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: Cellular and Molecular Life Sciences: CMLS

Article Title: OSGIN1 is a novel TUBB3 regulator that promotes tumor progression and gefitinib resistance in non-small cell lung cancer

doi: 10.1007/s00018-023-04931-4

Figure Lengend Snippet: OSGIN1 promotes NSCLC growth in vitro and in vivo. A, C Effect of OSGIN1 knockdown or overexpression on cell proliferation was measured by MTT assay. B, D Anchorage-independent growth from different cells with OSGIN1 knockdown or overexpression. Colonies were counted using Image J-Plus. E Effect of OSGIN1 knockdown on NSCLC patient-derived xenograft tumor growth in vivo. Mice were divided into 3 groups as follows: (1) shControl group, (2) shOSGIN1 #2 group and (3) shOSGIN1 #7 group. NSCLC PDX tissues were treated by direct injection of each viral particle at three time points when the average tumor volume reached approximately 100 mm3. The tumor volumes of the HLG77 (upper panel) and HLG80 (lower panel) NSCLC PDX cases were measured on the indicated days. F Tumor photographs and relevant tumor weight. G Expression of OSGIN1 in PDX tumor tissues. Data from in vitro experiments were presented as means ± SD. All data statistical differences were evaluated using Student’s t-test or one way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: The pLKO.1-puro non-target shRNA Control Plasmid DNA (shControl) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: In Vitro, In Vivo, Over Expression, MTT Assay, Derivative Assay, Injection, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: OSGIN1 is a novel TUBB3 regulator that promotes tumor progression and gefitinib resistance in non-small cell lung cancer

doi: 10.1007/s00018-023-04931-4

Figure Lengend Snippet: OSGIN1 promote phosphorylation of TUBB3 at serine 172 by DYRK1A via enhance interaction of DYRK1A and TUBB3. A The expression of TUBB3 and p-TUBB3 in control and OSGIN1 knockdown cells. The expression of TUBB3 and p-TUBB3 was measured by Western blotting. B TUBB3 phosphorylation-dependent cell growth in OSGIN1 knockdown A549 cells. shControl or shOSGIN1 cells were transfected with TUBB3-WT, -172A, or -172D, and cell proliferation was subsequently evaluated by MTT assay. C OSGIN1 interacts with DYRK1A in NSCLC cells. Cells expressing OSGIN1-flag and DYRK1A-his were immunoprecipitated using his beads. The expression of flag and his was detected by Western blotting. D OSGIN1 interacts with DYRK1A in vitro. Recombinant OSGIN1 and/or DYRK1A proteins were mixed and IP using GST beads. Binding between OSGIN1 and DYRK1A was detected by Western blotting. E In vitro kinase assay to check effect of OSGIN1 on phosphorylation of TUBB3 by DYRK1A. F Flag IP from Lentix-293T cells expressing Flag-tagged DYRK1A together with the different Myc-tagged OSGIN1 domain mutants. G OSGIN1 enhances the interaction between TUBB3 and DYRK1A. Recombinant TUBB3 and DYRK1A proteins were co-incubated with or without OSGIN1 protein. Incubated proteins were immunoprecipitated using an anti-DYRK1A antibody. TUBB3, OSGIN1 and DYRK1A proteins were detected by Western blotting. H Modeling of OSGIN1 bind with TUBB3 and DYRK1A. Blue: OSGIN1; yellow: DYRK1A; Pink: TUBB3. The three-dimensional (3D) structures of TUBB3 and DYRK1A are derived from the Protein Data Bank (PDB Accession Number 6S8L and 2WO6, respectively). DYRK1A catalytic residue Asp287 was specified to be within 8 Å to the TUBB3 Ser172 in the docking process. The DYRK1A-TUBB3 model with the best docking score was chosen as the receptor molecule to further dock OSGIN1 into it. AlphaFold model (AF-Q9UJX0-F1-model_v4) of OSGIN1 was used. The final model is consistent with experimental data and has a docking score < − 200, which is similar to the values of complex tructures in the PDB, indicating a high-confidence model. I Proteins of the MKK3/6-p38 signaling pathway and phosphorylated TUBB3 were measured in NSCLC cells expressing stable shDYRK1A by Western blotting. All data statistical differences were evaluated using Student’s t-test. *P < 0.05, ***P < 0.001

Article Snippet: The pLKO.1-puro non-target shRNA Control Plasmid DNA (shControl) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Western Blot, Transfection, MTT Assay, Immunoprecipitation, In Vitro, Recombinant, Binding Assay, Kinase Assay, Incubation, Derivative Assay, Residue

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: OSGIN1 is a novel TUBB3 regulator that promotes tumor progression and gefitinib resistance in non-small cell lung cancer

doi: 10.1007/s00018-023-04931-4

Figure Lengend Snippet: Suppression of OSGIN1 expression increases gefitinib sensitivity in vitro and in vivo. A, C HCC827R cells expressing shOSGIN1 or shTUBB3 and vector control cells were treated with the indicated doses of gefitinib for 72 h, and cell viability was analyzed by MTT assay. B Foci formation of the indicated cells in the presence or absence of gefitinib was analyzed. D H1650 cells overexpressing OSGIN1 or Mock were treated with the indicated concentrations of gefitinib for 72 h. Cell proliferation was then analyzed by MTT assay. E Effect of OSGIN1 knockdown on gefitinib resistant NSCLC patient-derived xenograft tumor growth in vivo. Mice were divided into 4 groups as follows: (1) shControl + vehicle group, (2) shControl + 50 mg/kg gefitinib group, (3) shOSGIN1 #2 + vehicle group and (4) shOSGIN1 #2 + 50 mg/kg gefitinib group. Gefitinib resistant NSCLC PDX tissues were treated by direct injection of each viral particle at three time points when the average tumor volume reached approximately 100 mm3. And oral treatment for indicated gefitinib when the average tumor volume reached approximately 300 mm3. The tumor volumes of LG1GR NSCLC PDX cases were measured on the indicated days. F Tumor photographs. G Tumor weight. H Expression of OSGIN1, EGFR, and MKK3/6-p38 signaling proteins in LG1GR NSCLC PDX tumor tissues. Data from in vitro experiments were presented as means ± SD from triplicate experiments. All data statistical differences were evaluated using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: The pLKO.1-puro non-target shRNA Control Plasmid DNA (shControl) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, In Vitro, In Vivo, Plasmid Preparation, MTT Assay, Derivative Assay, Injection