Journal: Oncology Letters

Article Title: Restoration of the ATG5-dependent autophagy sensitizes DU145 prostate cancer cells to chemotherapeutic drugs

doi: 10.3892/ol.2021.12899

Figure Lengend Snippet: DU145 cells lack ATG5 expression and are defective in the induction of autophagy. (A) Detection of ATG5 protein expression in cell lysates derived from three prostate cancer cell lines, DU145, PC3 and 22RV1, and three lung cancer cell lines, A549, NCI-H1650 and NCI-H1975, using western blotting. (B) Detection of LC3-II and SQSTM1 protein expression in PC3, DU145 and NCI-H1650 cell lysates following treatment of the corresponding cells with the lysosomal inhibitor chloroquine (50 µM) or the proteasomal inhibitor bortezomib (10 µM) for 24 h. (C) Detection of LC3-II protein expression in PC3, DU145 and 22RV1 cell lysates following treatment of the cells with the lysosomal inhibitor chloroquine (50 µM) or the hydroxylmethylglutaryl Co-enzyme A reductase inhibitor atorvastatin (10 µM) for 48 h. β-actin was used as a loading control. ATG5, autophagy related 5; SQSTM1, sequesteome 1.

Article Snippet: A total of 2.5×10 4 DU145 cells [transfected with the empty vector control FUW-Mr2tTA (Addgene, Inc.; cat. no. 20342) or FUW-HA-ATG5] were seeded in each well of a 12-well culture plate.

Techniques: Expressing, Derivative Assay, Western Blot, Control

Journal: Oncology Letters

Article Title: Restoration of the ATG5-dependent autophagy sensitizes DU145 prostate cancer cells to chemotherapeutic drugs

doi: 10.3892/ol.2021.12899

Figure Lengend Snippet: Restoration of ATG5 expression restores ATG5-dependent autophagy in DU145 cells. (A) Ectopic expression of ATG5 in DU145 cells induced formation of the autophagosomal marker protein LC3-II. ATG5 was ectopically expressed in DU145 cells using a lentiviral expression system. The ATG5-expressing and vector control cells were lysed and the expression levels of LC3-II, SQSTM1 and ATG5 were detected by western blot analysis. β-actin was used as the lysate loading control. (B) Ectopic expression of ATG5 restored the autophagic flux in DU145 cells. The ATG5-expressing cells and the vector control cells were treated with the lysosomal inhibitor chloroquine (50 µM) for 12 h and the expression levels of LC3-II, SQSTM1 and ATG5 were detected by western blot analysis. Clathrin-HC was used as a lysate loading control. (C) Autophagy was induced by the mTOR inhibitor rapamycin in the ATG5-expressing DU145 cells, but not in the vector control cells. The ATG5-expressing and the vector control DU145 cells were treated with rapamycin (1 µM) for 12 h. The cells were fixed, and the autophagosomes and ATG5 were visualized by immunofluorescent staining with an anti-LC3 antibody and an anti-ATG5 antibody, respectively. White arrows indicate the induced autophagosomes. Scale bar, 20 µm. ATG5, autophagy related 5; SQSTM1, sequesteome 1; Clathrin-HC, clathrin heavy chain.

Article Snippet: A total of 2.5×10 4 DU145 cells [transfected with the empty vector control FUW-Mr2tTA (Addgene, Inc.; cat. no. 20342) or FUW-HA-ATG5] were seeded in each well of a 12-well culture plate.

Techniques: Expressing, Marker, Plasmid Preparation, Control, Western Blot, Staining

Journal: Oncology Letters

Article Title: Restoration of the ATG5-dependent autophagy sensitizes DU145 prostate cancer cells to chemotherapeutic drugs

doi: 10.3892/ol.2021.12899

Figure Lengend Snippet: Restoration of the ATG5-dependent autophagy in DU145 cells promotes cell proliferation and migration. (A) ATG5-expressing DU145 cells exhibited a higher proliferation rate compared with the vector control cells. The data used for semi-quantification were from three independent experiments. ***P<0.001. (B and C) ATG5-expressing DU145 cells exhibited a higher migration rate compared with the vector control cells. (B) Wound healing assay images (magnification, ×100). (C) Semi-quantification of cell migration was based on three independent experiments using the wound healing assay. **P<0.01. ATG5, autophagy related 5.

Article Snippet: A total of 2.5×10 4 DU145 cells [transfected with the empty vector control FUW-Mr2tTA (Addgene, Inc.; cat. no. 20342) or FUW-HA-ATG5] were seeded in each well of a 12-well culture plate.

Techniques: Migration, Expressing, Plasmid Preparation, Control, Wound Healing Assay

Journal: Oncology Letters

Article Title: Restoration of the ATG5-dependent autophagy sensitizes DU145 prostate cancer cells to chemotherapeutic drugs

doi: 10.3892/ol.2021.12899

Figure Lengend Snippet: Restoration of ATG5-dependent autophagy in DU145 cells sensitizes them to the cytotoxicity of chemotherapeutic drugs and to ER stressors. (A) Light phase microscopic images of cells treated with the indicated concentrations of docetaxel for 72 h (left panel; magnification, ×200) and semi-quantification of the inhibition of proliferation by docetaxel treatment in ATG5-expressing or vector control cells (right panel). (B) Light phase microscopic images of the cells treated with the indicated concentrations of VPA for 48 h (left panel; magnification, ×200) and semi-quantification of the inhibition of proliferation by docetaxel treatment in ATG5-expressing or vector control cells (right panel). (C) Semi-quantification of the inhibition of proliferation by the ER stressors brefeldin A (500 nM), tunicamycin (2.0 µg/ml) and thapsigargin (250 nM) for 24 h in the ATG5-expressing or the vector control cells. In all the quantification figures, the data were from three independent experiments. *P<0.05; **P<0.01; ***P<0.001. ATG5, autophagy related 5; VPA, valproic acid.

Article Snippet: A total of 2.5×10 4 DU145 cells [transfected with the empty vector control FUW-Mr2tTA (Addgene, Inc.; cat. no. 20342) or FUW-HA-ATG5] were seeded in each well of a 12-well culture plate.

Techniques: Inhibition, Expressing, Plasmid Preparation, Control

Journal: Oncology Letters

Article Title: Restoration of the ATG5-dependent autophagy sensitizes DU145 prostate cancer cells to chemotherapeutic drugs

doi: 10.3892/ol.2021.12899

Figure Lengend Snippet: Restoration of ATG5-dependent autophagy in DU145 cells enhances apoptosis induced by docetaxel or thapsigargin. (A) ATG5-expressing or vector control cells were treated with the indicated concentration of docetaxel or thapsigargin for 48 h. The number of apoptotic cells was visualized using fluorescent staining with FITC-conjugated Annexin V. ‘Phase’ refers to light microscopic images (magnification, ×100). ‘Annexin V’ refers to images of the FITC-Annexin V fluorescence (magnification, ×100). The FITC-Annexin V fluorescent dots represent the apoptotic cells. (B) Semi-quantification of the apoptotic cells. The data were from three independent experiments. **P<0.01; ***P<0.001. ATG5, autophagy related 5.

Article Snippet: A total of 2.5×10 4 DU145 cells [transfected with the empty vector control FUW-Mr2tTA (Addgene, Inc.; cat. no. 20342) or FUW-HA-ATG5] were seeded in each well of a 12-well culture plate.

Techniques: Expressing, Plasmid Preparation, Control, Concentration Assay, Staining, Fluorescence

Journal: bioRxiv

Article Title: Successful Correction of ALD Patient-derived iPSCs Using CRISPR/Cas9

doi: 10.1101/2020.02.23.962118

Figure Lengend Snippet: A Scheme of oligodendrocyte (OL) differentiation with lentiviral transduction B Successful differentiation of intermediate oligodendrocytes from control (WT), ALD patient (DS) and mutation-corrected (DSC1, DSC2 and DSC3) iPSCs. There was double positive staining of MBP and O4.

Article Snippet: The lentiviral vector Teto-O-FUW-Sox10 (Addgene, MA, USA; Cat. No. 45843) and FUW-M2-rtTA (Addgene, MA, US; Cat. No. 20342) were co-transfected with the envelope plasmids pRSV-Rev (Addgene, MA, US; Cat. No. 12253), pMDLg/pRRE (Addgene, MA, US; Cat. No. 12251) and pMD2.

Techniques: Transduction, Control, Mutagenesis, Staining

Journal: bioRxiv

Article Title: Multi-omic rejuvenation of human cells by maturation phase transient reprogramming

doi: 10.1101/2021.01.15.426786

Figure Lengend Snippet: (A) Mean DNA methylation age (calculated using the multi-tissue clock ) throughout the reprogramming process where cells were transduced with our tetO-GFP-hOKMS vector and treated continuously with 2 µg/mL of doxycycline. Reprogramming is divided in three distinct phases: initiation phase (IP); maturation phase (MP) and stabilisation phase (SP). DNA methylation age decreased substantially during the maturation phase of reprogramming in cells that were successfully reprogramming (magenta line) but not in control cells (yellow and orange lines represent non-transduced cells and cells expressing hOKMS but failing to reprogram as indicated by cell surface markers, respectively). Points represent the mean and error bars the standard deviation. N = 3 biological replicates per condition, where fibroblasts were derived from different donors. N = 2 biological replicates for the iPSC timepoint (day 51). (B) Experimental scheme for maturation phase transient reprogramming (MPTR). The tetO-GFP-hOKMS reprogramming construct was introduced into fibroblasts from older donors by lentiviral transduction. Alternatively, cells were ‘mock infected’ as a negative control. Following this, cells were grown in the presence of 2 µg/mL doxycycline to initiate reprogramming. At several timepoints during the maturation phase, cells were flow sorted and successfully reprogramming cells (CD13-SSEA4+) and cells that were failing to reprogram (CD13+ SSEA4-) were collected for analysis. These were termed “transient reprogramming intermediate” and “failing to transiently reprogram intermediate”, respectively. Sorted cells were also further cultured, and grown in the absence of doxycycline for at least four weeks - these were termed “transiently reprogrammed” (CD13-SSEA4+) or “failed to transiently reprogram” (CD13+ SSEA4-). (C) Phase-contrast microscope images of cells after doxycycline treatment (transient reprogramming intermediate) and after withdrawal of doxycycline (transiently reprogrammed) as described in B. The morphology of some cells changed after doxycycline treatment. These cells appeared to form colonies, which became larger with longer exposure to doxycycline. After sorting, these cells were cultured in medium no longer containing doxycycline, and appeared to return to their initial fibroblast morphology. (D) Principal component analysis of transient reprogramming and reference reprogramming sample transcriptomes (light blue to dark blue, data from , and our novel Sendai reprogramming dataset). Reference samples form a reprogramming trajectory along PC1. Transiently reprogrammed samples and control samples were located at the beginning of this trajectory. (E) Mean gene expression levels for the fibroblast specific gene FSP1 and the iPSC specific gene NANOG . Transiently reprogrammed samples expressed these genes at levels similar to control fibroblasts. Bars represent the mean and error bars the standard deviation. Samples transiently reprogrammed for 13, 15 or 17 days were pooled. The number of samples in each group is indicated in brackets. (F) Principal component analysis of transient reprogramming (magenta points) and reference reprogramming sample methylomes (light blue to dark blue, data from , and our novel Sendai reprogramming dataset). Reference samples formed a reprogramming trajectory along PC1. Transient reprogramming samples moved along this trajectory with continued exposure to doxycycline (light magenta points) and returned to the beginning of the trajectory after withdrawal of doxycycline (magenta points). Control samples (yellow and orange points) remained at the beginning of the trajectory throughout the experiment. (G) Mean DNA methylation levels across the fibroblast specific gene FSP1 and the iPSC specific gene POU5F1 (encoding OCT4). Transiently reprogrammed samples had methylation profiles across these genes that resemble those found in fibroblasts. Grey bars and black bars indicate the locations of Ensembl annotated promoters and genes, respectively. Samples transiently reprogrammed for 10, 13, 15 or 17 days were pooled for visualisation purposes. The number of samples in each group is indicated in brackets.

Article Snippet: Viral particles were generated by transfecting HEK293T cells with the packaging plasmids pMD2.G (Addgene 12259) and psPAX2 (Addgene 12260) and either FUW-tetO-GFP-hOKMS or FUW-M2rtTA.

Techniques: DNA Methylation Assay, Transduction, Plasmid Preparation, Expressing, Standard Deviation, Derivative Assay, Construct, Infection, Negative Control, Cell Culture, Microscopy, Methylation

Journal: bioRxiv

Article Title: Multi-omic rejuvenation of human cells by maturation phase transient reprogramming

doi: 10.1101/2021.01.15.426786

Figure Lengend Snippet: (A) Mean DNA methylation age (calculated using the skin and blood clock ) throughout the reprogramming process where cells were transduced with our tetO-GFP-hOKMS vector and treated continuously with 2 µg/mL of doxycycline. Reprogramming is divided into three distinct phases: initiation phase (IP); maturation phase (MP) and stabilisation phase (SP). DNA methylation age decreased substantially during the maturation phase of reprogramming in cells that were successfully reprogramming (magenta line) but not in control cells (yellow and orange lines represent non-transduced cells and cells expressing hOKMS but failing to reprogram as indicated by cell surface markers, respectively). Points represent the mean and error bars the standard deviation. N = 3 biological replicates per condition, where fibroblasts were derived from different donors. N = 2 biological replicates for the iPSC timepoint (day 51). (B) The percentage of cells measured in each quadrant during the flow sort for successfully reprogramming cells (top panel). Cells were classified as CD13 only (CD13+ SSEA4-), double negative (“DN”, CD13-SSEA4-), double positive (“DP”, CD13+ SSEA4+) or SSEA4 only (CD13-SSEA4+). Cells that were collected are colour coded (light yellow = negative control intermediate, light orange = failed to transiently reprogram intermediate, light magenta = transient reprogramming intermediate). Bars represent the mean and error bars the standard deviation. Representative flow cytometry plots (bottom panel) show where gates were placed for determining presence/absence of surface markers.

Article Snippet: Viral particles were generated by transfecting HEK293T cells with the packaging plasmids pMD2.G (Addgene 12259) and psPAX2 (Addgene 12260) and either FUW-tetO-GFP-hOKMS or FUW-M2rtTA.

Techniques: DNA Methylation Assay, Transduction, Plasmid Preparation, Expressing, Standard Deviation, Derivative Assay, Negative Control, Flow Cytometry

Journal: bioRxiv

Article Title: Multi-omic rejuvenation of human cells by maturation phase transient reprogramming

doi: 10.1101/2021.01.15.426786

Figure Lengend Snippet: (A) Mean DNA methylation age of samples after transient reprogramming calculated using skin and blood clock . DNA methylation age substantially reduced after 13 days of transient reprogramming. Shorter and longer lengths of transient reprogramming led to smaller reductions in DNA methylation age. Bars represent the mean and error bars represent the standard deviation. Significance was calculated with the Mann-Whitney U test. The number of samples in each group is indicated in brackets. (B) Mean telomere length (calculated using the telomere length clock ) throughout the reprogramming process where cells were transduced with our tetO-GFP-hOKMS vector and treated continuously with 2 µg/mL of doxycycline. Reprogramming is divided into three distinct phases: initiation phase (IP); maturation phase (MP) and stabilisation phase (SP). Telomere length decreases during the initiation and maturation phases and begins to increase during the stabilisation phase. Points represent the mean and error bars the standard deviation. N = 3 biological replicates per condition, where fibroblasts were derived from different donors. N = 2 biological replicates for the iPSC timepoint (day 51). (C) The location of rejuvenated CpG sites after 13 days of transient reprogramming. Sites rejuvenated by demethylation are coloured in blue and sites rejuvenated by methylation are coloured in pink.

Article Snippet: Viral particles were generated by transfecting HEK293T cells with the packaging plasmids pMD2.G (Addgene 12259) and psPAX2 (Addgene 12260) and either FUW-tetO-GFP-hOKMS or FUW-M2rtTA.

Techniques: DNA Methylation Assay, Standard Deviation, MANN-WHITNEY, Transduction, Plasmid Preparation, Derivative Assay, Methylation