Journal: Neoplasia (New York, N.Y.)

Article Title: A Preclinical Study Combining the DNA Repair Inhibitor Dbait with Radiotherapy for the Treatment of Melanoma 1

doi: 10.1016/j.neo.2014.08.008

Figure Lengend Snippet: Effect of Dbait on H2AX phosphorylation. (A) SK28 and 501mel melanoma cells were transfected with an inactive control oligonucleotide or Dbait ± NU7026 (DNA-PK inhibitor). Immunofluorescence of γ-H2AX (red) and chromatin (DAPI; blue) was visualized. Dbait treatment led to non-localized pan-nuclear H2AX phosphorylation evidencing Dbait activity. This activity was dependent on DNA-PK activation. Bar, 50 μm. (B) SK28 melanoma cells were transfected with an inactive control oligonucleotide or Dbait. Immunofluorescence of γ-H2AX (red) and chromatin (DAPI; blue) was visualized immediately after irradiation and/or Dbait treatment. Irradiation alone resulted in localized γ-H2AX foci representing radio-induced DNA DSBs; Dbait treatment with or without irradiation led to non-localized pan-nuclear H2AX phosphorylation evidencing Dbait activity. Bar, 30 μm. (C) SK28 cells were transduced with lentiviruses that express either control, non-targeting shRNA, or shRNA targeting DNA-PKcs. After Dbait transfection, cells were immunostained with mouse monoclonal anti–DNA-PKcs or anti–γ-H2AX. Dbait activity was not detected in cells transduced with shRNA targeting DNA-PKcs. Bar, 50 μm.

Article Snippet: Subconfluent SK28 cells were transduced with lentiviruses that expressed either the control, non-targeting shRNA (shCTL; sc-108080; Santa Cruz Biotechnology, (Dallas, Texas, USA)), or shRNA targeting DNA-PKcs (shDNA-PK; sc-35200-V; Santa Cruz Biotechnology) at a multiplicity of infection of 3 using polybrene (5 μg/ml).

Techniques: Phospho-proteomics, Transfection, Control, Immunofluorescence, Activity Assay, Activation Assay, Irradiation, Transduction, shRNA

Journal: Molecular Therapy

Article Title: GTSE1-driven ZEB1 stabilization promotes pulmonary fibrosis through the epithelial-to-mesenchymal transition

doi: 10.1016/j.ymthe.2024.09.029

Figure Lengend Snippet: GTSE1 is upregulated in IPF patient tissues (A) Schematic view of comprehensive bioinformatic analyses using IPF patient cohorts (left panels). DEG lists of IPF tissues were obtained from the GEO database and annotated in volcano plots (middle panels); highlighted genes are significantly differentially expressed at a default adjusted p value cutoff (red = upregulated, blue = downregulated). DEG lists were subjected to GSEA to find the significantly enriched gene sets (right panels). FDR-q < 0.05 was used to set the significance threshold. NESs indicate that the genes in the annotated gene sets are enriched in IPF lung tissues (NES >0) or in normal control lung tissues (NES <0). (B) Genes commonly upregulated in fibrotic tissues from three independent IPF patient cohorts. (C) Genes commonly upregulated in IPF patient tissues and BLM-/IR-induced PF mouse models. (D) mRNA levels of GTSE1 in lungs from IPF patients and normal lungs from healthy controls. (E) Masson’s trichrome (MT) staining and IHC staining for GTSE1 in IPF patient tissues and normal lung tissues. Blue patches indicate collagen deposition. The yellow dotted line distinguishes between the zones with strong and weak blue color. (F) Correlation between GTSE1 protein levels and fibrosis severity in IPF patient tissues (normal: n = 24, IPF: n = 24). All graphs indicate the mean ± SEM. The fold change was calculated relative to the control. ∗, ∗∗, ∗∗∗, ∗∗∗∗ indicates p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively.

Article Snippet: Non-targeting control shRNA (shCTRL) lentiviral particles (#108080), GTSE1 shRNA lentiviral particles (#75216-V), and polybrene (#134220) were obtained from Santa Cruz Biotechnology.

Techniques: Control, Staining, Immunohistochemistry

Journal: Molecular Therapy

Article Title: GTSE1-driven ZEB1 stabilization promotes pulmonary fibrosis through the epithelial-to-mesenchymal transition

doi: 10.1016/j.ymthe.2024.09.029

Figure Lengend Snippet: GTSE1 is required for profibrotic phenotypic change in fibroblasts and epithelial cells (A) Western blots for analyzing FMT or (B) EMT markers and (C) IF staining for F-actin in cells transfected with shCTRL or sh GTSE1 with or without TGF-β treatment. Small values above each blot reflect the relative intensity of the target proteins normalized to the endogenous control, β-actin. (D and E) Cell migration and fibrotic remodeling of siRNA-transfected cells were assessed with and without TGF-β treatment using (D) wound-healing and (E) gel contraction assays after 24 or 48 h. Results are presented as a fold change of wound closure or gel contraction compared with the original volume. (F) EpCAM (green) was used to identify epithelial adhesion markers co-stained with GTSE1 (red) and α-SMA (violet). All graphs indicate the mean ± SEM ( n = 3/group). The fold change was calculated relative to the control. ∗, ∗∗, ∗∗∗, ∗∗∗∗ indicates p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively.

Article Snippet: Non-targeting control shRNA (shCTRL) lentiviral particles (#108080), GTSE1 shRNA lentiviral particles (#75216-V), and polybrene (#134220) were obtained from Santa Cruz Biotechnology.

Techniques: Western Blot, Staining, Transfection, Control, Migration

Journal: Molecular Therapy

Article Title: GTSE1-driven ZEB1 stabilization promotes pulmonary fibrosis through the epithelial-to-mesenchymal transition

doi: 10.1016/j.ymthe.2024.09.029

Figure Lengend Snippet: GTSE1 interacts with ZEB1, increasing its protein level Western blots showing the EMT-TFs in cells transfected with shCTRL or sh GTSE1 with and without (A) IR or (B) TGF-β treatment. Small values above each blot reflect the relative intensity of the target proteins normalized to β-actin. (C) Heatmaps show the protein levels of EMT-TFs in IR-treated L132 and TGF-β-treated HPF cells as a fold change from the naive cell control (left). The IP heatmap (right) shows the GTSE1-binding ratio of EMT-TF proteins as a fold change to the total protein levels. (D) Immunoblots showing the interaction between Flag-tagged GTSE1 and ZEB1 in HEK 293T cells (left) and the interaction between endogenous GTSE1and ZEB1 in L132 cells (right). (E) Immunofluorescence staining for GTSE1 and ZEB1 in L132 cells 24 h after TGF- β treatment. (F) PLA confirms endogenous GTSE1–ZEB1 interactions in cells transfected with shCTRL or sh GTSE1 and treated with TGF-β. Interactions with the target proteins are indicated as red dots. Cell nuclei were counterstained with DAPI. Representative fluorescence images (G) from BLM- or IR-induced PF mouse tissues and (H) from IPF patient tissues. (I) Correlation between GTSE1 and ZEB1 expression in IPF patients (Normal: n = 24, IPF: n = 24). All graphs indicate the mean ± SEM. The fold change was calculated relative to the control. ∗, ∗∗, ∗∗∗, ∗∗∗∗ indicates p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively.

Article Snippet: Non-targeting control shRNA (shCTRL) lentiviral particles (#108080), GTSE1 shRNA lentiviral particles (#75216-V), and polybrene (#134220) were obtained from Santa Cruz Biotechnology.

Techniques: Western Blot, Transfection, Control, Binding Assay, Immunofluorescence, Staining, Fluorescence, Expressing

Journal: Molecular Therapy

Article Title: GTSE1-driven ZEB1 stabilization promotes pulmonary fibrosis through the epithelial-to-mesenchymal transition

doi: 10.1016/j.ymthe.2024.09.029

Figure Lengend Snippet: Increase protein stability of ZEB1 mediates GTSE1-induced EMT (A) RT-PCR in cells transfected with shCTRL or sh GTSE1 with and without 8 Gy of IR. Small values above each blot reflect the relative intensity of the target mRNAs normalized to the endogenous control, gapdh . (B) Measurement of ZEB1 protein stability under a protein synthesis blockade from CHX. L132 cells were transfected with sh GTSE1 or Flag-GTSE1 and treated with 100 μg/mL CHX for various time periods. The graph shows the relative levels of ZEB1 protein as a fold change from its initial level. (C and D) The extent of ubiquitination of the ZEB1 protein was confirmed by immunoprecipitation and immunoblot analyses. (C) HEK 293T cells transfected with Flag-GTSE1 at 24 h. Cells were treated with 10 μM MG132 for 8 h. (D) siGTSE1 was co-transfected with HA-tagged ubiquitin in HEK 293T cells. (E) CDH1 promoter activity in HEK 293T cells was measured using a luciferase reporter assay after siGTSE1 transfection for 24 h. (F) ChIP assay was performed in GTSE1-deficient L132 cells. The chromatin was immunoprecipitated with an anti-ZEB1 antibody. Each precipitated DNA was analyzed by quantitative PCR using promoter-specific primers for CDH1 . (G) Immunoblots for EMT markers and (H) IF staining for F-actin in L132 cells co-transfected with Flag-tagged GTSE1 and si ZEB1 . (I) Cell migration of GTSE1-deficient L132 cells was assessed using a wound-healing assay after 24 and 48 h, with and without transfection with Flag-tagged ZEB1. Results are presented as a percentage of wound closure compared with the original volume. All graphs indicate the mean ± SEM. The fold change was calculated relative to the control. ∗, ∗∗, ∗∗∗, ∗∗∗∗ indicates p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively.

Article Snippet: Non-targeting control shRNA (shCTRL) lentiviral particles (#108080), GTSE1 shRNA lentiviral particles (#75216-V), and polybrene (#134220) were obtained from Santa Cruz Biotechnology.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Activity Assay, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Staining, Migration, Wound Healing Assay

Journal: Molecular Therapy

Article Title: GTSE1-driven ZEB1 stabilization promotes pulmonary fibrosis through the epithelial-to-mesenchymal transition

doi: 10.1016/j.ymthe.2024.09.029

Figure Lengend Snippet: GTSE1 binds to the non-phosphorylated form of ZEB1 and blocks its degradation (A) A diagram illustrating the ZEB1 mutants. The phospho-deficient mutant (S585A) replaces serine 585 with alanine, and the phosphor-mimetic mutant (S585E) replaces serine 585 with aspartic acid. (B) V5-tagged GTSE1 was co-transfected with the Flag-tagged ZEB1 mutant forms in HEK 293T and (C) L132 cells. Phosphorylation of each ZEB1 mutant was analyzed by immunoprecipitation with an anti-V5 agarose affinity gel antibody and detected with a western blot analysis. PLA confirmed the interaction between the V5-tagged GTSE1 and Flag-tagged ZEB1 mutant forms. Interactions with the target proteins are indicated as red dots. Cell nuclei were counterstained with DAPI (blue). (D) Ubiquitination of Flag-tagged ZEB1 was analyzed by immunoprecipitation with an anti-HA agarose affinity gel antibody and detected with a western blot analysis. V5-tagged GTSE1 and the Flag-tagged ZEB1 mutant forms were co-transfected with HA-tagged ubiquitin into HEK 293T cells for 24 h. (E) HEK 293T cells were transfected with Flag-tagged ZEB1 mutant forms and siCTRL or si GTSE1 and treated with 100 μg/mL CHX for various time periods. The graph shows the relative protein levels as a fold change from the initial Flag-tagged ZEB1 protein level. (F) L132 cells were transfected with siCTRL, si ATM , or si GTSE1 and exposed to 10 Gy of IR. Protein expression was then analyzed by western blotting. (G) The interaction between Flag-tagged ZEB1 and ATM or GTSE1 was confirmed by immunoprecipitation and immunoblot analyses. Flag-tagged ZEB1 and siATM or siGTSE1 were co-transfected with HEK 293T cells for 24 h. (H) L132 cells were transfected with siCTRL, siATM, or siGTSE1 and treated with 100 μg/mL of CHX for various time periods. The graph shows the relative levels of ZEB1 protein. (I) Immunoblots indicate protein level variations in response to IR. All graphs indicate the mean ± SEM ( n = 3/group). The fold change was calculated relative to the control. ∗, ∗∗, ∗∗∗, ∗∗∗∗ indicates p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively.

Article Snippet: Non-targeting control shRNA (shCTRL) lentiviral particles (#108080), GTSE1 shRNA lentiviral particles (#75216-V), and polybrene (#134220) were obtained from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Transfection, Phospho-proteomics, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Expressing, Control

Journal: Molecular Therapy

Article Title: GTSE1-driven ZEB1 stabilization promotes pulmonary fibrosis through the epithelial-to-mesenchymal transition

doi: 10.1016/j.ymthe.2024.09.029

Figure Lengend Snippet: GTSE1-ZEB1 interaction serves as a therapeutic biomarker for monitoring disease status in preclinical models (A) Schematic of the experimental schedule for the in vivo studies (BLM-induced PF model and IR-induced PF model). Mannose LNPs containing siRNA (0.5 mg/kg) were delivered intratracheally twice to assess the effect of Gtse1 silencing in murine PF models. (B) Sirius red staining of collagen in lung tissues from BLM- or IR-induced PF model mice indicates severity of lung fibrosis. (C) Representative immunohistochemistry images of GTSE1 and ZEB1 in mouse lung tissues. Graphs show scores quantifying the target protein-positive area. (D) Correlation between GTSE1or ZEB1 protein expression and the fibrotic area in mouse PF treated with mannose LNPs. (E) Correlation between GTSE1 and ZEB1 expression in mouse PF treated with mannose LNPs. All graphs indicate the mean ± SEM ( n = 3/group). The fold change was calculated relative to the control. ∗, ∗∗, and ∗∗∗ indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

Article Snippet: Non-targeting control shRNA (shCTRL) lentiviral particles (#108080), GTSE1 shRNA lentiviral particles (#75216-V), and polybrene (#134220) were obtained from Santa Cruz Biotechnology.

Techniques: Biomarker Discovery, In Vivo, Staining, Immunohistochemistry, Expressing, Control

Journal: Molecular Therapy

Article Title: GTSE1-driven ZEB1 stabilization promotes pulmonary fibrosis through the epithelial-to-mesenchymal transition

doi: 10.1016/j.ymthe.2024.09.029

Figure Lengend Snippet: Schematic of the proposed mechanism of GTSE1 in IR-induced PF GTSE1 protein levels increase during PF development, and it binds to the relatively unstable form of ZEB1 (non-phosphorylated at S585) and translocates it into the nucleus, hindering its degradation. ZEB1 stabilization by GTSE1 leads to an increase in the EMT signature, driving PF.

Article Snippet: Non-targeting control shRNA (shCTRL) lentiviral particles (#108080), GTSE1 shRNA lentiviral particles (#75216-V), and polybrene (#134220) were obtained from Santa Cruz Biotechnology.

Techniques:

Journal: Biological Research

Article Title: REC8 suppresses tumor angiogenesis by inhibition of NF-κB-mediated vascular endothelial growth factor expression in gastric cancer cells

doi: 10.1186/s40659-020-00307-1

Figure Lengend Snippet: IHC assay showed REC8 expression and MVD in gastric cancer. a Representative images of immunohistochemistry staining for REC8 expression and abundance of angiogenesis (CD31 marker) in gastric cancer tissue and paired control. Sar bar = 100 nm; b Quantification of MVD in clinical tissue microarrays between cancer tissues (n = 59) and paired controls (n = 16) were analyzed by t-test. **p < 0.01

Article Snippet: The lentivirus control shRNA (shCTL: sc-108080) and REC8 shRNA (shREC8: sc-106878-V) were purchased from Santacruz Biotechnology, INC. Puromycin was purchased from sigma and used to select for stably infected cells.

Techniques: Expressing, Immunohistochemistry, Staining, Marker, Control

Journal: Biological Research

Article Title: REC8 suppresses tumor angiogenesis by inhibition of NF-κB-mediated vascular endothelial growth factor expression in gastric cancer cells

doi: 10.1186/s40659-020-00307-1

Figure Lengend Snippet: Gastric cancer cells with REC8 depletion promoted chemotactic migration and tube formation of endothelial cells. a Real-time and b western blotting were performed to examine the REC8 expression at mRNA and protein level. c , d Conditional medium (CM) from BGC823 or AGS-1 cells infected with shCTL and shREC8 were used to be chemoattractant for HUVECs migration by transwell assays. ***p < 0.001 versus shCTL group by t-tests. Sar bar = 50um; e , f HUVECs were treated with conditional medium from BGC823 cells treated as indicated. Tube formation of HUVEC cells were visualized by phase contrast inverted microscope (100 ×), **p < 0.01 versus shCTL group by t-tests

Article Snippet: The lentivirus control shRNA (shCTL: sc-108080) and REC8 shRNA (shREC8: sc-106878-V) were purchased from Santacruz Biotechnology, INC. Puromycin was purchased from sigma and used to select for stably infected cells.

Techniques: Migration, Western Blot, Expressing, Infection, Inverted Microscopy

Journal: Biological Research

Article Title: REC8 suppresses tumor angiogenesis by inhibition of NF-κB-mediated vascular endothelial growth factor expression in gastric cancer cells

doi: 10.1186/s40659-020-00307-1

Figure Lengend Snippet: Depletion of REC8 enhanced HUVECs migration and tube formation through upregulation of VEGF in gastric cancer cells. a – c The mRNA and protein level of VEGF were analyzed by real-time qPCR, western blotting and ELISA in BGC823 and AGS-1 cells. ***p < 0.001, **p < 0.01 verus shCTL. d – g Transwell assay and tube formation assay were employed to detect HUVEC migration and tube formation with or without neutralizing VEGF antibody in CM from indicated group in BGC823 cells. Quantification was performed to analyze the statistical significance. ***p < 0.001

Article Snippet: The lentivirus control shRNA (shCTL: sc-108080) and REC8 shRNA (shREC8: sc-106878-V) were purchased from Santacruz Biotechnology, INC. Puromycin was purchased from sigma and used to select for stably infected cells.

Techniques: Migration, Western Blot, Enzyme-linked Immunosorbent Assay, Transwell Assay, Tube Formation Assay

Journal: Biological Research

Article Title: REC8 suppresses tumor angiogenesis by inhibition of NF-κB-mediated vascular endothelial growth factor expression in gastric cancer cells

doi: 10.1186/s40659-020-00307-1

Figure Lengend Snippet: NF-κB is required for VEGF transcription in gastric cancer cell with ablation of REC8. a Western blotting was used to detect phosphorylation of NF-κB. b A putative binding sequence of NF-κB p65 in VEGF promoter by UCSC analysis. c ChIP analysis of the binding of NF-κB to VEGF gene promoter in BGC823 cells depleted with REC8. Two-way ANOVA, **p < 0.01, the experiment was repeated three times. Error bars indicate mean ± S.D. d Western blotting and e – f tube formation were applied to measure VEGF in conditional medium from BGC823 cell infected with indicated lentivirus in present with or without NF-κB inhibitor BAY11-7082 treatment. g Schematic diagram for the mechanistic role of REC8 in tumor angiogenesis by regulation NF-κB-mediated VEGF in gastric cancer

Article Snippet: The lentivirus control shRNA (shCTL: sc-108080) and REC8 shRNA (shREC8: sc-106878-V) were purchased from Santacruz Biotechnology, INC. Puromycin was purchased from sigma and used to select for stably infected cells.

Techniques: Western Blot, Phospho-proteomics, Binding Assay, Sequencing, Infection

Journal: Neoplasia (New York, N.Y.)

Article Title: A Preclinical Study Combining the DNA Repair Inhibitor Dbait with Radiotherapy for the Treatment of Melanoma 1

doi: 10.1016/j.neo.2014.08.008

Figure Lengend Snippet: Effect of Dbait on H2AX phosphorylation. (A) SK28 and 501mel melanoma cells were transfected with an inactive control oligonucleotide or Dbait ± NU7026 (DNA-PK inhibitor). Immunofluorescence of γ-H2AX (red) and chromatin (DAPI; blue) was visualized. Dbait treatment led to non-localized pan-nuclear H2AX phosphorylation evidencing Dbait activity. This activity was dependent on DNA-PK activation. Bar, 50 μm. (B) SK28 melanoma cells were transfected with an inactive control oligonucleotide or Dbait. Immunofluorescence of γ-H2AX (red) and chromatin (DAPI; blue) was visualized immediately after irradiation and/or Dbait treatment. Irradiation alone resulted in localized γ-H2AX foci representing radio-induced DNA DSBs; Dbait treatment with or without irradiation led to non-localized pan-nuclear H2AX phosphorylation evidencing Dbait activity. Bar, 30 μm. (C) SK28 cells were transduced with lentiviruses that express either control, non-targeting shRNA, or shRNA targeting DNA-PKcs. After Dbait transfection, cells were immunostained with mouse monoclonal anti–DNA-PKcs or anti–γ-H2AX. Dbait activity was not detected in cells transduced with shRNA targeting DNA-PKcs. Bar, 50 μm.

Article Snippet: Subconfluent SK28 cells were transduced with lentiviruses that expressed either the control, non-targeting shRNA (shCTL; sc-108080; Santa Cruz Biotechnology, (Dallas, Texas, USA)), or shRNA targeting DNA-PKcs (shDNA-PK; sc-35200-V; Santa Cruz Biotechnology) at a multiplicity of infection of 3 using polybrene (5 μg/ml).

Techniques: Phospho-proteomics, Transfection, Control, Immunofluorescence, Activity Assay, Activation Assay, Irradiation, Transduction, shRNA

Journal: BMC Cancer

Article Title: NAD(P)-dependent steroid dehydrogenase-like is involved in breast cancer cell growth and metastasis

doi: 10.1186/s12885-020-06840-2

Figure Lengend Snippet: NSDHL knockdown suppresses total cholesterol level and promotes erlotinib response in MDA-MB-231 cell. a Total cholesterol levels measured in BT-20 and MDA-MB-231 cells transfected with NSDHL siRNA or control siRNA (20 nM); b Dose-response curve of erlotinib in BT-20 and MDA-MB-231 cells transfected with NSDHL siRNA or control siRNA (20 nM); c Representative western blot images of NSDHL, EGFR, and precursor and mature SREBP-1 and data of relative expression levels of NSDHL, EGFR, and precursor SREBP-1 in BT-20 and MDA-MB-231 cells transfected with NSDHL siRNA or control siRNA (20 nM). Data represent the mean ± standard deviation of three independent experiments. * p < 0.05, *** p < 0.001

Article Snippet: Precisely, 4 × 10 5 MDA-MB-231 cells were seeded in each well of 12-well plate 24 h prior to viral infection and were replaced with media containing 5 μg/ml polybrene® (sc-134220, Santa Cruz) and 20 μl of NSDHL-targeting shRNA lentiviral particles (shNSDHL) (sc-90849-V, Santa Cruz) or control shRNA lentiviral particles (shCtrl) (sc-108080, Santa Cruz).

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, Standard Deviation

Journal: BMC Cancer

Article Title: NAD(P)-dependent steroid dehydrogenase-like is involved in breast cancer cell growth and metastasis

doi: 10.1186/s12885-020-06840-2

Figure Lengend Snippet: NSDHL knockdown suppressed tumor growth and lung metastasis of MDA-MB-231 xenograft mice. a, b Data of relative NSDHL mRNA level analyzed by real-time RT-PCR and representative western blot images of NSDHL in MDA-MB-231 cells transduced with NSDHL shRNA or control shRNA lentivirus; c Data of tumor volume measured weekly in NSDHL shRNA or control shRNA mice; d Gross images and wet weight of tumors removed from NSDHL shRNA or control shRNA mice at 44 days post-injection; e Representative NSDHL immunohistochemistry image and scores analyzed from NSDHL shRNA or control shRNA tumor tissues; f Gross and H&E images of lungs and data of metastatic foci analyzed from NSDHL shRNA or control shRNA lung tissues. In vitro data represent the means ± standard deviations of three independent experiments. Animal data represent the means ± standard deviations of five mice per group. * p < 0.05, ** p < 0.01

Article Snippet: Precisely, 4 × 10 5 MDA-MB-231 cells were seeded in each well of 12-well plate 24 h prior to viral infection and were replaced with media containing 5 μg/ml polybrene® (sc-134220, Santa Cruz) and 20 μl of NSDHL-targeting shRNA lentiviral particles (shNSDHL) (sc-90849-V, Santa Cruz) or control shRNA lentiviral particles (shCtrl) (sc-108080, Santa Cruz).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Transduction, shRNA, Control, Injection, Immunohistochemistry, In Vitro

Journal: Communications biology

Article Title: Compressive stress triggers fibroblasts spreading over cancer cells to generate carcinoma in situ organization.

doi: 10.1038/s42003-024-05883-6

Figure Lengend Snippet: Fig. 3 | Cell–cell and cell–matrix adhesions are required for fibroblast spreading. a Western blot showing N-cadherin expression level in Control fibroblasts (Ctrl, transfected with scrambled shRNA) and fibroblast depleted from N-cadherin (shN-cadh). GAPDH is used as a loading control. b Frequency of capsules in which fibroblasts envel- oped cancer cells. Capsules contain cancer cells with control or N-cadherin-depleted fibroblasts. t = 0 corresponds to the confluent stage. n ≥30 capsules per condition. c. Western blot showing Fibronectin expression level in Control fibroblasts (shCtrl, transfected with scrambled shRNA) and fibroblast depleted from fibronectin (shFN) using five differ- ent shRNA probes. GAPDH is used as a loading control. d Frequency of capsules in which fibroblasts enveloped cancer cells. Capsules contain fibroblasts showing different degrees of fibronectin depletion. t = 0 corresponds to the confluent stage. n = 40 capsules. e Confocal images of co-culture at day 5, which corresponds to the early stage, before con- fluency (two upper rows) and 10 days, which cor- responds to the final stage with fully spread fibroblasts (two bottom rows). Both cell types are labeled with phalloidin (F-actin, red), fibroblasts express GFP (green), fibronectin is labeled with antibodies (magenta). Scale bar: 100 μm. f Confocal images of co-culture at the onset of fibroblasts spreading. Fibroblasts expressed GFP (green), can- cer cells are unstained, and fibronectin is labeled with antibodies (magenta). Insets, higher magnifi- cation of boxed regions. Scale bars: 20 μm.

Article Snippet: To inhibit cell contractility, spheroids were incubated in a culture medium using 50 μM myosin II inhibitor, blebbistatin (MERCK), or 100 μM ROCK inhibitor, Y-27632 (MERCK). siRNA and shRNA knockdowns HT29 cells and NIH3T3 were infected with MyH9 shRNA lentiviral particles (aYAP, Gift), control shRNA Lentiviral Particles (Santa Cruz Biotechnology, sc-108080), N-cadherin shRNA lentiviral particles (Santa Cruz Biotechnology sc-35999-V) and Fibronectin shRNA lentiviral particles (SHCLNG,MERCK) and shCtrl (SHC002, MERCK).

Techniques: Western Blot, Expressing, Control, Transfection, shRNA, Capsules, Co-Culture Assay, Labeling