non targeting control shrna shcontrol  (Addgene inc)

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: Ribavirin has synergistic effects on 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) downregulation. (A) Three AMPK-targeting short hairpin RNAs (shRNAs) were screened to evaluate their knockdown efficiency and suppress AMPK expression in lung cancer cells. The data shown are representative of n = 3 biological replicates. (B) Tumor anatomy of mouse Lewis lung carcinoma (LLC) cells subcutaneous tumor-bearing mice in different treatment groups. (C) Tumor volume of LLC subcutaneous tumor-bearing mice in different treatment groups. Statistical significance between datasets was assessed by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± standard error of the mean (SEM); n = 8 mice per group, with differences denoted by ∗ P < 0.05 and ∗∗ P < 0.01. (D) Tumor weights of LLC subcutaneous tumor-bearing mice in different treatment groups. Statistical significance between datasets was assessed by one-way ANOVA, followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± SEM; n = 8 mice per group, with differences denoted by ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (E) Schematic illustration of the mechanism by which Ribavirin regulates AMPK activation. AMPK is phosphorylated in response to an increased AMP/adenosine triphosphate (ATP) or adenosine diphosphate (ADP)/ATP ratio, influencing cell growth and proliferation by inactivating mechanistic target of rapamycin complex 1 (mTORC1). Ribavirin exerts its regulatory effect by inhibiting AMPK phosphorylation. Created with www.BioRender.com . shControl: non-targeting control short hairpin RNA; shAMPKα: AMPKα-targeting short hairpin RNA; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PBS: phosphate buffered saline; mTOR: mechanistic target of rapamycin; Raptor: regulatory-associated protein of mTOR; Deptor: DEP domain-containing mTOR-interacting protein; PRAS40: proline-rich Akt substrate of 40 kDa; mLST8: mammalian lethal with SEC13 protein 8; 4EBP1:eukaryotic translation initiation factor 4E-binding protein 1; P: phosphorylation.

Article Snippet: The mice were randomly divided into four groups ( n = 8): the non-targeting control short hairpin RNA (shControl)-LLC + phosphate buffered saline (PBS) (Sangon Biotech, Shanghai, China) group, shControl-LLC + Ribavirin group, shAMPKα-LLC + PBS group, and shAMPKα-LLC + Ribavirin group.

Techniques: Knockdown, Expressing, Activation Assay, Phospho-proteomics, Control, shRNA, Saline, Binding Assay

Journal: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: CXCR4 regulates TSCC cell proliferation, migration, invasion, and EMT. a Cell proliferation assessed by CCK-8 assay in CAL-27 cells with different CXCR4 expression levels (Control, shControl, shRNA-CXCR4-KD, and shRNA-CXCR4-OE). b Colony formation assay of CAL-27 cells with different CXCR4 expression levels. Representative images (left) and quantification (right). c Transwell migration assay of CAL-27 cells with different CXCR4 expression levels. Representative images (left) and quantification (right). Scale bars, 100 μm. d Transwell invasion assay of CAL-27 cells with different CXCR4 expression levels. Representative images (left) and quantification (right). Scale bars, 100 μm. e Quantification of apoptotic cells measured by flow cytometry. f Representative flow cytometry plots showing apoptosis in CAL-27 cells with different CXCR4 expression levels. g Western blot analysis of EMT markers (E-cadherin, N-cadherin, and Vimentin) in CAL-27 cells with different CXCR4 expression levels. h qRT-PCR analysis of EMT markers in CAL-27 cells with different CXCR4 expression levels. i Wound healing assay showing the effect of CXCL12 on CAL-27 cell migration. Representative images (left) and quantification (right). Scale bars, 100 μm. j Real-time cell analysis (RTCA) showing the effect of CXCL12 on CAL-27 cell migration. Data are presented as mean ± SD from three independent experiments. *p < 0.05 , **p < 0.01 , ***p < 0.001 , ****p < 0.0001 (compared to Control or Serum-Free); # p < 0.05 , ## p < 0.01 , #### p < 0.0001 (compared to shControl or Blank-Serum); && p < 0.01 , &&&& p < 0.0001 (compared to shControl- CXCR4-KD)

Article Snippet: Lentiviral vectors expressing CXCR4 shRNA (shCXCR4) or non-targeting control shRNA (shControl) were constructed by cloning the target sequences into the pLKO.1 vector (Addgene, Cat10878).

Techniques: Migration, CCK-8 Assay, Expressing, Control, shRNA, Colony Assay, Transwell Migration Assay, Transwell Invasion Assay, Flow Cytometry, Western Blot, Quantitative RT-PCR, Wound Healing Assay, Cell Analysis

Journal: bioRxiv

Article Title: Galectin-1 Modulates Hepatocellular Carcinoma Response to Thermal Ablation Through Regulating Glycolysis

doi: 10.1101/2024.12.12.628238

Figure Lengend Snippet: A-B) SNU449 survival rates (n=3 each) (A) post-thermal exposure at 37°C and 47°C at 24, 48, and 72 hours. Corresponding cell growth (B) . C) Western blot of Gal-1 in SNU449, SNU423, and HepG2/C3a (positive control). β-Tubulin was used to as loading control. D-E) SNU449 cell growth (n=3 each) (D) post-thermal exposure at 37°C and 47°C with Gal-1 inhibitor OTX or DMSO at 24, 48, and 72 hours. Corresponding cell survival rates (E) . F) Western blot of Gal-1 in shGal-1-SNU449 (SNU449 with Gal-1 knockdown) and respective shControl-SNU449. G-H) shControl and shGal-1-SNU449 cell growth (n=3) (G) post-thermal exposure at 37°C and 47°C at 24, 48, and 72 hours. Corresponding cell survival rates (H) . p-values were calculated using one-tailed-unpaired student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The following day, cells were transduced with lentiviral shRNA particles carrying three to five expression constructs each encoding target-specific 19-25 nucleotides (plus hairpin) shRNA designed to silence the gene expression of galectin-1 (sc-35441-V, scbt) or respective shControl particles (sc-108080, scbt).

Techniques: Western Blot, Positive Control, Control, Knockdown, One-tailed Test