Journal: Oncology Letters

Article Title: Inhibition of autophagy enhances apoptosis induced by bortezomib in AML cells

doi: 10.3892/ol.2020.12370

Figure Lengend Snippet: Downregulation of Beclin-1 enhances apoptosis and reduces cell viability induced by bortezomib in NB4 cells. Cells were infected with lentiviruses expressing shRNAs (non-targeting control or Beclin-1). Puromycin-resistant cells were pooled after each infection. (A) Cells transfected with the control shRNA and shBeclin-1 were treated with or without bortezomib (20 nM) for 24 h, and the expression levels of cleaved caspase-3, cleaved PARP, Bcl-2, p62 and Beclin-1, and LC3-I to LC3-II conversion were determined by western blotting. (B) Ratio of Bcl-2 and β-actin expression levels. Data are presented as the mean ± standard deviation of three independent repeats. *P<0.05 and **P<0.01. (C) Ratio of LC3-II and β-actin expression levels. Data are presented as the mean ± standard deviation of three independent repeats. **P<0.01. (D) Cells transfected with the control shRNA and shBeclin-1 were treated with bortezomib (20 nM) for 0 and 24 h, and cell viability was assessed using a water-soluble tetrazolium salts-8 assay. Data are presented as the mean ± standard deviation of three independent repeats. ***P<0.001. shRNA/sh, short hairpin RNA; CTRL, control; PARP, poly(ADP-ribose) polymerase.

Article Snippet: Lentiviral particles containing short hairpin RNA (shRNA/sh) Beclin-1 (cat. no. 131209BZ; 5′-CCGACTTGTTCCTTACGGAAA-3′) and shRNA negative control (shCTRL; cat. no. 131127CZ; 5′-TTCTCCGAACGTGTCACGTTTC-3′) expression vectors were obtained from Shanghai GenePharma Co., Ltd., and were then cloned into pGLV3/H1/GFP-Puro vector (Shanghai GenePharma Co., Ltd.).

Techniques: Infection, Expressing, Transfection, shRNA, Western Blot, Standard Deviation

Journal: Theranostics

Article Title: The LRP4/YAP axis drives the radiation-tolerant persister (RTP) cell state in breast cancer

doi: 10.7150/thno.101393

Figure Lengend Snippet: A RNAi screen for the identification of functional regulator of radiation-induced cell plasticity. A . schematic representation of RNAi screening strategy. B-C . Proportion of ALDH br cells in BFP + ( B ) and RFP + SUM159 cells ( C ) following silencing of each individual gene contained in the RNAi library and normalized with non-targeting siRNA (siCTRL). Genes targeted by a siRNA inducing a significant reduction of the ALDH br cell proportion are highlighted. Statistical test used is Student's t-test. Data represent mean ± SD D . Representative images of the high-content screening captures. ALDEFLUOR cellular staining is represented in green. Dead cells are labeled by DRAQ5 in red. RFP+ cells are in yellow and BFP+ cells in blue. E-F . Validation of candidate genes using FACS analysis. Proportion of ALDH br cells in BFP + ( E ) and RFP + SUM159 cells ( F ) are represented normalized with non-targeting siRNA (siCTRL). siRNA targeting ALDH1A1 was used as positive control and siRNA targeting TEXT12 or VDACL were used as negative control. Statistical test used is Student's t-test. Data represent mean ± SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: To evaluate the effect of LRP4 silencing on radiotherapy-response, SUM159, S68, and MDA-MB-231 cells were first transduced with a lentivirus expressing shRNA constructs (shLRP4, #SHCLNV or shCTRL, #SHC201VN (empty vector); Merck Sigma-Aldrich)..Cells shCTRL or shLRP4 were plated at low-density (between 75 and 2000 cells per well, depending on the dose) in 12-well plates (Falcon, 353043), with 3.5mL of culture medium and irradiated between 0 and 6Gy using Synergy linear accelerator (Elekta).

Techniques: Functional Assay, High Content Screening, Staining, Labeling, Biomarker Discovery, Positive Control, Negative Control

Journal: Theranostics

Article Title: The LRP4/YAP axis drives the radiation-tolerant persister (RTP) cell state in breast cancer

doi: 10.7150/thno.101393

Figure Lengend Snippet: LRP4/YAP axis inhibition radiosensitizes iCSC. A-B . Proportion of cells ( A , SUM159; B , S68) presenting a nuclear location of YAP in each cell subpopulations five days post-irradiation, with representative images of YAP staining (in green) in each cell subpopulations on the right panel. Nuclei are counterstained with DAPi (in blue). C . Pre-ranked GSEA interrogating differential expression between iCSC and late non-CSC and YAP/TAZ target genes. D . Western blot of markers related to YAP/TAZ signaling and its activation in SUM159 and S68 cells silenced for LRP4 (shLRP4) compared to the non-targeting shRNA (shCTRL). The mean intensities are indicated below each band for each condition. E . Heat map representing the mRNA expression of the LRP4, ALDH1A1, and YAP/TAZ target genes in SUM159 and S68 silenced for LRP4 (siLRP4) compared to the non-targeting siRNA (siCTRL). Each row represents three independent replicates per conditions (R1, R2, and R3). F . Proportion of SUM159 cells presenting a nuclear location of YAP in late non-CSC and iCSC following LRP4 silencing (siLRP4) compared to a non-targeting siRNA (siCTRL). G-H . SUM159 ( G ) and S68 cells ( H ), following LRP4 silencing (shLRP4) compared to a non-targeting siRNA (shCTRL), were exposed to various dose of radiation therapy and subjected to clonogenic survival assays ( G ), with representative images (right panels). I . Patient-derived xenograft organoid (PDXO) size distribution for CRCM389 cells WT (shCTRL) or silenced for LRP4 (shLRP4) following irradiation (RT) and compared to untreated condition (CTRL) (left panel). Representative pictures of PDXO-CRCM389 7 days post-treatment (right panel). Statistical test used is Student's t-test. Data represent mean ± SD. ns (not significant), *p<0.05, **p<0.01, ***p<0.001. J . Kaplan-Meier tumor-free survival curves of mice xenografted with 100,000-200,000 SUM159 irradiated cells silenced for LRP4 (shLRP4) compared to the control (shCTRL). p-value and hazard ration (HR) estimated according to Log-rank (Mantel-Cox) test.

Article Snippet: To evaluate the effect of LRP4 silencing on radiotherapy-response, SUM159, S68, and MDA-MB-231 cells were first transduced with a lentivirus expressing shRNA constructs (shLRP4, #SHCLNV or shCTRL, #SHC201VN (empty vector); Merck Sigma-Aldrich)..Cells shCTRL or shLRP4 were plated at low-density (between 75 and 2000 cells per well, depending on the dose) in 12-well plates (Falcon, 353043), with 3.5mL of culture medium and irradiated between 0 and 6Gy using Synergy linear accelerator (Elekta).

Techniques: Inhibition, Irradiation, Staining, Quantitative Proteomics, Western Blot, Activation Assay, shRNA, Expressing, Derivative Assay, Control

Journal: Scientific Reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro

doi: 10.1038/srep43539

Figure Lengend Snippet: ( A ) Knockdown of Tet2 by using shRNA. Upper : C2C12 cells were transfected with Tet2-specific shRNA plasmid (shTet2) or a control shRNA plasmid (shCtrl) and then selected with puromycin. The mRNA levels of Tet family genes or myoblast differentiation-associated genes in puromycin-selected cells were detected by qRT-PCR. Lower : the cells were induced to differentiation and the mRNA levels of Tets or myoblast differentiation-associated genes were detected 4 d after differentiation induction. Myog, myogenin; MyoM, myomaker. ( B ) Western blot confirmation of Tet2 protein decline in shRNA- or siRNA-mediated Tet2 knockdown cells. β-actin was used as an internal control. The expected Tet2 band (NW: 224 kDa) is shown. Full-length blots are presented in . ( C ) Evaluation of myotube formation of Tet2 knockdown C2C12 cells. Myotubes were visualized by immunostaining for MyHC 6 d after differentiation induction (left). Nuclei were stained with DAPI. Scale bar, 100 μm. The fusion index was calculated as the ratio of the number of nuclei in MyHC-positive cells to the total number of nuclei present in the observation field. The quantitative analysis revealed a significant decrease in the fusion index of shTet2 C2C12 as compared with shCtrl cells (right). Data are presented as means ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: shRNA, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Immunostaining, Staining

Journal: Scientific Reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro

doi: 10.1038/srep43539

Figure Lengend Snippet: C2C12 cells transfected with Tet2 shRNA (shTet2) or control shRNA (shCtrl) were cultured in growth medium. Methylation status of promoters of myogenin ( A ), Myf6 ( B ) or myomaker ( C ) was analyzed by using bisulfite sequencing. Numbers with a minus sign indicate the position of CpG relative to the transcription starting site. Each row represents an individual clone sequenced; black and white circles represent methylated and unmethylated CpGs, respectively. Percentage of methylation indicates the proportion of methylated CpG sites relative to the whole CpG sites examined.

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: Transfection, shRNA, Cell Culture, Methylation, Methylation Sequencing

Journal: Scientific Reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro

doi: 10.1038/srep43539

Figure Lengend Snippet: C2C12 cells transfected with Tet2 shRNA (shTet2) or control shRNA (shCtrl) were cultured in growth medium with or without vitamin C (VC; 500 μM). ( A ) Immunostaining for 5hmC in shTet2 vs shCtrl C2C12 cells with or without VC treatment. Nuclei were stained with DAPI. Scale bar, 50 μm. ( B ) Quantification of fluorescence intensities of 5hmC. The quantitative analysis revealed that Tet2 knockdown decreased the ability of VC to enhance 5hmC formation. ( C ) qRT-PCR analysis for the expression of myogenin, Myf6 and myomaker in shTet2 vs shCtrl C2C12 cells with or without VC treatment. Gapdh was used as an internal control. Data are presented as means ± SEM (n = 3). Asterisks above columns represent significant difference among groups (p < 0.05).

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: Transfection, shRNA, Cell Culture, Immunostaining, Staining, Fluorescence, Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro

doi: 10.1038/srep43539

Figure Lengend Snippet: C2C12 cells transfected with Tet2 shRNA (shTet2) or control shRNA (shCtrl) were subdivided into two experimental groups: non-vitamin C treatment group (indicated as VC−), in which VC was absent in both growth medium and differentiation medium, and VC-treatment group (indicated as VC+), in which the cells were first cultured for 48 h in growth medium containing 500 μM VC, and then were shifted to differentiated medium containing 500 μM VC. ( A ) Evaluation of myoblast differentiation in different treatment groups. Cells after 6 d of differentiation induction were immunostained with anti-MyHC antibodies to mark the myotubes. Nuclei were stained with DAPI. Scale bar, 100 μm. ( B ) Quantification analysis for differentiation efficiency in different treatment groups. The fusion index was calculated as the ratio of the number of nuclei in MyHC-positive cells to the total number of nuclei present in the observation field. ( C ) qRT-PCR analysis for the expression of myogenin, Myf6 and myomaker in C2C12 cells after 4 d of differentiation induction in different treatment groups. Gapdh was used as an internal control. Data are presented as means ± SEM (n = 3). Asterisks above columns represent significant difference among groups (p < 0.05).

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: Transfection, shRNA, Cell Culture, Staining, Quantitative RT-PCR, Expressing