Journal: Signal transduction and targeted therapy

Article Title: Human adipose and umbilical cord mesenchymal stem cell-derived extracellular vesicles mitigate photoaging via TIMP1/Notch1.

doi: 10.1038/s41392-024-01993-z

Figure Lengend Snippet: Fig. 2 AMSC-EV and HUMSC-EV mitigated photoaging of HaCaTs in vitro. a Representative image of MSC-EV uptake by HaCaTs pretreated with or without UVB irradiation (scale bar, 20 μm). b Representative immunofluorescence staining images of positive cells of ROS (green) and DAPI (scale bar, 20 μm). c Fluorescence intensity of ROS levels. n = 3, ****P < 0.0001. d Representative images of SA-β-gal staining in HaCaTs (scale bar, 100 μm). e Quantitation of SA-β-gal positive cells in HaCaTs. f Representative immunofluorescence staining images of positive cells of γ-H2Ax (red) and DAPI (scale bar, 20 μm). g Quantitation of the mean number of γ-H2Ax foci/cell. n = 3, ****P < 0.0001. h Quantitation of HaCaTs proliferation detected by CCK8 assay with the OD value on Day 1, Day 2, Day 3, Day 4, and Day 5. n = 3, ***P < 0.001, ****P < 0.0001. i Representative images of transwell assays of HaCaTs (scale bar, 100 μm). j Quantitation of transwell assays of HaCaTs. n = 3, ****P < 0.0001. k Representative images of migration assay and the image was taken at the indicated times (scale bar, 100 μm). l Quantitation of migration assays of HaCaTs. n = 3, *P < 0.05, **P < 0.01. m Quantitation of IL-1β, IL-6, and TNF-α released by HaCaTs was detected through qRT-PCR. n = 3, ****P < 0.0001. n Quantitation of IL-1β, IL-6, and TNF-α released by HaCaTs was detected through ELISA. n = 3, **P < 0.01, ****P < 0.0001

Article Snippet: After UVB, HDFs were incubated with ADAM10 inhibitor GI254023X (HY-19956, MedChemExpress) for 2 h, and then cells were incubated with fibroblast medium for an additional 24 h. After UVB, 2 mM Notch activator Valproic acid (Sodium Valproate) sodium (HY-10585A, MedChemExpress) were used with MSC-EV or TIMP1 for 24 h. Signal Transduction and Targeted Therapy (2024) 9:294 Isolation of EV We isolated MSC-EV following the MIEV2018 guidelines.

Techniques: In Vitro, Irradiation, Staining, Fluorescence, Quantitation Assay, CCK-8 Assay, Migration, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Signal transduction and targeted therapy

Article Title: Human adipose and umbilical cord mesenchymal stem cell-derived extracellular vesicles mitigate photoaging via TIMP1/Notch1.

doi: 10.1038/s41392-024-01993-z

Figure Lengend Snippet: Fig. 3 AMSC-EV and HUMSC-EV mitigated photoaging of HDFs in vitro. a Representative images of MSC-EV uptake by HDFs pretreated with or without UVB irradiation (scale bar, 20 μm). b Representative immunofluorescence staining images ROS (green) and DAPI (scale bar, 20 μm). c Fluorescence intensity of ROS levels. n = 3, ***P < 0.001, ****P < 0.0001. d Representative images of SA-β-gal staining in HDFs (scale bar, 100 μm). e Quantitation of SA-β-gal positive cells in HDFs. n = 3, ****P < 0.0001. f Representative immunofluorescence staining images of positive cells of γ-H2Ax (red) and DAPI (scale bar, 20 μm). g Quantitation of a mean number of γ-H2Ax foci/cell. h Quantitation of HDFs proliferation detected by CCK8 assay with the OD value on Day 1, Day 2, Day 3, Day 4, and Day 5. n = 3, ***P < 0.001, ****P < 0.0001. i Quantitative of COL31A in HDFs by qRT-PCR. n = 3, *P < 0.05, **P < 0.01. j Quantitation of TIMP1, MMP1, and MMP9 released by HDFs by qRT- PCR. n = 3, *P < 0.05, ***P < 0.001, ****P < 0.0001. k Western blot analysis showing the change of COL3, TIMP1, MMP1 and MMP9 in HDFs. l Quantitative of COL3, TIMP1, MMP1, and MMP9 in HDFs by western blot. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. m Quantitation of IL-1β, IL-6, and TNF-α released by HDFs by qRT-PCR. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n Quantitation of IL-1β, IL-6, and TNF-α released by HDFs was detected through ELISA. n = 3, **P < 0.01, ****P < 0.0001

Article Snippet: After UVB, HDFs were incubated with ADAM10 inhibitor GI254023X (HY-19956, MedChemExpress) for 2 h, and then cells were incubated with fibroblast medium for an additional 24 h. After UVB, 2 mM Notch activator Valproic acid (Sodium Valproate) sodium (HY-10585A, MedChemExpress) were used with MSC-EV or TIMP1 for 24 h. Signal Transduction and Targeted Therapy (2024) 9:294 Isolation of EV We isolated MSC-EV following the MIEV2018 guidelines.

Techniques: In Vitro, Irradiation, Staining, Fluorescence, Quantitation Assay, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Signal transduction and targeted therapy

Article Title: Human adipose and umbilical cord mesenchymal stem cell-derived extracellular vesicles mitigate photoaging via TIMP1/Notch1.

doi: 10.1038/s41392-024-01993-z

Figure Lengend Snippet: Fig. 4 AMSC-EV and HUMSC-EV mitigated photoaging in constructed full-thickness skin model. a Schematic representation of establishment and treatment of T-Skin model (Created with BioRender.com). b Representative images of histological analysis and biomarker staining of the T-Skin model (scale bar, 15 μm). c Representative images of SA-β-gal staining (scale bar, 20 μm). d Quantitation of SA-β-gal positive cells. n = 3, **P < 0.01, ***P < 0.001, ****P < 0.0001. e Representative images of ROS staining (scale bar, 20 μm). f Quantitation of ROS-positive cells. n = 3, ****P < 0.0001. g Representative images of γ-H2Ax staining (scale bar, 20 μm). h Quantitation of γ-H2Ax positive cells. n = 3, **P < 0.01, ****P < 0.0001. i Representative images of MSC-EV uptake by T-Skin model after 48 and 72 h (left, scale bar, 150 μm; right, scale bar, 25 μm). j Quantitation of IL-1β, IL-6, and TNF-α released by T-Skin model was detected through ELISA. n = 3, ****P < 0.0001

Article Snippet: After UVB, HDFs were incubated with ADAM10 inhibitor GI254023X (HY-19956, MedChemExpress) for 2 h, and then cells were incubated with fibroblast medium for an additional 24 h. After UVB, 2 mM Notch activator Valproic acid (Sodium Valproate) sodium (HY-10585A, MedChemExpress) were used with MSC-EV or TIMP1 for 24 h. Signal Transduction and Targeted Therapy (2024) 9:294 Isolation of EV We isolated MSC-EV following the MIEV2018 guidelines.

Techniques: Construct, Biomarker Discovery, Staining, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Signal transduction and targeted therapy

Article Title: Human adipose and umbilical cord mesenchymal stem cell-derived extracellular vesicles mitigate photoaging via TIMP1/Notch1.

doi: 10.1038/s41392-024-01993-z

Figure Lengend Snippet: Fig. 7 MSC-EV rescues HDFs and HaCaTs photoaging by upregulating TIMP1. a Venn diagram of the protein contents of AMSC-EV and HUMSC-EV. b Heatmap of the protein contents of AMSC-EV and HUMSC-EV. c Heatmap of extracellular matrix organization. d Western blot analysis showing COL1, COL3, COL6, and TIMP1 expression in AMSC-EV and HUMSC-EV. e Quantitation of TIMP1 in AMSC-EV and HUMSC- EV was detected by Western blot. n = 3. f Representative immunofluorescence staining images of positive cells of ROS (green) and DAPI (scale bar, 35 μm). g Quantitation of ROS-positive cells. n = 3, ****P < 0.0001. h Representative immunofluorescence staining images of positive cells of γ-H2Ax (red) and DAPI (scale bar, 20 μm). i Quantitation of a mean number of γ-H2Ax foci/cell. n = 3, ***P < 0.001, ****P < 0.0001. j Representative images of SA-β-gal staining in HDFs and HaCaTs (scale bar, 100 μm). k Quantitation of SA-β-gal positive cells in HDFs and HaCaTs. n = 3, ***P < 0.001, ****P < 0.0001. l Representative images of transwell assays of HaCaTs (scale bar, 100 μm). m epresentative images of migration assay of HaCaTs (scale bar, 100 μm). n Western blot analysis showing the change of COL3, TIMP1, MMP1 and MMP9 in HDFs

Article Snippet: After UVB, HDFs were incubated with ADAM10 inhibitor GI254023X (HY-19956, MedChemExpress) for 2 h, and then cells were incubated with fibroblast medium for an additional 24 h. After UVB, 2 mM Notch activator Valproic acid (Sodium Valproate) sodium (HY-10585A, MedChemExpress) were used with MSC-EV or TIMP1 for 24 h. Signal Transduction and Targeted Therapy (2024) 9:294 Isolation of EV We isolated MSC-EV following the MIEV2018 guidelines.

Techniques: Western Blot, Expressing, Quantitation Assay, Staining, Migration

Journal: Signal transduction and targeted therapy

Article Title: Human adipose and umbilical cord mesenchymal stem cell-derived extracellular vesicles mitigate photoaging via TIMP1/Notch1.

doi: 10.1038/s41392-024-01993-z

Figure Lengend Snippet: Fig. 8 MSC-EV rescues HDFs photoaging by downregulating NOTCH signaling pathway. a Venn diagram showing the overlapped genes between photoaging (UVB/Control) and Treatment (EV/UVB) based on data from RNA-seq. Rev-photoaging-DEGs were defined as subsets of overlapped DEGs that were changed in the opposite direction in photoaging and treatment. b Representative GO terms of Rev-photoaging DEGs for Biological Process (green), Cellular Component (orange), and Molecular Function (blue) were shown. c Heatmaps showing the expression profile of genes in response to UVB and EV treatment in Notch signaling. d Quantitation of different mRNA levels of Hes1, Tle1, Lfng, Dll1, and HeyL. e Western blot analysis showing expression of NOTCH1, NICD1, HES1, SIRT1, P16, P21, P53 and GAPDH after EV treatment. f Western blot analysis showing expression of NOTCH1, NICD1, HES1, SIRT1, P16, P21, P53 and GAPDH after TIMP1 treatment. g Representative immunofluorescence staining images of positive cells of HES1 in nude mice dorsal skin injected PBS, AMSC-EV, or HUMSC-EV (top, scale bar, 30 μm; bottom, scale bar, 10 μm). h Representative immunofluorescence staining images of SA-β-gal (scale bar, 100 μm), ROS (green), and γ- H2Ax (red) (scale bar, 30 μm). i Luciferase-reporter assays of HES1 transcriptional activity in HDFs after UVB, FM (culture medium), EV, TIMP1, GI254023X, and VPA treatment. n = 3, **P < 0.01, ***P < 0.001, ****P < 0.0001. j Schematic of the role of EV in mediating UVB-induced photoaging of Notch signaling and cellular senescence. (Created with BioRender.com)

Article Snippet: After UVB, HDFs were incubated with ADAM10 inhibitor GI254023X (HY-19956, MedChemExpress) for 2 h, and then cells were incubated with fibroblast medium for an additional 24 h. After UVB, 2 mM Notch activator Valproic acid (Sodium Valproate) sodium (HY-10585A, MedChemExpress) were used with MSC-EV or TIMP1 for 24 h. Signal Transduction and Targeted Therapy (2024) 9:294 Isolation of EV We isolated MSC-EV following the MIEV2018 guidelines.

Techniques: Control, RNA Sequencing, Expressing, Quantitation Assay, Western Blot, Staining, Injection, Luciferase, Activity Assay

Journal: Signal transduction and targeted therapy

Article Title: Human adipose and umbilical cord mesenchymal stem cell-derived extracellular vesicles mitigate photoaging via TIMP1/Notch1.

doi: 10.1038/s41392-024-01993-z

Figure Lengend Snippet: Fig. 9 Schematic illustrations of AMSC-EV and HUMSC-EV repairments on photoaging in vitro and in vivo via regulation of the TIMP1/Notch pathway (Created with BioRender.com). AMSC-EV and HUMSC-EV improve photoaging in UVB-irradiated nude mice, HaCaTs, HKCs, HDFs, and T-Skin models through anti-inflammatory effects, ECM remodeling, and anti-senescence properties. Mechanistically, proteomic analysis revealed that TIMP1 is highly expressed in both AMSC-EV and HUMSC-EV and exerts similar effects to MSC-EV. TIMP1 inhibits MMPs, thereby altering extracellular matrix remodeling. Additionally, TIMP1 downregulates the activated Notch pathway and inhibits its downstream targets Hes1, P16, P21, and P53

Article Snippet: After UVB, HDFs were incubated with ADAM10 inhibitor GI254023X (HY-19956, MedChemExpress) for 2 h, and then cells were incubated with fibroblast medium for an additional 24 h. After UVB, 2 mM Notch activator Valproic acid (Sodium Valproate) sodium (HY-10585A, MedChemExpress) were used with MSC-EV or TIMP1 for 24 h. Signal Transduction and Targeted Therapy (2024) 9:294 Isolation of EV We isolated MSC-EV following the MIEV2018 guidelines.

Techniques: In Vitro, In Vivo, Irradiation

Journal: Critical Care Medicine

Article Title: Reversal of Acute Kidney Injury–Induced Neutrophil Dysfunction

doi: 10.1097/ccm.0000000000001472

Figure Lengend Snippet: Figure 5. Resistin in murine models of nephrotoxic and rhabdomyolysis-induced acute kidney injury (AKI). Plasma resistin levels are elevated as early as 4 hr after injection in mice with rhabdomyolysis-induced AKI (A) and remain elevated at 24 hr after induction (B). In nephrotoxic AKI, elevated plasma resistin levels are seen after 24 hr (B). (C) In vitro pretreatment with resistin significantly decreases neutrophil transwell migration rates, similar to that seen with either nephrotoxic or rhabdomyolysis-induced AKI. PMN = neurophils, Gly = glycerol, FA = folic acid.

Article Snippet: A subset of isolated neutrophils from untreated animals was incubated in phosphate-buffered saline (PBS) with or without 40 ng/mL of recombinant mouse resistin (R&D Systems) for 1 hour (37°C; 5% Co 2 ) prior to transwell migration.

Techniques: Clinical Proteomics, Injection, In Vitro, Migration

Journal: Critical Care Medicine

Article Title: Reversal of Acute Kidney Injury–Induced Neutrophil Dysfunction

doi: 10.1097/ccm.0000000000001472

Figure Lengend Snippet: Figure 6. Immunofluorescence staining for intracellular F-actin. Intracellular F-actin shows a normal pattern of distribution in neutrophils from control mice (A) or neutrophils undergoing control pretreatment (C). F-actin is primarily localized to the leading edge of the migrating cell, that is, neutrophils exhibit one short and narrow lamellipodia of F-actin at one pole of the cell (arrow). Contrary to that, F-actin localization is nearly lost in neutrophils from mice with acute kidney injury (AKI) (B) or neutrophils pretreated with resistin (D), that is, neutrophils are characterized by a lack of distinctly localized F-actin formation (arrow). No formation of a leading edge can be seen in either cell type, suggesting impaired migration of neutrophils during AKI or after pretreatment with resistin. fMLP = N-formyl-l-methionyl-l-leucyl-l phenylalanine.

Article Snippet: A subset of isolated neutrophils from untreated animals was incubated in phosphate-buffered saline (PBS) with or without 40 ng/mL of recombinant mouse resistin (R&D Systems) for 1 hour (37°C; 5% Co 2 ) prior to transwell migration.

Techniques: Immunofluorescence, Staining, Control, Migration

Journal: Critical Care Medicine

Article Title: Reversal of Acute Kidney Injury–Induced Neutrophil Dysfunction

doi: 10.1097/ccm.0000000000001472

Figure Lengend Snippet: Figure 7. The effects of acute kidney injury (AKI) in septic shock on plasma resistin and cell transwell migration. A, As to be expected, patients with AKI during septic shock show significantly elevated plasma creatinine concentrations when compared with those without AKI. Renal replacement therapy (RRT) significantly reduces plasma creatinine levels by day 4. B, Plasma resistin levels are also significantly elevated in patients with septic shock and AKI. Contrary to serum creatinine levels, our modes of RRT do not significantly lower plasma resistin levels. C, Neutrophilic-differentiated NB4 cells (NB4PMN) transwell migration is significantly reduced in patients with septic shock and AKI. RRT fails to restore normal NB4PMN transwell migration.

Article Snippet: A subset of isolated neutrophils from untreated animals was incubated in phosphate-buffered saline (PBS) with or without 40 ng/mL of recombinant mouse resistin (R&D Systems) for 1 hour (37°C; 5% Co 2 ) prior to transwell migration.

Techniques: Clinical Proteomics, Migration

Journal: Critical Care Medicine

Article Title: Reversal of Acute Kidney Injury–Induced Neutrophil Dysfunction

doi: 10.1097/ccm.0000000000001472

Figure Lengend Snippet: Figure 8. The effects of different resistin concentrations of neutrophilic-differentiated NB4 cells (NB4PMN) transwell migration and intracellular F-actin formation. A, Resistin at a physiologic concentration has no effect on NB4PMN transwell migration when compared with a resistin-free control. However, resistin at concentrations seen during (severe) acute kidney injury (AKI) significantly inhibits transwell migration (n = 8). B, Resistin at a physiologic concentration also has no effect on intracellular F-actin formation in NB4PMN cells. By contrast, resistin at concentrations seen during (severe) AKI significantly reduces intracellular F-actin formation in NB4PMN cells (n = 8). fMLP = N-formyl-l-methionyl-l-leucyl-l phenylalanine.

Article Snippet: A subset of isolated neutrophils from untreated animals was incubated in phosphate-buffered saline (PBS) with or without 40 ng/mL of recombinant mouse resistin (R&D Systems) for 1 hour (37°C; 5% Co 2 ) prior to transwell migration.

Techniques: Migration, Concentration Assay, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: NRF2 Regulates HER1 Signaling Pathway to Modulate the Sensitivity of Ovarian Cancer Cells to Lapatinib and Erlotinib

doi: 10.1155/2017/1864578

Figure Lengend Snippet: Pharmacological (bexarotene) and genetic inhibition (siRNA) of NRF2 causes transcriptional and translational downregulation of HER1. Luciferase assay showing transcriptional downregulation of HER1 following NRF2 inhibition by (a) bexarotene or (b) siRNA in PEO1, OVCAR3, and SKOV3 cell lines. Exponentially growing PEO1, SKOV3, and OVCAR3 excluding MCF7-AREc32 cell lines were transfected with either empty PGL3 basic vector or 1 μ g PGL3 basic vector with cloned HER1 driving the expression of luciferase gene. Cotransfection with 0.2 μ g pRL-CMV plasmid was performed as an internal transfection control as described in the Materials and Methods. At 24 h posttransfection, cells were (a) either left untreated or treated with 2.5 μ M bexarotene. (b) Cells were either transfected with scrambled siRNA (Sc) or transfected with 20 pmol of NRF2 siRNA (Si) for 24 h. Following treatments, lysates were prepared and luciferase activity was measured using dual luciferase reporter assay (Promega) in multiplate reader (MODULUS, Promega). (c) Immunoblot analysis following treatment with bexarotene demonstrated protein downregulation of HER1 receptor and decrease of NRF2, HO-1, and HER1. Exponentially growing cells were either left untreated (UT) or treated with 2.5 μ M bexarotene for 24 h before being harvested and processed for immunoblotting using relevant antibodies. Bar chart showing total NRF2, HO-1, and total HER1 levels in PEO1, OVCAR3, and SKOV3 cell lines by quantifying immunoblot signal intensities obtained in (c). (d) Immunoblot analysis following knockdown of NRF2 demonstrated protein downregulation of both HER1 receptor and decrease of NRF2 and HO-1 in PEO1, OVCAR3, and SKOV3 cell lines. Cells were either transfected with scrambled siRNA (Sc) or transfected with 75 pmol of NRF2 siRNA (Si). After 24 h and 48 h, cells were harvested and processed for immunoblotting using relevant antibodies. β -Actin of the same blot was used as loading control. Bar chart shows the levels of relevant proteins by quantifying immunoblot signal intensities obtained and expressed as fold change. Data shown in (a) and (b) are the means ± S.D. of triplicates, normalised to UT or scramble expressed in fold change with statistical significance determined by Student's t -test ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: Kinase inhibitors targeting HER1 receptor, lapatinib and erlotinib, were used by directly diluting the drugs in media to a final concentration of 5 μ M. tert-Butylhydroquinone (tBHQ; Sigma) and bexarotene (Carbosynth) stock solutions were made with dimethylsulfoxide (Fisher) and diluted to a final concentration as required with media.

Techniques: Inhibition, Luciferase, Transfection, Plasmid Preparation, Clone Assay, Expressing, Cotransfection, Control, Activity Assay, Reporter Assay, Western Blot, Knockdown

Journal: Oxidative Medicine and Cellular Longevity

Article Title: NRF2 Regulates HER1 Signaling Pathway to Modulate the Sensitivity of Ovarian Cancer Cells to Lapatinib and Erlotinib

doi: 10.1155/2017/1864578

Figure Lengend Snippet: Inhibition and knockdown of NRF2 by bexarotene and siRNA, respectively, elevate the level of ROS and depletion of total glutathione level. (a) Bexarotene treatment and knockdown of NRF2 by siRNA cause increase in ROS levels. Exponentially growing cells were seeded in triplicates in opaque flat bottom black-walled 96-well plates for 24 h. Following this, cells were either left untreated (UT) or treated with 2.5 μ M bexarotene or 7 pmol of siRNA (scrambled or targeted) for different time points as indicated. Following incubations, cells were loaded with DCFDA fluorescent stain for 45 min and assayed for ROS by measuring fluorescence as described in Materials and Methods. (b) Bexarotene and siRNA cause depletion of total glutathione. Exponentially growing cells were seeded in 60 mm tissue culture plates for 24 h and either left untreated (UT) or treated with media containing 1 nM heregulin alone (HRG) or with cotreatment of 2.5 μ M bexarotene or 100 pmol siRNA for 24 h before being harvested to prepare protein lysates and processed for glutathione assay as described in Materials and Methods. Data is shown as fold change of bexarotene or siRNA-treated cells to UT or scrambled siRNA, respectively, with statistical significance determined by one-way ANOVA followed by Tukey's post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Kinase inhibitors targeting HER1 receptor, lapatinib and erlotinib, were used by directly diluting the drugs in media to a final concentration of 5 μ M. tert-Butylhydroquinone (tBHQ; Sigma) and bexarotene (Carbosynth) stock solutions were made with dimethylsulfoxide (Fisher) and diluted to a final concentration as required with media.

Techniques: Inhibition, Knockdown, Staining, Fluorescence, Glutathione Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: NRF2 Regulates HER1 Signaling Pathway to Modulate the Sensitivity of Ovarian Cancer Cells to Lapatinib and Erlotinib

doi: 10.1155/2017/1864578

Figure Lengend Snippet: Treatment with bexarotene causes inhibition of NRF2-dependent antioxidant response pathway and generates ROS. (A) Western analysis showing repression of NRF2 and HO-1 levels following bexarotene treatment in PEO1, OVCAR3, and SKOV3 cell lines. Exponentially growing cells were either left untreated, treated with 2.5 μ M bexarotene, or a combination of 2.5 μ M bexarotene together with 5 μ M of lapatinib and erlotinib for 24 h before being harvested to prepare protein lysates and processed as described in Materials and Methods. β -Actin was used as loading control. The bars indicate NRF2 and HO-1 levels following quantification of immunoblot signal intensities obtained in (a) and normalised to the value of UT and expressed as fold change. The signal intensities of bands were quantified through integrated optical densitometry measurement. (b) Bexarotene treatment causes inhibition of NRF2-dependent transcription. Exponentially growing AREc32 cell line stably expressing 8× cis -antioxidant response elements driving the expression of luciferase gene in an NRF2-dependent manner were either left untreated (UT), treated with bexarotene alone, or with bexarotene and combination of lapatinib and erlotinib for different time points as indicated. Following this, cell lysates were prepared and assayed for luciferase activity (BrightGlo Luciferase System, Promega). (c) Bexarotene treatment causes increase in ROS levels. Exponentially growing AREc32 cell lines stably expressing 8× cis -antioxidant response elements driving the expression of luciferase gene in an NRF2-dependent manner were seeded in triplicates in opaque flat bottom black-walled 96-well plates for 24 h. Following this, cells were either left untreated (UT), treated with bexarotene alone, or with bexarotene and combination of lapatinib or erlotinib for different time points as indicated. Following incubations, cells were loaded with DCFDA fluorescent stain for 45 min and assayed for ROS as described in Materials and Methods. Data are the mean values ± S.D of quadruplicates, normalised to untreated (UT) and expressed as fold change with statistical significance determined by one-way ANOVA followed by Tukey's post hoc test according to the scale ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Kinase inhibitors targeting HER1 receptor, lapatinib and erlotinib, were used by directly diluting the drugs in media to a final concentration of 5 μ M. tert-Butylhydroquinone (tBHQ; Sigma) and bexarotene (Carbosynth) stock solutions were made with dimethylsulfoxide (Fisher) and diluted to a final concentration as required with media.

Techniques: Inhibition, Western Blot, Control, Stable Transfection, Expressing, Luciferase, Activity Assay, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: NRF2 Regulates HER1 Signaling Pathway to Modulate the Sensitivity of Ovarian Cancer Cells to Lapatinib and Erlotinib

doi: 10.1155/2017/1864578

Figure Lengend Snippet: Treatment with lapatinib, erlotinib, and bexarotene causes inhibition of NRF2-dependent transcription and depletion of total glutathione levels. (a) Single and combination of lapatinib and erlotinib cause inhibition of NRF2-dependent transcription. Exponentially growing MCF7-AREc32 cell lines stably expressing cis -regulatory antioxidant response elements driving the expression of luciferase gene in an NRF2-dependent manner were treated with 1 nM HRG alone or with cotreatment of 5 μ M lapatinib and erlotinib either individually or in combination for different time points as indicated. Following this, cell lysates were prepared and assayed for luciferase activity as described in Materials and Methods. Data shown are mean values ± S.D of quadruplicates, normalised to untreated (UT) and expressed as fold change with statistical significance determined by one-way ANOVA followed by Tukey's post hoc test. Asterisks indicate significant differences between individual groups as indicated and according to the scale ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (b) Single and combination of lapatinib and erlotinib cause decrease in glutathione level. Exponentially growing cells were seeded in 60 mm tissue culture plates for 24 h and either left untreated (UT) or treated with media containing 1 nM heregulin alone (HRG) or with cotreatment of 5 μ M lapatinib and erlotinib or their combination with 2.5 μ M bexarotene (COMB) for 72 h before being harvested to prepare protein lysates and processed for glutathione assay. Data are mean values ± S.D of triplicates and expressed as fold change to the UT. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test according to the scale ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Kinase inhibitors targeting HER1 receptor, lapatinib and erlotinib, were used by directly diluting the drugs in media to a final concentration of 5 μ M. tert-Butylhydroquinone (tBHQ; Sigma) and bexarotene (Carbosynth) stock solutions were made with dimethylsulfoxide (Fisher) and diluted to a final concentration as required with media.

Techniques: Inhibition, Stable Transfection, Expressing, Luciferase, Activity Assay, Glutathione Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: NRF2 Regulates HER1 Signaling Pathway to Modulate the Sensitivity of Ovarian Cancer Cells to Lapatinib and Erlotinib

doi: 10.1155/2017/1864578

Figure Lengend Snippet: NRF2 inhibition with bexarotene sensitises EGFR signalling pathway to HER1 inhibitors (a) lapatinib and (b) erlotinib. Immunoblot analysis showing bexarotene-dependent repression of EGFR signalling following its combination with lapatinib and erlotinib. Exponentially growing cells were either left untreated in media containing 1 nM heregulin (UT) or treated with the same media containing in the presence of 1 nM heregulin with lapatinib (Lap) alone or erlotinib (Erl) alone, each at 5 μ M, or with cotreatment of 2.5 μ M bexarotene (Lap + Bex) or (Erl + Bex) for 24 h before and processed for immunoblotting using relevant antibodies, and β -actin was used as loading control. (c) NRF2 knockdown and inhibition increase the chance of cytotoxicity of HER family-targeted agents, lapatinib and erlotinib in ovarian cancer cells. Exponentially growing cells were seeded in triplicates in 96-well plates for 24 h. Following this, cells were either left untreated in media containing 1 nM heregulin (H) or treated with same media containing in the presence of 1 nM HRG with 5 μ M each of lapatinib (H + L) or erlotinib (H + E) or treated with combination 5 μ M lapatinib and 2.5 μ M bexarotene (H + L + BEX) or combination of 5 μ M erlotinib and 2.5 μ M bexarotene (H + E + BEX). Cell number was assessed indirectly by use of the cell titre glo assay. Data shown are means ± S.D. of triplicates, normalised to (H), expressed in fold change with statistical significance was calculated by one-way ANOVA followed by Tukey's post hoc test according to the scale: ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).

Article Snippet: Kinase inhibitors targeting HER1 receptor, lapatinib and erlotinib, were used by directly diluting the drugs in media to a final concentration of 5 μ M. tert-Butylhydroquinone (tBHQ; Sigma) and bexarotene (Carbosynth) stock solutions were made with dimethylsulfoxide (Fisher) and diluted to a final concentration as required with media.

Techniques: Inhibition, Western Blot, Control, Knockdown, Glo Assay