stable knockdown cells  (Obio Technology Corp Ltd)

Journal: Cell Death & Disease

Article Title: HOXA9 orchestrates EMT and metastasis in oral cancer via transcriptional activation of vimentin and β-catenin signaling

doi: 10.1038/s41419-026-08664-7

Figure Lengend Snippet: A Bar plot showing the results of qRT-PCR showing the efficient knockdown of HOXA9 in HSC-3 and CAL-27 cells, at the mRNA level. B Representative western blot images confirming the knockdown of HOXA9 in HSC-3 and CAL-27 cells with β-actin as an endogenous control. Bar plot showing the results of quantitative densitometric analysis confirming the HOXA9-knockdown in HSC-3 and CAL-27 cells at protein level. C Bright field images showing the changes in the morphology of HSC-3 and CAL-27 cells upon successful HOXA9-knockdown (enlarged 10X magnification). D Line plots showing the gradual reduction in the proliferation rate upon HOXA9-knockdown with increase in the cell-doubling time of HSC-3 cells (32.68 h) and E in CAL-27 cells (46.90 h), compared to the corresponding scrambled cells (23.7 hours and 32.27 h), respectively. F Representative images of the anchorage-dependent colony formation assay (upper panel) and its quantitative analysis confirming the reduction in the number and size of the colonies upon HOXA9-knockdown in HSC-3 and CAL-27 cells, when compared to the scrambled cells (lower panel). G Cell cycle analysis of scrambled and HOXA9-knockdown cells using flow cytometry. Silencing of HOXA9 in HSC-3 cells showed a significant increase in the number of cells undergoing apoptosis, and G0/G1 cell cycle arrest (Scr v/s Sh-HOXA9: apoptotic phase 5.461% ± 0.80 v/s 8.58% ± 0.57; G0/G1 arrest 22.39% ± 3.80 v/s 36.60% ± 2.56). H In CAL-27 cells, the silencing of HOXA9 has significantly induced apoptosis (Scr v/s Sh-HOXA9: apoptotic phase 4.84% ± 0.12 v/s 13.70% ± 1.86), compared to the scrambled cells. I Confocal images of actin-phalloidin staining showing substantial changes in actin cytoskeletal rearrangements and the cell morphology upon HOXA9-knockdown in OC cells. The arrows indicate the presence of filopodia (63X magnification). J Box plots confirming the significant reduction in the number and length of filopodia upon HOXA9-knockdown in HSC-3 and CAL-27, respectively. Quantitatively, the number of filopodia in HSC-3 cells decreased from 22 ± 3 (mean ± SD) in scrambled (Scr) to 13 ± 2 in knockdown cells (Sh-HOXA9), and the length of the filopodia decreased from 0.26 µm±0.006 to 0.11 µm ± 0.009. In CAL-27 cells, the number of filopodia decreased from 82 ± 6 (mean ± SD) to 22 ± 2.64, and the length of the filopodia decreased from 0.37 µm ± 0.07 to 0.16 µm ± 0.003 following HOXA9 knockdown. The data represents mean ± SD of the experiments performed in duplicates, repeated twice and P < 0.05 was considered statistically significant.

Article Snippet: To generate a stable knockdown cell line, HOXA9 shRNA retroviral plasmids (pGFP-V-RS) were purchased from Origene Technologies (TG307647) and transfected into Phoenix packaging cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific, USA).

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Control, Colony Assay, Cell Cycle Assay, Flow Cytometry, Staining

Journal: Cell Death & Disease

Article Title: HOXA9 orchestrates EMT and metastasis in oral cancer via transcriptional activation of vimentin and β-catenin signaling

doi: 10.1038/s41419-026-08664-7

Figure Lengend Snippet: A Representative images of tumors extracted from nude mice ( n = 4). The mice that received scrambled HSC-3 cells showed progressively growing tumors. B The line graph represents the difference in the tumor volume of nude mice of scrambled and knockdown group as measured at regular time intervals. C Representative images of H&E staining and IHC staining for the tumor tissues stained with Pan-cytokeratin (CK-Pan) antibody. The tissues of scrambled group showing higher positive staining than that of knockdown group. D Bar graph showing the significant reduction in the IHC optical density score of the tissues of knockdown group mice when compared to scrambled group, stained with CK-Pan antibody. E Representative images of metastasized lung tissues in the presence and absence of HOXA9 expression. The arrows represent the metastatic nodules in mice lungs. F Bar graph showing the quantitative analysis of number of metastatic nodules in nude mice receiving scrambled and HOXA9-knockdown HSC-3 cells. G Representative images of H&E staining performed for mice lung tissues under 10X magnification. Mice that received scrambled cells exhibited extensive lung metastasis. P < 0.05 was considered statistically significant.

Article Snippet: To generate a stable knockdown cell line, HOXA9 shRNA retroviral plasmids (pGFP-V-RS) were purchased from Origene Technologies (TG307647) and transfected into Phoenix packaging cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific, USA).

Techniques: Knockdown, Staining, Immunohistochemistry, Expressing

Journal: Journal of Translational Medicine

Article Title: Glutamine metabolism reprogramming promotes bladder cancer progression via PYCR1: a multi-omics and functional validation study

doi: 10.1186/s12967-025-07386-2

Figure Lengend Snippet: PYCR1 knockdown impairs proline synthesis and suppresses the PI3K/AKT/mTOR signaling pathway in BLCA cells. ( A ) Quantification of intracellular proline levels in T24 and J82 cells after PYCR1 knockdown. ( B ) Correlation analysis showing positive associations between PYCR1 expression and SLC1A5 (left) and P5CS (right) in BLCA samples. ( C ) Western blot analysis confirming that knockdown of PYCR1 reduces protein expression levels of P5CS and SLC1A5. ( D ) Heatmap of differentially expressed genes in BLCA cells following PYCR1 knockdown from RNA-seq data. ( E - F ) Pathway enrichment analyses of downregulated ( E ) and upregulated ( F ) genes after PYCR1 knockdown, indicating involvement in PI3K-AKT and immune-related signaling pathways. ( G ) Western blot analysis validating the downregulation of PI3K, AKT, and mTOR pathway components upon PYCR1 knockdown in T24 and J82 cells. ( H ) Western blot analysis of PI3K/AKT/mTOR pathway activation following PYCR1 overexpression with or without LY294002 treatment in T24 and J82 cells. PYCR1 overexpression increased phosphorylation of PI3K, AKT, and mTOR, which was reversed by the PI3K inhibitor. Vinculin served as the loading control. Data are presented as mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01; oe: Overexpression

Article Snippet: To generate stable gene knockdown cells, lentiviral vectors expressing specific short hairpin RNA (shRNA) sequences targeting the desired gene, along with corresponding negative controls, were sourced from Genechem (Shanghai, China).

Techniques: Knockdown, Expressing, Western Blot, RNA Sequencing, Protein-Protein interactions, Activation Assay, Over Expression, Phospho-proteomics, Control