Journal: Cell Death & Disease

Article Title: Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition

doi: 10.1038/cddis.2012.63

Figure Lengend Snippet: Knockdown of Twist1 enhances NSCLC cell sensitivity to cisplatin. ( a ) A549 and H1299 cells were treated with the indicated concentrations of cisplatin for 24 h. Cell viability was measured with the MTT assay. The viability of control cells was set at 100%, and survival relative to the control presented. ( b ) A549 and H1299 cells were transfected with two different Twist1 or control siRNAs for 30 h. Twist1 mRNA levels were measured using RT-PCR analysis. ( c and d ) A549 and H1299 cells were transfected with siCTL or siTwist1 for 20 h, followed by 8 (A549 cells) or 6 μ M (H1299 cells) cisplatin for 24 h. Cell viability was measured with the MTT assay ( c ) and protein levels determined using western blot analysis ( d ). Data shown are representative of three independent experiments. All graphs are shown as the mean of each group and error bars represent S.D. ** P <0.01 versus the siCTL/cisplatin-treated group or siTwist1-treated group

Article Snippet: Twist1 (no. 1, sc-38604), Mcl-1 (sc-35877), AMPK α 1/2 (sc-45312), p21 (sc-29427) and control (no. 1, sc-37007) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Twist1 (no. 2, s14523) and control (no. 2, 4611) siRNAs from Ambion (Austin, TX, USA), S6K1 (Hs_RPS6KB1_5) and control (1022076) siRNAs from Qiagen (Valencia, CA, USA) and mTOR (6381) and control (6568) siRNAs from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown, MTT Assay, Control, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Cell Death & Disease

Article Title: Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition

doi: 10.1038/cddis.2012.63

Figure Lengend Snippet: Knockdown of Twist1 induces a decrease in Mcl-1 protein expression. ( a ) H1299 cells were transfected with control or Twist1 siRNA for 24 h. Cells were reseeded at a density of 2000 cells per well in three 60-mm plates. Colony formation was monitored over the following 10 days. Colonies were stained using the Diff-Quick kit. ( b and c ) A549 and H1299 cells were transfected with control or Twist1 siRNA for 30 h. The indicated protein and mRNA levels were measured using western blot ( b and c ) and RT-PCR ( c ) analyses. Data shown are representative of at least two independent experiments

Article Snippet: Twist1 (no. 1, sc-38604), Mcl-1 (sc-35877), AMPK α 1/2 (sc-45312), p21 (sc-29427) and control (no. 1, sc-37007) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Twist1 (no. 2, s14523) and control (no. 2, 4611) siRNAs from Ambion (Austin, TX, USA), S6K1 (Hs_RPS6KB1_5) and control (1022076) siRNAs from Qiagen (Valencia, CA, USA) and mTOR (6381) and control (6568) siRNAs from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown, Expressing, Transfection, Control, Staining, Diff-Quik, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition

doi: 10.1038/cddis.2012.63

Figure Lengend Snippet: Mcl-1 is essential for NSCLC survival. ( a and b ) A549 and H1299 cells were transfected with control or Mcl-1 siRNA for 48 h. Cell morphology images were obtained under a microscope ( b ). ( c ) H1299 cells were transfected with control or Mcl-1 siRNA for 24 h. Cells were reseeded at a density of 2000 (1 × ) and 3000 (1.5 × ) cells per well in three 60-mm plates. Colony formation was monitored over the following 10 days. Colonies were stained using the Diff-Quick kit. ( d ) H1299 cells were transfected with plasmids for empty vector control (pcDNA3) or His-tagged Mcl-1 (His-Mcl-1) or control or Twist1 siRNAs for 24 h, followed by treatment with 5 μ M cisplatin for 24 h. The indicated protein levels were measured using western blot analysis ( a and d ). In all panels, representative of three independent experiments is shown

Article Snippet: Twist1 (no. 1, sc-38604), Mcl-1 (sc-35877), AMPK α 1/2 (sc-45312), p21 (sc-29427) and control (no. 1, sc-37007) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Twist1 (no. 2, s14523) and control (no. 2, 4611) siRNAs from Ambion (Austin, TX, USA), S6K1 (Hs_RPS6KB1_5) and control (1022076) siRNAs from Qiagen (Valencia, CA, USA) and mTOR (6381) and control (6568) siRNAs from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Control, Microscopy, Staining, Diff-Quik, Plasmid Preparation, Western Blot

Journal: Cell Death & Disease

Article Title: Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition

doi: 10.1038/cddis.2012.63

Figure Lengend Snippet: Knockdown of Twist1 knockdown inhibits mTOR-mediated Mcl-1 downregulation. ( a ) H1299 cells were transfected with two different Twist1 or control siRNAs for 30 h. ( b ) H1299 cells were transfected with control or mTOR siRNA for 30 h. ( c ) H1299 cells were transfected with control or mTOR siRNA for 20 h, followed by treatment with 5 μ M cisplatin for 24 h. Cell viability was measured with the MTT assay ( c ). The viability of control siRNA (siCTL)-treated group was set at 100%, and survival relative to the siCTL presented. The indicated protein levels were measured using western blot analysis ( a – c ). The blot shown is representative of two independent experiments. Cell viability data are shown as the mean of triplicate samples, and error bars reflect S.D. * P <0.05 versus the siCTL/cisplatin-treated group or simTOR-treated group

Article Snippet: Twist1 (no. 1, sc-38604), Mcl-1 (sc-35877), AMPK α 1/2 (sc-45312), p21 (sc-29427) and control (no. 1, sc-37007) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Twist1 (no. 2, s14523) and control (no. 2, 4611) siRNAs from Ambion (Austin, TX, USA), S6K1 (Hs_RPS6KB1_5) and control (1022076) siRNAs from Qiagen (Valencia, CA, USA) and mTOR (6381) and control (6568) siRNAs from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown, Transfection, Control, MTT Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition

doi: 10.1038/cddis.2012.63

Figure Lengend Snippet: Twist1 knockdown-induced Mcl-1 decrease is mediated by S6K1 downregulation. ( a ) H1299 cells were transfected with control or S6K1 siRNA for 30 h. ( b and c ) H1299 cells were transfected with control or S6K1 siRNA for 20 h, followed by treatment with 5 μ M cisplatin for 24 h. Cell viability of control siRNA (siCTL)-treated group was set at 100%, and survival relative to the siCTL presented ( b ). ( d ) H1299 cells were transfected with increasing concentrations of Myc-tagged S6K1, along with Twist1 siRNA for 48 h. The indicated protein levels were measured using western blot analysis ( a , c and d ). Data shown are representative of at least two independent experiments. Cell viability data are shown as the mean of each group and error bars reflect S.D. * P <0.05 versus the siCTL/cisplatin-treated group or siS6K1-treated group

Article Snippet: Twist1 (no. 1, sc-38604), Mcl-1 (sc-35877), AMPK α 1/2 (sc-45312), p21 (sc-29427) and control (no. 1, sc-37007) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Twist1 (no. 2, s14523) and control (no. 2, 4611) siRNAs from Ambion (Austin, TX, USA), S6K1 (Hs_RPS6KB1_5) and control (1022076) siRNAs from Qiagen (Valencia, CA, USA) and mTOR (6381) and control (6568) siRNAs from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown, Transfection, Control, Western Blot

Journal: Cell Death & Disease

Article Title: Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition

doi: 10.1038/cddis.2012.63

Figure Lengend Snippet: Knockdown of Twist1 induces mTOR activity inhibition through AMPK. ( a and b ) H1299 and A549 cells were transfected with control, Twist1 or Mcl-1 siRNA for 48 h. ADP/ATP ratios were determined using the Enzylight ADP/ATP ratio assay kit ( a ). Data are expressed as mean of three independent experiments values and error bars reflect S.D. ( c ) H1299 cells were transfected with control, Twist1 or AMPK α 1/2 siRNA for 48 h. The indicated protein levels were measured using western blot analysis ( b and c ) and blots shown are representative of two independent experiments

Article Snippet: Twist1 (no. 1, sc-38604), Mcl-1 (sc-35877), AMPK α 1/2 (sc-45312), p21 (sc-29427) and control (no. 1, sc-37007) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Twist1 (no. 2, s14523) and control (no. 2, 4611) siRNAs from Ambion (Austin, TX, USA), S6K1 (Hs_RPS6KB1_5) and control (1022076) siRNAs from Qiagen (Valencia, CA, USA) and mTOR (6381) and control (6568) siRNAs from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown, Activity Assay, Inhibition, Transfection, Control, Western Blot

Journal: Cell Death & Disease

Article Title: Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition

doi: 10.1038/cddis.2012.63

Figure Lengend Snippet: Knockdown of p21 Waf1/CIP1 promotes the cell death response to cisplatin and Twist1 siRNA. ( a and b ) A549 and H1299 cells were treated with the indicated concentrations of cisplatin for 24 h. ( c ) A549 and H1299 cells were transfected with control or Twist1 siRNA for 48 h. ( d ) A549 cells were transfected with control, Twist1 or p21 Waf1/CIP1 siRNA for 24 h, followed by treatment with 6 μ M cisplatin for 24 h. The indicated protein and mRNA levels were measured using western blot ( a , c and d ) and RT-PCR ( b and c ) analyses. Cell viability was measured with the MTT assay ( d ). Cell viability of control siRNA (siCTL)-treated group was set at 100%, and survival relative to the siCTL presented. The graph is shown as the mean of each group and error bars reflect S.D. * P <0.05 versus the siTwist1/cisplatin-treated group

Article Snippet: Twist1 (no. 1, sc-38604), Mcl-1 (sc-35877), AMPK α 1/2 (sc-45312), p21 (sc-29427) and control (no. 1, sc-37007) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Twist1 (no. 2, s14523) and control (no. 2, 4611) siRNAs from Ambion (Austin, TX, USA), S6K1 (Hs_RPS6KB1_5) and control (1022076) siRNAs from Qiagen (Valencia, CA, USA) and mTOR (6381) and control (6568) siRNAs from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown, Transfection, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, MTT Assay

Journal: Frontiers in Physiology

Article Title: Twist1 -Haploinsufficiency Selectively Enhances the Osteoskeletal Capacity of Mesoderm-Derived Parietal Bone Through Downregulation of Fgf23

doi: 10.3389/fphys.2018.01426

Figure Lengend Snippet: Differential postnatal expression of Twist1 in frontal and parietal bones. Time-course RT-qPCR analysis performed on frontal and parietal bone tissues ( n = 10) revealed a differential expression of Twist1 with higher levels in parietal bone. The relative mRNA level in each sample is normalized to housekeeping gene Gapdh . Values are presented as relative to Gapdh expression. Values: ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001.

Article Snippet: Cells were transduced with either Fgf23 shRNA lentiviral particles (sc-39487-V) or Twist1 shRNA lentiviral particles (sc-38605-V) Santa Cruz Biotechnology, Santa Cruz, CA, United States) and shRNA (scramble) lentiviral particles-A (sc-108080) was used as control (Santa Cruz Biotechnology, Santa Cruz, CA, United States).

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics

Journal: Frontiers in Physiology

Article Title: Twist1 -Haploinsufficiency Selectively Enhances the Osteoskeletal Capacity of Mesoderm-Derived Parietal Bone Through Downregulation of Fgf23

doi: 10.3389/fphys.2018.01426

Figure Lengend Snippet: Twist1 haploinsufficiency enhances the osteoskeletal potential of mesoderm-derived parietal bone. (A) FOb and POb cultured under osteogenic conditions for 28 days and stained with Alizarin Red. (B) Quantification of Alizarin Red staining revealing a more intense mineralization of the extracellular matrix in Twist1 +/− POb as compared to wild-type POb. (C) Number of large bone nodules formation identified by von-Kossa staining at osteogenic differentiation day 28. (D) Relative mRNA expression of early ( Osx ) and late ( Bglap ) osteogenic markers in wild-type and Twist1 +/− osteoblasts cultures. Results are representative of three experiments performed. (E) Time course of frontal and parietal bone healing defects, quantification of defect repair ( n ? = ?5/each bone) based on μCT results are analyzed using magic wand tool in Photoshop after standardization of threshold for regenerating calvarial bone. Results are representative of two independent experiments performed. Statistical analysis was performed using the Mann–Whitney Test. ∗ P ≤ 0.05. (F) Quantification of bone thickness in uninjured wild type and Twist1 +/− Frontal and Parietal bones ∗ P ≤ 0.05. (G) Bone formation in wild-type and Twist1 +/− frontal and parietal bones assessed by culturing skulls for 48 h in presence of calcein (1 μg/ml). (H) Quantification of calcein incorporation. Histomorphometric analysis of calcein accumulation was obtained using Image J64 densitometry analysis. Scale bar, 10 μm. Results are representative of three independent experiments performed. Values: ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001.

Article Snippet: Cells were transduced with either Fgf23 shRNA lentiviral particles (sc-39487-V) or Twist1 shRNA lentiviral particles (sc-38605-V) Santa Cruz Biotechnology, Santa Cruz, CA, United States) and shRNA (scramble) lentiviral particles-A (sc-108080) was used as control (Santa Cruz Biotechnology, Santa Cruz, CA, United States).

Techniques: Derivative Assay, Cell Culture, Staining, Expressing, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: Twist1 -Haploinsufficiency Selectively Enhances the Osteoskeletal Capacity of Mesoderm-Derived Parietal Bone Through Downregulation of Fgf23

doi: 10.3389/fphys.2018.01426

Figure Lengend Snippet: Transcriptomic profiles of wild-type and Twist +/− frontal and parietal bones. RNA-Seq analysis to assess gene expression changes caused by Twist1 haploinsufficiency in calvarial bone of pN21 mice revealed significant differences. (A) Venn diagram showing the comparison of top genes found differentially expressed (up and down regulated) in wild-type and Twist +/− Frontal (F) and Parietal (P) bones for FDR < 0.1 and log 2 (fold change) >3, with fold change as ratios of gene-level FPKM values. Their gene expression levels are affected ≥3 fold. (B) Scatterplot representation showing the fold change Scatter plots of gene expression of genes [based on log 2 (FPKM + 1) expression values with each dot representing a gene]. In the bottom of each graph the pair-wise Pearson’s correlation (r) for all genes is shown. (C) Heatmap showing Fgf23 as a component of a unique signature of genes that are exclusively upregulated in wild-type parietal bones. (upregulation in Red; downregulation in Green).

Article Snippet: Cells were transduced with either Fgf23 shRNA lentiviral particles (sc-39487-V) or Twist1 shRNA lentiviral particles (sc-38605-V) Santa Cruz Biotechnology, Santa Cruz, CA, United States) and shRNA (scramble) lentiviral particles-A (sc-108080) was used as control (Santa Cruz Biotechnology, Santa Cruz, CA, United States).

Techniques: RNA Sequencing, Gene Expression, Comparison, Expressing

Journal: Frontiers in Physiology

Article Title: Twist1 -Haploinsufficiency Selectively Enhances the Osteoskeletal Capacity of Mesoderm-Derived Parietal Bone Through Downregulation of Fgf23

doi: 10.3389/fphys.2018.01426

Figure Lengend Snippet: Validation of Fgf23 in calvarial bones and derived osteoblasts. (A) Validation of Fgf23 downregulation obtained by performing PCR analysis on bone tissues. (B) RT-qPCR analysis showing Fgf23 down-regulation in osteoblast primary cultures (depleted of pericranium and dura mater- derived cells). (C) ELISA assay performed on media collected from osteoblast cells and concentrated by 20-fold. Elevated level of secreted FGF23 protein is detected in the medium of wild-type POb, whereas in Twist +/− POb protein levels are sharply decreased to values similar to that of wild-type and Twist +/− FOb. Value: ∗∗∗ p ≤ 0.001.

Article Snippet: Cells were transduced with either Fgf23 shRNA lentiviral particles (sc-39487-V) or Twist1 shRNA lentiviral particles (sc-38605-V) Santa Cruz Biotechnology, Santa Cruz, CA, United States) and shRNA (scramble) lentiviral particles-A (sc-108080) was used as control (Santa Cruz Biotechnology, Santa Cruz, CA, United States).

Techniques: Biomarker Discovery, Derivative Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Physiology

Article Title: Twist1 -Haploinsufficiency Selectively Enhances the Osteoskeletal Capacity of Mesoderm-Derived Parietal Bone Through Downregulation of Fgf23

doi: 10.3389/fphys.2018.01426

Figure Lengend Snippet: Inhibition of osteogenesis and bone mineralization by exogenous FGF23 protein. (A) Treatment with FGF23 protein (100 ng/ml) suppresses markedly osteogenic differentiation of Twist +/− POb as revealed by Alizarin red staining performed at differentiation day 28. (B) Quantification of Alizarin red staining for the osteogenic assay shown in panel A (C) RT-qPCR analysis of the osteogenic marker osteocalcin ( Bglap ) confirms that FGF23 treatment abrogates the enhanced osteogenic of Twist1 +/− POb. (D) Validation of effective Fgf23 silencing monitored by RT-qPCR (left panel) and immunoblotting analysis (right panel). (E) Fgf23 silencing enhances the osteogenic capacity of wild-type POb mirroring that of Twist1 +/− POb as revealed by Alizarin red staining. (F) Quantification of Alizarin red staining for the osteogenic assay shown in panel E . (G) osteocalcin ( Bglap ) expression by RT-qPCR analysis confirms increased terminal differentiation in sh Fgf23 POb compared to scramble POb. (H) Exogenous added FGF23 also decreases dramatically bone mineralization in parietal bone organ culture. Calcein was added to the cultures for 48 h. Immunofluorescence showing calcein accumulation into the bone. (I) Histomorphometric analysis of calcein accumulation. Scale bar, 10 μm. Results are representative of three independent experiments performed. Values: ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001.

Article Snippet: Cells were transduced with either Fgf23 shRNA lentiviral particles (sc-39487-V) or Twist1 shRNA lentiviral particles (sc-38605-V) Santa Cruz Biotechnology, Santa Cruz, CA, United States) and shRNA (scramble) lentiviral particles-A (sc-108080) was used as control (Santa Cruz Biotechnology, Santa Cruz, CA, United States).

Techniques: Inhibition, Staining, Quantitative RT-PCR, Marker, Biomarker Discovery, Western Blot, Expressing, Organ Culture, Immunofluorescence

Journal: Frontiers in Physiology

Article Title: Twist1 -Haploinsufficiency Selectively Enhances the Osteoskeletal Capacity of Mesoderm-Derived Parietal Bone Through Downregulation of Fgf23

doi: 10.3389/fphys.2018.01426

Figure Lengend Snippet: Twist1 silencing phenocopies the osteoskeletal profile of Twist1 haploinsufficiency in POb by downregulating Fgf23 . (A) Osteogenic differentiation assay performed on sh Twist1 POb revealed a robust extracellular matrix mineralization. (B) Quantification of Alizarin red staining for the osteogenic assay shown in panel A . (C) RT-qPCR analysis showing significant upregulation of Bglap as compared to scramble POb. (D) RT-qPCR analysis performed on stable transduced cells upon puromicin selection reveals that sh-mediated Twist1 silencing triggers a significant downregulation of Fgf23 expression. (E) Immunoblotting analysis using anti-FGF23 antibody confirms decreased FGF23 at protein level in sh Twist1 POb compared to scramble POb. (F) Bulk RNA-seq analysis showing the effect of Twist1 haploinsufficiency on some of its target genes such Zeb , Snail1 , and Cdh1 and comparison in wild-type versus Twist1 haploinsufficiency parietal bones. (G) As assessed by RT-qPCR analysis a similar pattern is observed in stable transduced sh Twist1 POb and scramble POb upon puromicin selection and culturing them. Values: ∗ p ≤ 0.05, ∗∗ p ≤ 0.01.

Article Snippet: Cells were transduced with either Fgf23 shRNA lentiviral particles (sc-39487-V) or Twist1 shRNA lentiviral particles (sc-38605-V) Santa Cruz Biotechnology, Santa Cruz, CA, United States) and shRNA (scramble) lentiviral particles-A (sc-108080) was used as control (Santa Cruz Biotechnology, Santa Cruz, CA, United States).

Techniques: Differentiation Assay, Staining, Quantitative RT-PCR, Selection, Expressing, Western Blot, RNA Sequencing, Comparison

Journal: Frontiers in Physiology

Article Title: Twist1 -Haploinsufficiency Selectively Enhances the Osteoskeletal Capacity of Mesoderm-Derived Parietal Bone Through Downregulation of Fgf23

doi: 10.3389/fphys.2018.01426

Figure Lengend Snippet: Twist1 -haploinsufficiency- Fgf23 -downregulation axis. Schematic representation of the molecular mechanism mediating the effect of Twist1 haploinsufficiency in enhancing the osteoskeletal ability of mesoderm-derived parietal bone via downregulation of Fgf23.

Article Snippet: Cells were transduced with either Fgf23 shRNA lentiviral particles (sc-39487-V) or Twist1 shRNA lentiviral particles (sc-38605-V) Santa Cruz Biotechnology, Santa Cruz, CA, United States) and shRNA (scramble) lentiviral particles-A (sc-108080) was used as control (Santa Cruz Biotechnology, Santa Cruz, CA, United States).

Techniques: Derivative Assay