; *** P < 0.001, ** P < 0.01 (two-way ANOVA). D Effect of FOSL1 knockdown on clonogenic survival. Representative images of colony formation assays for PC-3 cells treated as in (C). E Effect of FOSL1 knockdown on apoptosis. (Left) Representative flow cytometry plots of Annexin V/PI staining. Quadrants: viable cells (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), and necrotic (Annexin V⁻/PI⁺). (Right) Quantification of total apoptotic cells (early + late apoptosis). Data are mean ± SD; *** P < 0.001, ** P < 0.01 (one-way ANOVA). F Identification of FOSL1-regulated DNPC genes. Venn diagram showing the overlap between predicted FOSL1 target genes (from SCENIC) and the DNPC gene signature, yielding 8 candidate genes. G FOSL1 binding to candidate gene promoters (ChIP-qPCR). ChIP-qPCR analysis showing enrichment of FOSL1 at the promoters of the 8 candidate genes in control vs. FOSL1-knockdown PC-3 cells. Results are presented as % input. Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-tailed Student’s t-test). H Expression of candidate genes upon FOSL1 knockdown (qRT-PCR). mRNA expression levels of four selected candidate genes in PC-3 cells after FOSL1 knockdown. Data are normalized to control and presented as mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-tailed Student’s t-test). I Correlation between FOSL1 and HMGA1 expression. Scatter plot showing a significant positive correlation between FOSL1 and HMGA1 expression across all malignant epithelial cells (n = 152,872). The red line indicates the linear regression fit (R² = 0.212, P < 0.0001, Pearson correlation). J Spatial co-expression of FOSL1 and HMGA1. (Left three panels) Spatial mapping of FOSL1 and HMGA1 expression and their co-localization in a representative section (Patient 01). Red spots indicate high co-expression, yellow indicates high FOSL1 alone, green indicates high HMGA1 alone, and gray indicates low expression of both. (Right panel) Correlation analysis of FOSL1 and HMGA1 expression specifically within the co-expressing spots (red spots, left panel), showing a strong positive correlation (R = 0.923, P = 1.28e-10, Pearson correlation). K - M Western blot analysis of downstream pathways. Protein levels of (K) stemness markers (CD44, OCT4, SOX2, NANOG), (L) EMT markers (ZO-1, N-cadherin, E-cadherin, Vimentin, SNAIL), and (M) DNPC markers (DSG3, KRT6A, KRT5), along with FOSL1 and HMGA1, in PC-3 cells under four conditions: Control, FOSL1 knockdown (siFOSL1), HMGA1 overexpression (oeHMGA1), and FOSL1 knockdown combined with HMGA1 rescue (siFOSL1 + oeHMGA1). GAPDH served as a loading control. N Rescue of proliferation by HMGA1 overexpression. CCK-8 proliferation assay of PC-3 cells under the four conditions described in (K-M). Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-way ANOVA). O Rescue of apoptosis by HMGA1 overexpression. Quantification of total apoptosis (Annexin V⁺ cells) by flow cytometry under the four conditions described in (K-M). Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (one-way ANOVA). P Pharmacological inhibition of FOSL1 reduces HMGA1. Western blot showing dose-dependent decrease of FOSL1 and its downstream target HMGA1 in PC-3 cells treated with increasing concentrations (0, 0.2, 2, 5, 10 µM) of the FOSL1 degrader T-5224. Q In vivo combination therapy schematic. Workflow of the xenograft study. Nude mice bearing PC-3 subcutaneous tumors were treated starting at day 13 post-inoculation with vehicle, Docetaxel (1 mg/kg), FOSL1 degrader T-5224 (10 mg/kg), or the combination via intraperitoneal injection every 48 h. Treatment continued until day 28. R Representative images of resected tumors. Photographs of excised tumors from each treatment group at the study endpoint. S Tumor growth curves. Tumor volume (mm³) was measured over time for each treatment group. Data are presented as mean ± SEM; *** P < 0.001 (two-way ANOVA). T Tumor weight at endpoint. Final tumor weights (g) for each group. Data are mean ± SEM; *** P < 0.001, ** P < 0.01 (one-way ANOVA). U Assessment of tumor cell proliferation (IHC). Representative immunohistochemistry images showing Ki67 expression in tumor sections from each treatment group. Scale bar, 100 μm

Article Snippet: HMGA1 overexpression plasmid (Cat: HG11613-NF), and the empty vector plasmid pCMV3-N-FLAG were purchased from SinoBiological.

Techniques: Functional Assay, Activity Assay, Derivative Assay, Knockdown, CCK-8 Assay, Transfection, Control, Flow Cytometry, Staining, Binding Assay, ChIP-qPCR, Two Tailed Test, Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Proliferation Assay, Inhibition, In Vivo, Injection, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Oncogenic ZEB2/miR-637/HMGA1 signaling axis targeting vimentin promotes the malignant phenotype of glioma

doi: 10.1016/j.omtn.2020.12.029

Figure Lengend Snippet: miR-637 directly targeted HMGA1 in glioma (A) Venn diagram indicating the intersection of miR-637 target genes predicted in public databases (TargetScan, miRDB, and PICA) overlapping with microarray results. (B) Quantitative real-time PCR assay demonstrated decreased HMGA1 levels under miR-637 mimics transfection. (C) Western blot demonstrated decreased HMGA1 levels under miR-637 mimics transfection. (D) The putative miR-637-binding sites in the 3′-UTR region of HMGA1 are marked in red. (E) Relative luciferase activity was examined after the transfection of reporter vector with HMGA1 wild-type 3ʹ-UTR or mutant 3ʹ-UTR under co-transfection with miR-637 mimics or inhibitor in 293T cells. (F) IHC analysis of HMGA1 in glioma tissues was performed. (G) Expression of HMGA1 was negatively correlated with that of miR-637 in glioma tissues. Data are presented as mean ± SD (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001). WT, wild type; Mut, mutant.

Article Snippet: An expression plasmid for ZEB2 and HMGA1 was designed and synthesized by Vigenebio (Jinan, People’s Republic of China).

Techniques: Microarray, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Binding Assay, Luciferase, Activity Assay, Plasmid Preparation, Mutagenesis, Cotransfection, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Oncogenic ZEB2/miR-637/HMGA1 signaling axis targeting vimentin promotes the malignant phenotype of glioma

doi: 10.1016/j.omtn.2020.12.029

Figure Lengend Snippet: miR-637 suppresses the malignant phenotype of glioma by downregulating HMGA1 in vitro (A) The cell viability of U251 or U87 cells transfected with miR-637 lentiviral expression particles and/or a HMGA1 expression plasmid was evaluated by the MTT assay. (B–G) The proliferative rate, migration, and invasion of U251 or U87 cells transfected with miR-637 mimics and/or a HMGA1 expression plasmid were evaluated by the EdU incorporation assay (B and C), Transwell assay (D and E), and Boyden assay (F and G), respectively. Scale bar indicates 100 μm. Data are presented as mean ± SD (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).

Article Snippet: An expression plasmid for ZEB2 and HMGA1 was designed and synthesized by Vigenebio (Jinan, People’s Republic of China).

Techniques: In Vitro, Transfection, Expressing, Plasmid Preparation, MTT Assay, Migration, Transwell Assay, Boyden Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Oncogenic ZEB2/miR-637/HMGA1 signaling axis targeting vimentin promotes the malignant phenotype of glioma

doi: 10.1016/j.omtn.2020.12.029

Figure Lengend Snippet: miR-637 modulates xenograft tumor growth by targeting HMGA1 in vivo (A) Images of xenograft tumor models of mice injected with indicated U87 cells are shown. In the first group of four mice, U87/NC cells were injected in the left flank, and U87/HMGA1 cells were injected in the right flank; in the second group of four mice, U87/miR-637 cells were injected in the left flank, and U87/miR-637/HMGA1 cells were injected in the right flank. Scale bar indicates 1 cm. (B) Tumor weight was measured at the 28th day after inoculation in each group. (C and D) IHC analysis and quantification of Ki-67 expression in xenograft tumors from mice injected with indicated U87 cells were performed. Scale bar indicates 100 μm. Data are presented as mean ± SD (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001). NC, negative control.

Article Snippet: An expression plasmid for ZEB2 and HMGA1 was designed and synthesized by Vigenebio (Jinan, People’s Republic of China).

Techniques: In Vivo, Injection, Expressing, Negative Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Oncogenic ZEB2/miR-637/HMGA1 signaling axis targeting vimentin promotes the malignant phenotype of glioma

doi: 10.1016/j.omtn.2020.12.029

Figure Lengend Snippet: HMGA1 promotes glioma migration and invasion through vimentin in vitro (A) The expression of crucial EMT-related proteins was examined by western blot assays. (B) The interaction between HMGA1 and vimentin was examined by coIP assay. (C) IHC analysis of vimentin in glioma tissues was performed. (D) Expression of vimentin was positively correlated with that of miR-637 in glioma tissues. (E–H) Migration and invasion of U251 or U87 cells transfected with an HMGA1 expression plasmid and/or siRNA targeting vimentin were evaluated by the Transwell assay (E and F) and Boyden assay (G and H), respectively. Scale bar indicates 100 μm. Data are presented as mean ± SD (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001). NC, negative control.

Article Snippet: An expression plasmid for ZEB2 and HMGA1 was designed and synthesized by Vigenebio (Jinan, People’s Republic of China).

Techniques: Migration, In Vitro, Expressing, Western Blot, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Transwell Assay, Boyden Assay, Negative Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Oncogenic ZEB2/miR-637/HMGA1 signaling axis targeting vimentin promotes the malignant phenotype of glioma

doi: 10.1016/j.omtn.2020.12.029

Figure Lengend Snippet: Correlation between the clinicopathological factors and expression of ZEB2/miR-637/HMGA1/vimentin axis

Article Snippet: An expression plasmid for ZEB2 and HMGA1 was designed and synthesized by Vigenebio (Jinan, People’s Republic of China).

Techniques: Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Oncogenic ZEB2/miR-637/HMGA1 signaling axis targeting vimentin promotes the malignant phenotype of glioma

doi: 10.1016/j.omtn.2020.12.029

Figure Lengend Snippet: HMGA1 and vimentin are associated with unfavorable prognosis in glioma (A) Expression level of HMGA1 in glioma with different IDH status in CGGA cohort was examined. (B) Expression level of HMGA1 in glioma with different 1p/19q status in CGGA cohort was examined. (C) Expression level of vimentin in glioma with different IDH status in CGGA cohort was examined. (D) Expression level of vimentin in glioma with different 1p/19q status in CGGA cohort was examined. (E–G) HMGA1 was associated with an unfavorable prognosis in Nanfang Hospital, CGGA, and TCGA cohorts. (H–J) Vimentin was associated with an unfavorable prognosis in Nanfang Hospital, CGGA, and TCGA cohorts.

Article Snippet: An expression plasmid for ZEB2 and HMGA1 was designed and synthesized by Vigenebio (Jinan, People’s Republic of China).

Techniques: Expressing