Journal: bioRxiv

Article Title: Acod1 negatively impacts osteoclastogenesis via GPR91-mediated NFATc1 activation

doi: 10.1101/2022.04.07.487467

Figure Lengend Snippet: (A,B) Irg1 gene expression analysis by qPCR. BMDMs (A) and RAW 264.7 macrophages (B) were stimulated with 10 ng/mL LPS and 50 ng/mL RANKL for 6 h, respectively. BMDMs were additionally treated with 25 ng/mL M-CSF. Relative Irg1 expression levels were normalized to the reference gene Hprt1 (n=4). (C) FACS analysis of lentiviral-transduced Mock and Acod1 overexpression cells for eGFP fluorescence. Parental RAW264.7 macrophages were included as a negative control. (D) Irg1 gene expression analysis of Mock and Acod1 overexpressing cells by qPCR. Relative Irg1 expression levels were normalized to reference gene Hprt1 (n=4) (E) The ATP content in Mock and Acod1 overexpressing cells was assessed by Cell TiterGlo ® Assay analysis after 4 or 24 h cultivation (n=4). (F) Cell viability of Mock and Acod1 cells was assessed by Zombie NIR™ staining. The percentage of live cells was shown. (G) Mitochondrial activity was evaluated by MitoTracker™ Deep Red. Both Mock and Acod1 cells were cultured for 24 h (n=4). (H) The mitochondrial DNA content was assessed by determining the mitochondrial copy number. Both Mock and Acod1 cells were stimulated with 50 ng/mL RANKL for four days. The number of mitochondria per cell were quantified by qPCR. Mitochondrial DNA (mtDNA) copies were normalized with nuclear DNA (nucDNA) (n=4). (I) Cellular oxidative stress was measured by CellROX™ Deep Red reagent. Mock and Acod1 cells were stimulated with RANKL. Cells were evaluated by FACS on day 3 after 30 min CellROX™ staining (n=4). Statistical analysis was performed using Mann-Whitney test.

Article Snippet: Recombinant lentiviral vectors (Acod1: pLV[Exp]-EGFP:T2A:Puro-EF1A>HA/mAcod1[nm_0008392.1] Mock: pLV[Exp]-EGFP:T2A:Puro-EF1A>mCherry) purchased from VectorBuilder (Chicago, IL, USA) were added at a MOI of 10 into the cells followed by 1 h spin infection at a speed of 300 g. Puromycin selection was performed after 48 h incubation in fresh medium.

Techniques: Gene Expression, Expressing, Over Expression, Fluorescence, Negative Control, Staining, Activity Assay, Cell Culture, MANN-WHITNEY

Journal: Journal of Cancer

Article Title: LASS2 impairs proliferation of glioma stem cells and migration and invasion of glioma cells mainly via inhibition of EMT and apoptosis promotion

doi: 10.7150/jca.71256

Figure Lengend Snippet: Analysis of LASS2 expression on a human glioma tissue microarry. (A) Representative immunohistochemical staining images of LASS2. Scale bar = 100 µm. (B) LASS2 levels in grade II to IV glioma samples were significantly lower than those in the normal adjancent tissue (NAT) (*P < 0.05 for grade II, and **P < 0.01 for both grade III and IV, n.s., no significance; one-way ANOVA). (C) The level of LASS2 in glioma of all grades was significantly lower than in NAT (*P < 0.05; unpaired two-tailed Student's t-test). (D) Representative images showing the immunuofluorescent co-staining of LASS2 and CD133 in the NAT and gliomas graded from I to IV. Scale bar = 20 µm.

Article Snippet: Plasmids pLV-Vec (pLV [Exp] -EGFP: T2A: Puro-EF1A), pLV-LASS2 (pLV [Exp] -EGFP: T2A: Puro-EF1A>hCERS2[NM_022075.4]) and pLV-shLASS2 (pLV [shRNA] -EGFP:T2A:Puro-U6>hCERS2[shRNA#1]) were purchased from VectorBuilder (Guangzhou, China).

Techniques: Expressing, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Journal of Cancer

Article Title: LASS2 impairs proliferation of glioma stem cells and migration and invasion of glioma cells mainly via inhibition of EMT and apoptosis promotion

doi: 10.7150/jca.71256

Figure Lengend Snippet: Isolation of GSCs from U-87 MG and U251 cells. (A) The proportion of CD133 + cells in U-87 MG and U251 cells. (B) The sorted CD133 + U-87 MG cells were retested by flow cytometry after 7 days of culture, and the percentages of CD133 + cells and CD133 - cells were shown. (C) CD133 + cells and CD133 - cells were maintained in the serum-free condition for 7 days. CD133 + cells formed spheres, whereas CD133 - cells showed degradation. Scale bar = 200 µm. (D) The sphere derived from sorted CD133 + cells was shown to express stem cell markers CD133 and Nestin. Scale bar = 20 µm. (E) CD133 and Nestin were overexpressed in proliferated sorted CD133 + cells compared with CD133 - cells, whereas CD133 - cells overexpressed LASS2 compared with CD133 + cells (*P < 0.05, vs. CD133 - group; unpaired two-tailed Student's t-test; n = 3). (F, G) The glioma xenografts derived from CD133 + cells were more malignant than those derived from CD133- cells. (H) The average final tumor weight of the xenograft derived from CD133 + cells was significantly higher than that from CD133 - cells in nude mice ( F, G, and H, *P < 0.05, **P < 0.01, and ***P < 0.001, vs. CD133 - group; unpaired two-tailed Student's t-test; n = 3 animals).

Article Snippet: Plasmids pLV-Vec (pLV [Exp] -EGFP: T2A: Puro-EF1A), pLV-LASS2 (pLV [Exp] -EGFP: T2A: Puro-EF1A>hCERS2[NM_022075.4]) and pLV-shLASS2 (pLV [shRNA] -EGFP:T2A:Puro-U6>hCERS2[shRNA#1]) were purchased from VectorBuilder (Guangzhou, China).

Techniques: Isolation, Flow Cytometry, Derivative Assay, Two Tailed Test

Journal: Journal of Cancer

Article Title: LASS2 impairs proliferation of glioma stem cells and migration and invasion of glioma cells mainly via inhibition of EMT and apoptosis promotion

doi: 10.7150/jca.71256

Figure Lengend Snippet: LASS2 inhibits sphere formation of GSCs. CD133 + U-87 MG cells were stably transfected with pLV and pLV- LASS2. (A) Downregulation of LASS2 promoted sphere formation of GSCs (**P < 0.01, vs. pLV control; unpaired two-tailed Student's t-test; n = 3). Scale bar = 200 µm. (B) Downregulation of LASS2 significantly reduced the protein level of LAAS2 while significantly increasing those of Notch1 and glioma stem cells stemness proteins CD133, Nestin and Sox2 (*P < 0.05, vs. pLV control; unpaired two-tailed Student's t-test; n = 3).

Article Snippet: Plasmids pLV-Vec (pLV [Exp] -EGFP: T2A: Puro-EF1A), pLV-LASS2 (pLV [Exp] -EGFP: T2A: Puro-EF1A>hCERS2[NM_022075.4]) and pLV-shLASS2 (pLV [shRNA] -EGFP:T2A:Puro-U6>hCERS2[shRNA#1]) were purchased from VectorBuilder (Guangzhou, China).

Techniques: Stable Transfection, Transfection, Two Tailed Test

Journal: Journal of Cancer

Article Title: LASS2 impairs proliferation of glioma stem cells and migration and invasion of glioma cells mainly via inhibition of EMT and apoptosis promotion

doi: 10.7150/jca.71256

Figure Lengend Snippet: Effect of LASS2 on cell migration, invasion and apoptosis. (A) Wound healing assay of pLV-vector or pLV-LASS2-transfected glioma/glioblastoma cells at 0 h, 12 h and 24 h after scratch. The images were taken from an inverted microscope under 10× magnification (*P < 0.05 and **P < 0.01, vs. pLV control; unpaired two-tailed Student's t-test; n = 3). Scale bar = 200 µm. (B) Colony formation assay in pLV-vector or pLV-LASS2-transfected U251 and U-87 MG cells. Images were acquired at 4× magnification (*P <0.05, vs. pLV control; unpaired two-tailed Student's t-test; n = 3). (C) Transwell assay demonstrated that LASS2 inhibits the migration of U251 and U-87 MG cells compared with the pLV control (*P <0.05; unpaired two-tailed Student's t-test; n = 3). Scale bar = 200 µm. (D) The immunofluorescence staining of MMP9 and SPHK1 was shown in both U251 and U-87 MG cells. Scale bar = 200 µm. (E) RNA-Seq shows that LASS2 influenced cell migration/invasion, apoptosis, epithelial- mesenchymal transition (EMT) conversion and cellular life activity. (F) Overexpression of LASS2 reduced the protein levels of MMP2, MMP9, and SPHK1 while increasing that of TIMP2 in both U251 and U-87 MG cells. (G) LASS2 overexpression increased the levels of Bax, cleaved Caspase-3, TNF-α, and p53 while reducing that of Bcl-2 in both U251 and U-87 MG cells. (H) Overexpression of LASS2 reduced the protein levels of Vimentin and N-cadherin while increasing that of E-cadherin in both U251 and U-87 MG cells (F, G, and H, *P <0.05 and **P < 0.01, vs. pLV control in each cell line; unpaired two-tailed Student's t-test; n = 3).

Article Snippet: Plasmids pLV-Vec (pLV [Exp] -EGFP: T2A: Puro-EF1A), pLV-LASS2 (pLV [Exp] -EGFP: T2A: Puro-EF1A>hCERS2[NM_022075.4]) and pLV-shLASS2 (pLV [shRNA] -EGFP:T2A:Puro-U6>hCERS2[shRNA#1]) were purchased from VectorBuilder (Guangzhou, China).

Techniques: Migration, Wound Healing Assay, Plasmid Preparation, Transfection, Inverted Microscopy, Two Tailed Test, Colony Assay, Transwell Assay, Immunofluorescence, Staining, RNA Sequencing Assay, Activity Assay, Over Expression

Journal: Journal of Cancer

Article Title: LASS2 impairs proliferation of glioma stem cells and migration and invasion of glioma cells mainly via inhibition of EMT and apoptosis promotion

doi: 10.7150/jca.71256

Figure Lengend Snippet: LASS2 inhibited tumor growth in a pLV-LASS2-U-87 MG glioblastoma xenograft nude mouse model. (A) Representative photographs showing the gross pLV-LASS2-U-87 MG and empty scramble control glioblastoma xenografts from the nude mouse. (B) The tumor volume was evaluated between the scrambled control and pLV-LASS2 groups (**P < 0.05 and **P < 0.01 vs. pLV control group; unpaired two-tailed Student's t-test; n = 5 animals). (C) The final tumor weight was measured after dissection. The average final weight of tumors derived from pLV-LASS2-transcfected U-87 MG cells was significantly lower than those derived from the scrambled control (**P < 0.01, vs. pLV control group; unpaired two-tailed Student's t-test; n = 5 animals). (D) Representative images for H&E staining from either group were shown. (E) IHC staining of LASS2, TIMP2, MMP9, and SPHK1 in xenografted tumors derived from U-87 MG cells transfected with either pLV-LASS2 or scrambled control. Scale bar = 20 µm. (F) Western blot analysis of LASS2, SPHK1, TIMP2, MMP2, and MMP9 in xenografted tumors derived from U-87 MG cells transfected with either pLV or pLV-LASS2. (G) Western blot analysis of Bax, Bcl-2, pro-Caspase-3, cleaved Caspase-3, TNF-α and p53 in xenografted tumors derived from U-87 MG cells transfected with either pLV or pLV-LASS2. (H) Western blot analysis of EMT conversion-related proteins Vimentin, E-cadherin, and N-cadherin in xenografted tumors derived from U-87 MG cells transfected with either pLV or pLV-LASS2 ( F, G, and H, *P <0.05, **P < 0.01, and ***P < 0.001, vs. pLV control; unpaired two-tailed Student's t-test; n = 3).

Article Snippet: Plasmids pLV-Vec (pLV [Exp] -EGFP: T2A: Puro-EF1A), pLV-LASS2 (pLV [Exp] -EGFP: T2A: Puro-EF1A>hCERS2[NM_022075.4]) and pLV-shLASS2 (pLV [shRNA] -EGFP:T2A:Puro-U6>hCERS2[shRNA#1]) were purchased from VectorBuilder (Guangzhou, China).

Techniques: Two Tailed Test, Dissection, Derivative Assay, Staining, Immunohistochemistry, Transfection, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Hypomethylation-associated ELF3 helps nasopharyngeal carcinoma to escape immune surveillance via MUC16-mediated glycolytic metabolic reprogramming

doi: 10.1152/ajpcell.00438.2024

Figure Lengend Snippet: Association between MUC16 level and clinicopathological characteristics in patients with NPC

Article Snippet: The lentiviral plasmids used to knockdown MUC16 (pLV[shRNA]-EGFP:T2A: Puro-U6>hMUC16[shRNA]), knockdown ELF3 (pLV[shRNA]-EGFP:T2A: Puro-U6>hELF3[shRNA]), or overexpress MUC16 (pLV[Exp]-mCherry:T2A: Puro-EF1A>hMUC16 [NM_001414686.1]) were designed by VectorBuilder (Guangzhou, Guangdong, PR China).

Techniques: Expressing

Journal: American Journal of Physiology - Cell Physiology

Article Title: Hypomethylation-associated ELF3 helps nasopharyngeal carcinoma to escape immune surveillance via MUC16-mediated glycolytic metabolic reprogramming

doi: 10.1152/ajpcell.00438.2024

Figure Lengend Snippet: Working model for regulation of nasopharyngeal carcinoma (NPC) progression by ELF3/MUC16 axis. Figure contains images from Servier Medical Art ( https://smart.servier.com ) licensed under a CC BY 4.0 license ( https://creativecommons.org/licenses/by/4.0/ ). ELF3, E74-like factor 3; MUC16, mucin-16.

Article Snippet: The lentiviral plasmids used to knockdown MUC16 (pLV[shRNA]-EGFP:T2A: Puro-U6>hMUC16[shRNA]), knockdown ELF3 (pLV[shRNA]-EGFP:T2A: Puro-U6>hELF3[shRNA]), or overexpress MUC16 (pLV[Exp]-mCherry:T2A: Puro-EF1A>hMUC16 [NM_001414686.1]) were designed by VectorBuilder (Guangzhou, Guangdong, PR China).

Techniques:

Journal: iScience

Article Title: Heterotypic cell-in-cell structures between cancer and NK cells are associated with enhanced anticancer drug resistance

doi: 10.1016/j.isci.2022.105017

Figure Lengend Snippet: Analysis of heterotypic CIC structures between hABCB1 overexpressing cancer and NK-92 cells (A and B) Cell proliferation of wild-type or hABCB1 overexpressing A549 or A375SM cells was measured by CCK-8 assay after treatment with various concentrations of doxorubicin and paclitaxel (0.5, 1, 2, 5 and 10 μM) for 24 h. p values were determined using the student’s t- tests. All experiments were performed in triplicate. ∗, p < 0.05; ∗∗, p < 0.01. (C) A549 and A375SM wild-type or hABCB1 overexpressing cells were co-cultured with NK-92 cells for 4 h to analyze the heterotypic CIC population. (D) Heterotypic CIC populations in NK-92 injected A549 and A375SM wild-type or hABCB1 overexpressing tumors of xenograft animal models were analyzed using flow cytometry. ∗, p < 0.05; ns, not significant. (E) Cell proliferation of wild-type or hABCB1 overexpressing cancer organoids (SNU-CO-1) were measured by CCK-8 assay after treatment with various concentrations of doxorubicin and paclitaxel (0.5, 1 and 5 μM) for 24 h. (F) The wild-type or hABCB1 overexpressing cancer organoids (SNU-CO-1) were co-cultured with NK-92 cells for 4 h to analyze the heterotypic CIC population. (G) Heterotypic CIC and non-CIC formed by co-culture of A549 and NK-92 cells were isolated, and the differences in NK susceptibility and drug resistance were analyzed. The mechanistic model of this study is that the heterotypic CIC populations exhibit higher activation of signaling proteins such as Akt, IkBα, Shc, Raf and Cyclin D1, increased cancer cell growth and drug resistance, and decreased NK sensitivity compared to non-CIC populations.

Article Snippet: After 24 h, cells were infected hABCB1 lentivirus particle (pLV[Exp]-Puro-EF1A-hABCB1 [NM_001348945.1] from Vectorbuilder, vector ID: VB900100-2568gcj) using a Trans-Dux MAX reagent (Systembio) and they were treated with puromycin 1 μg/mL for 2 weeks.

Techniques: CCK-8 Assay, Cell Culture, Injection, Flow Cytometry, Co-Culture Assay, Isolation, Activation Assay

Journal: iScience

Article Title: Heterotypic cell-in-cell structures between cancer and NK cells are associated with enhanced anticancer drug resistance

doi: 10.1016/j.isci.2022.105017

Figure Lengend Snippet:

Article Snippet: After 24 h, cells were infected hABCB1 lentivirus particle (pLV[Exp]-Puro-EF1A-hABCB1 [NM_001348945.1] from Vectorbuilder, vector ID: VB900100-2568gcj) using a Trans-Dux MAX reagent (Systembio) and they were treated with puromycin 1 μg/mL for 2 weeks.

Techniques: Virus, Recombinant, Staining, CCK-8 Assay, Ab Array, Software