Journal: Journal of Translational Medicine

Article Title: Stage-specific requirement for METTL3-dependent m 6 A modification during dental pulp stem cell differentiation

doi: 10.1186/s12967-022-03814-9

Figure Lengend Snippet: METTL3 is required for functional DPSC differentiation. A Two independent shRNA lentiviruses were used to inhibit METTL3 expression, as confirmed by qPCR. B Western blotting detected the protein expression of METTL3 in DPSCs after METTL3 shRNA treatment (n = 3). C qPCR analysis of the mRNA expression of ALP , RUNX2 and DSPP in differentiated DPSCs (n = 3). D The protein expression levels of METTL3, RUNX2 and DSPP in METTL3 knockdown DPSCs under odontogenic induction were evaluated by western blotting. E The mRNA expression level of METTL3 in DPSCs after adipogenic induction for 0, 3, 7, and 14 days. F The mRNA expression of PPAR and LPL after adipogenic induction as detected by qPCR (n = 3). G The odontogenic differentiation of shMETTL3-1 and shCTR-1 lentivirus-transduced DPSCs was evaluated by ALP and ARS staining (n = 5). H The differentiation capacity of shMETTL3-2- and shCTR-2-transduced DPSCs after induction. I Representative images of Oil Red O staining in METTL3 knockdown DPSCs. OM-DPSCs: DPSCs induced by odontogenic medium. Significance was determined via ANOVA or Student’s t test; the data are presented as the mean ± SD (n ≥ 3). * p < 0.05. ** p < 0.01. *** p < 0.001

Article Snippet: Two independent METTL3 shRNA sequences (shMETTL3-1 and shMETTL3-2) within lentiviral vectors were used. shMETTL3-1 lentivirus encoding METTL3-shRNA and shCTR-1 lentivirus encoding scrambled control (shCTR) sequences were constructed by GeneChem Company (Shanghai, China); shMETTL3-2 and shCTR-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, US). cDNA encoding the full-length METTL3 gene was amplified and used to construct a lentiviral vector for METTL3 overexpression (LV-METTL3) by GeneChem Company.

Techniques: Functional Assay, shRNA, Expressing, Western Blot, Staining

Journal: Cancers

Article Title: Estrogen Related Receptor Alpha (ERRα) a Bridge between Metabolism and Adrenocortical Cancer Progression

doi: 10.3390/cancers14163885

Figure Lengend Snippet: Metabolic changes in H295R cells related to ERRα expression levels. The metabolic profiles of H295R wild type (WT), shCTR, shERRα−/− and ERRα+/+ cells were assessed by Seahorse XFe96 Analyzer. ( a , b ) ATP Rate Assay was evaluated as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of Total ATP Production Rate (pmol/min) ( a ) and ATP production (%) ( b ) deriving from glycolysis and oxidative phosphorylation after the sequential addition of specific inhibitors; (* p < 0.05 vs. WT). ( c – e ) Mitochondrial Stress Analysis was performed as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of real-time oxygen consumption (OCR) rate (pmol/min/cells); (* p < 0.05 vs. shCTR). Mitochondrial Respiration ( c ), Basal Respiration ( d ), Maximal Respiration ( e ) were measured from OCR after the addition of specific inhibitors. ( f – h ) Glycolytic Stress Analysis was performed as indicated in “Materials and Methods”. Graph represents the mean ± SD of three independent experiments of Real-time extracellular acidification (ECAR) rate (mpH/min/cells); (* p < 0.05 vs. shCTR). Glycolitic function ( f ), Glycolysis ( g ) and Glycolytic Capacity ( h ) were measured from ECAR after the addition of specific inhibitors.

Article Snippet: After 48 h, cells were transfected with DNA plasmids: a plasmid encoding a scrambled short hairpin RNAs (shRNA) sequence (shCTR) (Santa Cruz, sc108060, Dallas, TX, USA), a plasmid expressing a shRNA to inhibit ERRα gene (shERRα−/−) (Santa Cruz, sc44706-sh), according to the manufacturer’s protocol (Santa Cruz Biotecnology, https://www.scbt.com/it/resources/protocols/shrna-plasmid-dna-mediated-inhibition-of-gene-expression (accessed on 7 January 2020)), or ERRα cDNA expression plasmid(ERRα+/+) (ADGENE, #10975), according to X-tremeGENETM HP DNA Transfection Reagent Protocol (Sigma).

Techniques: Expressing, Phospho-proteomics

Journal: Cancers

Article Title: Estrogen Related Receptor Alpha (ERRα) a Bridge between Metabolism and Adrenocortical Cancer Progression

doi: 10.3390/cancers14163885

Figure Lengend Snippet: ERRα modulates H295R cell motility and Vimentin expression. ( a , b ) H295R (WT), H295R clones, knock in (ERRα+/+) or knock out (shERRα−/−) for ERRα gene, and H295R cell stably transfected with control plasmid (shCTR) were used in Wound Healing ( a ) and Boyden Chamber ( b ) assays as reported in “Materials and Methods”. Images are from a representative experiment. ( c , d ) H295R cells were treated with vehicle (0) or XCT790 (1, 5, 10 μM) for 18 h and Wound Healing ( c ) and Boyden Chamber ( d ) assays were performed as reported in “Materials and Methods”. Images are from a representative experiment. ( c ) The wounds were observed under an inverted microscope immediately (0 h) and 18 h after the scratch (100× magnification). ( b , d ) Migrated cells were photographed under an inverted microscope and counted (see Material and Methods), 20× magnification. Graphs represent the mean ± SD of three independent experiments. The number of untreated cells (0) was set as 100% (* p < 0.05 vs. 0). ( e ) Total proteins from H295R clones (shCTR, shERRα−/−, ERRα+/+) were analyzed by western Blotting (WB) using antibodies against ERRα and Vimentin. GAPDH was used as a loading control. Blots are representative of three independent experiments with similar results. ( f ) H295R were transfected for 48 h with pcDNA3.1 non containing (EV) or containing ERRα coding sequence (pcDNA3.1-ERRα). After transfection cells were left untreated (−) or treated (+) for 24 h with XCT790 (10 μM). Total proteins were analyzed by WB using antibodies against ERRα and Vimentin. GAPDH was used as a loading control. ( g ) Cells were untreated (0) or treated with XCT790 (1, 5, 10 μM) for 24 h. Total proteins were analyzed by WB using antibodies against ERRα and Vimentin. GAPDH was used as a loading control. Original image of western blot can be found at .

Article Snippet: After 48 h, cells were transfected with DNA plasmids: a plasmid encoding a scrambled short hairpin RNAs (shRNA) sequence (shCTR) (Santa Cruz, sc108060, Dallas, TX, USA), a plasmid expressing a shRNA to inhibit ERRα gene (shERRα−/−) (Santa Cruz, sc44706-sh), according to the manufacturer’s protocol (Santa Cruz Biotecnology, https://www.scbt.com/it/resources/protocols/shrna-plasmid-dna-mediated-inhibition-of-gene-expression (accessed on 7 January 2020)), or ERRα cDNA expression plasmid(ERRα+/+) (ADGENE, #10975), according to X-tremeGENETM HP DNA Transfection Reagent Protocol (Sigma).

Techniques: Expressing, Clone Assay, Knock-In, Knock-Out, Stable Transfection, Transfection, Control, Plasmid Preparation, Inverted Microscopy, Western Blot, Sequencing

Journal: Cancers

Article Title: Estrogen Related Receptor Alpha (ERRα) a Bridge between Metabolism and Adrenocortical Cancer Progression

doi: 10.3390/cancers14163885

Figure Lengend Snippet: ERRα promotes H295R spheroids formation. ( a ) H295R were transfected for 48 h with pcDNA3.1 non containing (EV) or containing ERRα coding sequence (pcDNA3.1-ERRα) and then grown as 3D spheroids for 5 days. Spheroids were counted under an inverted microscope and results were expressed as fold change over control (EV) ± SD (TSFE, tumor spheroids formation efficiency); (* p < 0.05 vs. EV). Insert confirms ERRα overexpression. ( b ) H295R cells were left untreated (0) or treated with XCT790 (1, 5, 10 μM) for 24 h and TSFE was evaluated 5 days later (* p < 0.05 vs. 0). Images below graph are from a representative experiment (20× magnification). ( c ) Wild type H295R (WT) and H295R clones (shCTR, shERRα−/−, ERRα+/+) were used to evaluate 3D spheroids formation. TSFE was evaluated 5 days later (* p < 0.05 vs. WT). Images below graph are from a representative experiment (20× magnification). ( d ) H295R spheroids (H295R Sph-5) were allowed to grow for 5 days and then trypsinized and reseeded weekly in spheroid media for 5 weeks. Boyden Chamber Assay was performed as reported in the “Materials and Methods”. Migrated cells were randomly photographed and counted with ImageJ software (* p < 0.05 vs. WT). ( e ) H295R (WT) cells and H295R grown as spheroids for 5 weeks (H295R Sph-5), were analyzed by WB using antibody against Vimentin. GAPDH was used as a loading control. Blots are representative of three independent experiments with similar results. Original image of western blot can be found at .

Article Snippet: After 48 h, cells were transfected with DNA plasmids: a plasmid encoding a scrambled short hairpin RNAs (shRNA) sequence (shCTR) (Santa Cruz, sc108060, Dallas, TX, USA), a plasmid expressing a shRNA to inhibit ERRα gene (shERRα−/−) (Santa Cruz, sc44706-sh), according to the manufacturer’s protocol (Santa Cruz Biotecnology, https://www.scbt.com/it/resources/protocols/shrna-plasmid-dna-mediated-inhibition-of-gene-expression (accessed on 7 January 2020)), or ERRα cDNA expression plasmid(ERRα+/+) (ADGENE, #10975), according to X-tremeGENETM HP DNA Transfection Reagent Protocol (Sigma).

Techniques: Transfection, Sequencing, Inverted Microscopy, Control, Over Expression, Clone Assay, Boyden Chamber Assay, Software, Western Blot

Journal: Cancers

Article Title: Estrogen Related Receptor Alpha (ERRα) a Bridge between Metabolism and Adrenocortical Cancer Progression

doi: 10.3390/cancers14163885

Figure Lengend Snippet: ERRα expression levels and cholesterol influence H295R cell migration. ( a ) H295R clones (shCTR, ERRα+/+, shERRα−/−) were maintained in 5% FBS or 5% LpFS containing medium. Cells were used in Wound Healing ( a ) and Boyden Chamber ( b ) assays performed as reported in “Materials and Methods”. ( a ) Images are from a representative experiment (100× magnification). ( b ) Migrated cells were photographed under an inverted microscope (20× magnification) and counted with ImageJ software. Graphs represent the mean ± SD of three independent experiments (* p < 0.05 vs. FBS).

Article Snippet: After 48 h, cells were transfected with DNA plasmids: a plasmid encoding a scrambled short hairpin RNAs (shRNA) sequence (shCTR) (Santa Cruz, sc108060, Dallas, TX, USA), a plasmid expressing a shRNA to inhibit ERRα gene (shERRα−/−) (Santa Cruz, sc44706-sh), according to the manufacturer’s protocol (Santa Cruz Biotecnology, https://www.scbt.com/it/resources/protocols/shrna-plasmid-dna-mediated-inhibition-of-gene-expression (accessed on 7 January 2020)), or ERRα cDNA expression plasmid(ERRα+/+) (ADGENE, #10975), according to X-tremeGENETM HP DNA Transfection Reagent Protocol (Sigma).

Techniques: Expressing, Migration, Clone Assay, Inverted Microscopy, Software

Journal: Journal of Cancer

Article Title: PHLDA1 Modulates the Endoplasmic Reticulum Stress Response and is required for Resistance to Oxidative Stress-induced Cell Death in Human Ovarian Cancer Cells.

doi: 10.7150/jca.45262

Figure Lengend Snippet: Figure 2. H2O2-induced apoptosis of ovarian cancer cells after downregulation of PHLDA1. (A and B) Flow cytometric analysis of 2008 cells (A) and SKOV3 cells (B) expressing control (shctrl) or PHLDA1-targeting shRNAs (shPHLDA1). Lower right and upper right quadrants showed early apoptotic and late apoptotic/necrotic cells, respectively. n=3, *P<0.05, **P<0.001, compared with the shctrl group. (C and D) Western blot analysis of the apoptosis marker c-PARP1 in 2008 (C) and SKOV3 (D) cell lines expressing shctrl or shPHLDA1.

Article Snippet: Cells were infected by lentiviral vectors pLKO.1 encoding scrambled shctrl (Addgene, Cambridge, MA, USA) or one of the three PHLDA1-targeting shRNAs (all from Sigma, St. Louis, MO, USA): shPHLDA1-1 (CCG GCC TAA TCC GTA GTA ATT

Techniques: Expressing, Control, Western Blot, Marker

Journal: Journal of Cancer

Article Title: PHLDA1 Modulates the Endoplasmic Reticulum Stress Response and is required for Resistance to Oxidative Stress-induced Cell Death in Human Ovarian Cancer Cells.

doi: 10.7150/jca.45262

Figure Lengend Snippet: Figure 3. ER stress-induced apoptosis of ovarian cancer cells after downregulation of PHLDA1. Apoptosis assay of 2008 (A) and SKOV3 (B) cells expressing shctrl or shPHLDA1 after treatment with thapsigargin (Tg). **P<0.001 compared with the shctrl group. Mean ± SD, n = 3.

Article Snippet: Cells were infected by lentiviral vectors pLKO.1 encoding scrambled shctrl (Addgene, Cambridge, MA, USA) or one of the three PHLDA1-targeting shRNAs (all from Sigma, St. Louis, MO, USA): shPHLDA1-1 (CCG GCC TAA TCC GTA GTA ATT

Techniques: Apoptosis Assay, Expressing

Journal: Journal of Cancer

Article Title: PHLDA1 Modulates the Endoplasmic Reticulum Stress Response and is required for Resistance to Oxidative Stress-induced Cell Death in Human Ovarian Cancer Cells.

doi: 10.7150/jca.45262

Figure Lengend Snippet: Figure 5. Expression of autophagy-related proteins, anti-apoptosis proteins, ER stress-associated proteins after PHLDA1-downregulation in ovarian cancer cells. (A) Western blot analysis of Autophagy-related proteins (Beclin-1, P62, and LC3) and anti-apoptosis protein (Bcl-2) in 2008 cells expressing shctrl or shPHLDA1 after treatment with 0.2 mM H2O2. (B and C) Western blot analysis of ER stress-associated proteins (IRE1α, PERK, BIP, ERO1-Lα, and PDI) in 2008 (B) and SKOV3 (C) cells after treatment with 0.2 mM H2O2.

Article Snippet: Cells were infected by lentiviral vectors pLKO.1 encoding scrambled shctrl (Addgene, Cambridge, MA, USA) or one of the three PHLDA1-targeting shRNAs (all from Sigma, St. Louis, MO, USA): shPHLDA1-1 (CCG GCC TAA TCC GTA GTA ATT

Techniques: Expressing, Western Blot