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Corning Life Sciences 6 well plates
(A) Schematic illustration of mechanical vibration experiment in this work. Vertical mechanical vibration was applied to the <t>6-well</t> plate for 10 min every 24 h after the medium change. Phase-contrast microscopic images were acquired every 12 h. Cells were passaged continuously from P3 (Cycle 1) to P5 (Cycle 3). On the 7th day, the samples treated with vibration were collected, and seeded into a new 6-well plate. At the same time, partial samples were stained to evaluate the undifferentiation marker (B) Visualization of relative distances between designed conditions in this work. Four related parameters (acceleration, frequency, amplitude, energy) are visualized by principal component analysis. The proportion of variance for each principal component (PC) is PC1 (0.56), PC2 (0.27), and PC3 (0.16). The loadings are: acceleration: frequency: amplitude: energy = PC1 (−0.04, 0.42, −0.65, −0.64), PC2 (0.89, −0.43, 0.09, 0.13), PC3 (0.46, −0.80, −0.25, −0.30). The color representation is visualized in the legend. Representative four points from tapping and closing of incubator door condition measured in the preliminary examination is visualized with smaller dots to indicate the range of vibration.
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Images

1) Product Images from "Effect of mechanical vibration stress in cell culture on human induced pluripotent stem cells"

Article Title: Effect of mechanical vibration stress in cell culture on human induced pluripotent stem cells

Journal: Regenerative Therapy

doi: 10.1016/j.reth.2019.05.002

(A) Schematic illustration of mechanical vibration experiment in this work. Vertical mechanical vibration was applied to the 6-well plate for 10 min every 24 h after the medium change. Phase-contrast microscopic images were acquired every 12 h. Cells were passaged continuously from P3 (Cycle 1) to P5 (Cycle 3). On the 7th day, the samples treated with vibration were collected, and seeded into a new 6-well plate. At the same time, partial samples were stained to evaluate the undifferentiation marker (B) Visualization of relative distances between designed conditions in this work. Four related parameters (acceleration, frequency, amplitude, energy) are visualized by principal component analysis. The proportion of variance for each principal component (PC) is PC1 (0.56), PC2 (0.27), and PC3 (0.16). The loadings are: acceleration: frequency: amplitude: energy = PC1 (−0.04, 0.42, −0.65, −0.64), PC2 (0.89, −0.43, 0.09, 0.13), PC3 (0.46, −0.80, −0.25, −0.30). The color representation is visualized in the legend. Representative four points from tapping and closing of incubator door condition measured in the preliminary examination is visualized with smaller dots to indicate the range of vibration.
Figure Legend Snippet: (A) Schematic illustration of mechanical vibration experiment in this work. Vertical mechanical vibration was applied to the 6-well plate for 10 min every 24 h after the medium change. Phase-contrast microscopic images were acquired every 12 h. Cells were passaged continuously from P3 (Cycle 1) to P5 (Cycle 3). On the 7th day, the samples treated with vibration were collected, and seeded into a new 6-well plate. At the same time, partial samples were stained to evaluate the undifferentiation marker (B) Visualization of relative distances between designed conditions in this work. Four related parameters (acceleration, frequency, amplitude, energy) are visualized by principal component analysis. The proportion of variance for each principal component (PC) is PC1 (0.56), PC2 (0.27), and PC3 (0.16). The loadings are: acceleration: frequency: amplitude: energy = PC1 (−0.04, 0.42, −0.65, −0.64), PC2 (0.89, −0.43, 0.09, 0.13), PC3 (0.46, −0.80, −0.25, −0.30). The color representation is visualized in the legend. Representative four points from tapping and closing of incubator door condition measured in the preliminary examination is visualized with smaller dots to indicate the range of vibration.

Techniques Used: Staining, Marker

2) Product Images from "Titanium dioxide nanoparticles induce endoplasmic reticulum stress-mediated apoptotic cell death in liver cancer cells"

Article Title: Titanium dioxide nanoparticles induce endoplasmic reticulum stress-mediated apoptotic cell death in liver cancer cells

Journal: The Journal of International Medical Research

doi: 10.1177/0300060520903652

Influence of TiO 2 nanotubes on cell growth. L02 (a) and HepG2 (b) cells were incubated in TiO 2 nanotube-coated (2.5, 5.0, 7.5, and 10 µg/mL) 6-well plates for 48 hours at 60°C. Cell numbers were counted using cell counting chambers. Cells incubated in 6-well plates coated with 0 µg/mL TiO 2 nanotubes were used as a control. * P
Figure Legend Snippet: Influence of TiO 2 nanotubes on cell growth. L02 (a) and HepG2 (b) cells were incubated in TiO 2 nanotube-coated (2.5, 5.0, 7.5, and 10 µg/mL) 6-well plates for 48 hours at 60°C. Cell numbers were counted using cell counting chambers. Cells incubated in 6-well plates coated with 0 µg/mL TiO 2 nanotubes were used as a control. * P

Techniques Used: Incubation, Cell Counting

L02 (a) and HepG2 (b) cells were cultured in 6-well plates for 48 hours, then apoptosis of L02 cells and HepG2 cells was detected by flow cytometry. TiO 2 increased the percentage of apoptotic liver cancer cells. Annexin V-positive cells were considered as apoptotic liver cancer cells. Cells incubated with 0 µg/mL TiO 2 were used as a control. * P
Figure Legend Snippet: L02 (a) and HepG2 (b) cells were cultured in 6-well plates for 48 hours, then apoptosis of L02 cells and HepG2 cells was detected by flow cytometry. TiO 2 increased the percentage of apoptotic liver cancer cells. Annexin V-positive cells were considered as apoptotic liver cancer cells. Cells incubated with 0 µg/mL TiO 2 were used as a control. * P

Techniques Used: Cell Culture, Flow Cytometry, Incubation

3) Product Images from "c-Jun N-terminal kinase (JNK) is required for survival and proliferation of B-lymphoma cells"

Article Title: c-Jun N-terminal kinase (JNK) is required for survival and proliferation of B-lymphoma cells

Journal:

doi: 10.1182/blood-2004-10-3819

Inhibition of JNK activity induces growth arrest and apoptosis in B-lymphoma cells and can be overcome by the addition of anti-CD40 and IL-10. (A) BKS-2 murine B-lymphoma cells were cultured in 6-well plates at 1 × 10 6 cells/well in the presence
Figure Legend Snippet: Inhibition of JNK activity induces growth arrest and apoptosis in B-lymphoma cells and can be overcome by the addition of anti-CD40 and IL-10. (A) BKS-2 murine B-lymphoma cells were cultured in 6-well plates at 1 × 10 6 cells/well in the presence

Techniques Used: Inhibition, Activity Assay, Cell Culture

4) Product Images from "End-to-End Platform for Human Pluripotent Stem Cell Manufacturing"

Article Title: End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21010089

Quality of cryopreserved cells post thaw. ( A ) 6 × 10 5 cells from each vial were plated onto a well of a 6-well plate. Phase contrast images were taken 48–72 h post thaw. Magnification, 40×; ( B ) Cells were stained with AP staining kit three (vial #2) to five (all other vials) days post plating. Whole-well images are shown. Magnification, 1×.
Figure Legend Snippet: Quality of cryopreserved cells post thaw. ( A ) 6 × 10 5 cells from each vial were plated onto a well of a 6-well plate. Phase contrast images were taken 48–72 h post thaw. Magnification, 40×; ( B ) Cells were stained with AP staining kit three (vial #2) to five (all other vials) days post plating. Whole-well images are shown. Magnification, 1×.

Techniques Used: Staining

5) Product Images from "Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L"

Article Title: Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L

Journal: bioRxiv

doi: 10.1101/2020.07.27.223727

Antiviral activity of GC-376 analogues. a-d Antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay. a GC-376 ; b UAWJ246 ; c UAWJ247 ; d UAWJ248 . Vero E6 cells in a 96-well plate were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in the presence of the indicated concentrations of the tested compounds. At 48 hpi, the cells were fixed and stained with a rabbit monoclonal antibody against the SARS-CoV-2 NP and a secondary antibody conjugated with Alexa 488 (Green). The nuclei were counterstained with Hoechst dye (Blue). For each well, fluorescence images of approximately 10K cells were acquired and shown. The images are representatives of two repeats. e-h Antiviral activity of GC-376 analogues against SARS-CoV-2 in the plaque assay. e GC-376 ; f UAWJ246 ; g UAWJ247 ; h UAWJ248 . Vero E6 cells in 6-well plates were infected with approximately 40 PFU/well of SARS-CoV-2 (USA-WA1/2020 isolate). After 1 hour, the inoculum was removed, and the cells were overlaid with medium containing the indicated concentrations of the tested compounds and 1.2% Avicel RC-591. At 3 dpi, the overlay was removed, and the cells were stained with 0.2% crystal violet. The images are representatives of two repeats. Data fitting of the antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay ( i ) and the plaque assay ( j ).
Figure Legend Snippet: Antiviral activity of GC-376 analogues. a-d Antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay. a GC-376 ; b UAWJ246 ; c UAWJ247 ; d UAWJ248 . Vero E6 cells in a 96-well plate were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in the presence of the indicated concentrations of the tested compounds. At 48 hpi, the cells were fixed and stained with a rabbit monoclonal antibody against the SARS-CoV-2 NP and a secondary antibody conjugated with Alexa 488 (Green). The nuclei were counterstained with Hoechst dye (Blue). For each well, fluorescence images of approximately 10K cells were acquired and shown. The images are representatives of two repeats. e-h Antiviral activity of GC-376 analogues against SARS-CoV-2 in the plaque assay. e GC-376 ; f UAWJ246 ; g UAWJ247 ; h UAWJ248 . Vero E6 cells in 6-well plates were infected with approximately 40 PFU/well of SARS-CoV-2 (USA-WA1/2020 isolate). After 1 hour, the inoculum was removed, and the cells were overlaid with medium containing the indicated concentrations of the tested compounds and 1.2% Avicel RC-591. At 3 dpi, the overlay was removed, and the cells were stained with 0.2% crystal violet. The images are representatives of two repeats. Data fitting of the antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay ( i ) and the plaque assay ( j ).

Techniques Used: Activity Assay, Immunofluorescence, Infection, Staining, Fluorescence, Plaque Assay

6) Product Images from "Spatial control of oxygen delivery to 3D cultures alters cancer cell growth and gene expression"

Article Title: Spatial control of oxygen delivery to 3D cultures alters cancer cell growth and gene expression

Journal: bioRxiv

doi: 10.1101/522656

Bioreactor schematics. (a) Exploded view of the dual-chamber bioreactor. (b) Illustration of air-PDMS interface with Cell+Matrigel matrix. (c) Custom PDMS 6-well plates, with magnetic plates shown in blue, PDMS membrane in green, and spacer in white. (d) Diagram of direction of diffusion of oxygen from the 3% O 2 bottom chamber into the 0% O 2 top chamber. (e) Photo of bioreactor in use with two gradient plates and two 3% O 2 plates.
Figure Legend Snippet: Bioreactor schematics. (a) Exploded view of the dual-chamber bioreactor. (b) Illustration of air-PDMS interface with Cell+Matrigel matrix. (c) Custom PDMS 6-well plates, with magnetic plates shown in blue, PDMS membrane in green, and spacer in white. (d) Diagram of direction of diffusion of oxygen from the 3% O 2 bottom chamber into the 0% O 2 top chamber. (e) Photo of bioreactor in use with two gradient plates and two 3% O 2 plates.

Techniques Used: Diffusion-based Assay

7) Product Images from "The secretome of human dental pulp stem cells protects myoblasts from hypoxia-induced injury via the Wnt/β-catenin pathway"

Article Title: The secretome of human dental pulp stem cells protects myoblasts from hypoxia-induced injury via the Wnt/β-catenin pathway

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2020.4525

hDPSCs alleviate hypoxia-induced injury of C2C12 myoblasts in the coculture model. (A) Establishing the hDPSCs-C2C12 coculture system and preparing the hDPSC-CM. Upper: The indirect coculture system with hDPSCs and C2C12 myoblasts was established. C2C12 and cultured hDPSCs were seeded in 6-well plates and Transwell-Clear inserts, respectively, to study the paracrine effects. Lower: The hDPSC growth medium was replaced with serum-free medium, after which time it was collected and concentrated using an ultrafiltration unit (48 h) and then added to regular medium after diluting to different concentrations. (B) Optical micrographs of C2C12 cell morphology and quantity change at 24–72 h. Scale bar, 200 μm. (C) Cell proliferation was assessed through Ki-67 immunofluorescence staining and (E) statistical analysis of the Ki-67 (green)-positive rate. Scale bar, 100 μm. (D) Cell cycle distribution was monitored using flow cytometry. (F) Statistical analysis of the cell cycle in C2C12 myoblasts. ## P
Figure Legend Snippet: hDPSCs alleviate hypoxia-induced injury of C2C12 myoblasts in the coculture model. (A) Establishing the hDPSCs-C2C12 coculture system and preparing the hDPSC-CM. Upper: The indirect coculture system with hDPSCs and C2C12 myoblasts was established. C2C12 and cultured hDPSCs were seeded in 6-well plates and Transwell-Clear inserts, respectively, to study the paracrine effects. Lower: The hDPSC growth medium was replaced with serum-free medium, after which time it was collected and concentrated using an ultrafiltration unit (48 h) and then added to regular medium after diluting to different concentrations. (B) Optical micrographs of C2C12 cell morphology and quantity change at 24–72 h. Scale bar, 200 μm. (C) Cell proliferation was assessed through Ki-67 immunofluorescence staining and (E) statistical analysis of the Ki-67 (green)-positive rate. Scale bar, 100 μm. (D) Cell cycle distribution was monitored using flow cytometry. (F) Statistical analysis of the cell cycle in C2C12 myoblasts. ## P

Techniques Used: Cell Culture, Immunofluorescence, Staining, Flow Cytometry

Related Articles

Concentration Assay:

Article Title: Effect of mechanical vibration stress in cell culture on human induced pluripotent stem cells
Article Snippet: .. Cells dissociated into single cells were seeded at the concentration of 5000 cells/well in 6-well plates (Corning Life Sciences, Corning, NY, USA). .. To examine vibrational effects on the early passages of iPSCs, cells were examined after passage three (P3), which we labeled as cycle 1.

Incubation:

Article Title: Titanium dioxide nanoparticles induce endoplasmic reticulum stress-mediated apoptotic cell death in liver cancer cells
Article Snippet: .. Solutions of TiO2 nanoparticles (400 µL) at concentrations of 0, 2.5, 5.0, 7.5, and 10 µg/mL in 75% ethanol were added to the bottom layer of 6-well plates (Costar Corning Inc., Corning, NY, USA) and incubated in an electric thermostatically controlled incubator at 60°C (DHP-9162, Shanghai Yiheng Technical Co., Ltd., Shanghai, China). ..

Crocin Bleaching Assay:

Article Title: c-Jun N-terminal kinase (JNK) is required for survival and proliferation of B-lymphoma cells
Article Snippet: .. BKS-2 B-lymphoma cells, harvested from CBA/N mice and depleted of T cells, were treated with SP600125 at concentrations from 5 to 15 μM in 1-mL cultures of 1 × 106 cells in 6-well plates (Corning/Costar, Cambridge, MA). .. Cell lysates were prepared in 1 × sodium dodecyl sulfate (SDS) sample buffer or 1% Triton-X 100 as described earlier , and were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.

Infection:

Article Title: Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L
Article Snippet: .. Plaque assayVero E6 cells in 6-well plates (Corning) were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at approximately 40 PFU per well. .. After 1 hour of incubation at 37°C, the inoculum was removed and replaced with DMEM containing 1% FBS, 1.2% Avicel RC-591 (Dupont) and the tested compounds at different concentrations, in duplicate.

Mouse Assay:

Article Title: c-Jun N-terminal kinase (JNK) is required for survival and proliferation of B-lymphoma cells
Article Snippet: .. BKS-2 B-lymphoma cells, harvested from CBA/N mice and depleted of T cells, were treated with SP600125 at concentrations from 5 to 15 μM in 1-mL cultures of 1 × 106 cells in 6-well plates (Corning/Costar, Cambridge, MA). .. Cell lysates were prepared in 1 × sodium dodecyl sulfate (SDS) sample buffer or 1% Triton-X 100 as described earlier , and were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.

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    Corning Life Sciences ovca cells
    Metformin Represses SUCLG2 and TCA Intermediates to Inhibit HIF1α in Mesothelial Cells (A) Metabolomics. MS of metabolites extracted from mesothelial cells <t>(HPMCs)</t> treated with metformin (1 mM, 48 h)were analyzed using a two-sided t test with a FDR of 0.25 and an S 0 value of 0.125. Left: volcano plot showing significant metabolites by red dots (n = 2 patients in duplicate). Right: principal-component analysis (PCA) assessing metabolite profiles of HPMCs treated with metformin compared to control. Open circles represent each group (n = 2 patients in duplicate). (B) Proteomics. MS of proteins extracted from HPMCs treated with metformin were analyzed using a two-sided t test with an FDR of 0.05 and an S 0 value of 0.1. Left: volcano plot showing significant proteins by red dots (n = 4 patients in triplicate). Right: PCA assessing protein profiles of HPMCs treated with metformin compared to control. Open circles represent each group (n = 4 patients in triplicate). (C) Relative MS intensity of TCA metabolites in HPMCs treated with metformin from (A) was analyzed using a paired t test. (D) Relative protein expression of the TCA enzyme SUCLG2 in HPMCs treated with metformin from (B) was analyzed using a paired t test. (E) Western blot of HIF1α expression. HPMCs treated with metformin (250 μM, 72 h) followed by the addition of TYKnu <t>OvCa</t> CM ± succinate (suc, 5 mM) (representative blot from n = 2 patients in duplicate). (F) Prolyl hydroxylase (PHD) activity using a GFP reporter construct. A GFP-tagged HIF1α oxygen-dependent degradation domain construct (ODD-GFP) was expressed in HPMCs; cells were then treated with TYKnu OvCa CM ± metformin (1 mM) ± succinate (suc, 5 mM) for 16 h. An increased GFP signal indicates depleted PHD activity (n = 2 patients in duplicate). (G) Western blot of HIF1α expression. HPMCs were transfected with SUCLG2 and then treated with TYKnu OvCa CM ± metformin (1 mM, 48 h) (representative blot from n = 3 patients in duplicate). (H) SUCLG2 immunohistochemical staining in primary ovarian and omental metastasis from OvCa patients with diabetes using metformin compared to non-diabetic patients with OvCa not taking metformin. Quantification of primary tumor (n = 15 and 17 for control and metformin, respectively) or omental metastasis (n = 12 and 8 for control and metformin, respectively) was performed using ImageScope software. Images are representative and were taken at 20× magnification; scale bar represents 100 μm. Data represent mean values ± SDs. #p
    Ovca Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences 96 well culture plates
    C3larvin and C3 toxin cell entry experiments. Cell morphology assays were performed with J774A.1 mouse macrophage cells that were grown to confluence in 25-cm 2 culture flasks, the cells were resuspended, and 100 μl was transferred to 6- or <t>96-well</t>
    96 Well Culture Plates, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences isc 4
    Inhibition of Akt signaling in AML by <t>ISC-4.</t> (A) Western blot analysis of AML cells exposed to increasing concentrations of ISC-4 or GDC-0941 (1 μM) for 24 h. GAPDH was used as a loading control. (B) Densitometric quantification of western blot bands. (C) Detection of PI3K activation by flow cytometry in bulk primary human AML cells ( n = 3). (D,E) Flow cytometric detection of phospho-Akt (p-Akt) in CD34 + primary human AML cells ( n = 5) after treatment with DMSO or ISC-4 (1–10 μM). Results are mean ± standard error of the mean (SEM). * P
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    Corning Life Sciences collagen i
    IL-1 but not IL-33 affects fibroblast <t>collagen</t> I gel contraction and fiber formation. Primary airway fibroblasts (PAFs) from non-asthmatics and asthmatics were grown to confluence and seeded on 3D collagen I gels in the presence or absence of 1 ng/ml IL-1α, IL-1β or IL-33 and allowed to migrated into and contract the gel for 24 hours. ( a ) Representative gel contraction images after the 24 hour stimulation of non-asthmatic and asthmatic PAF-seeded gels with 1 ng/ml IL-1α, IL-1β or IL-33, ( b ) Percentage gel contraction comparing control non-stimulated rates of non-asthmatic and asthmatic -derived PAFs, ( c ) Percentage gel contraction after stimulation with IL-1α, IL-1β and IL-33, ( d ) semi-dry weight of contracted gels after stimulation with IL-1α, IL-1β and IL-33. ( e ) Representative images of fibrillar collagen taken with second harmonic generation (SHG) microscopy in gels, ( f ) SHG peak intensity of fibrillar collagen. ( g ) Entropy of collagen fibers, calculated using texturel analysis of SHG images. Representative images are 20X magnification and scale bars are provided. Exact P values indicated. SHG images are pseudo-coloured, showing fibrillar collagen in blue (un-labeled).
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    Metformin Represses SUCLG2 and TCA Intermediates to Inhibit HIF1α in Mesothelial Cells (A) Metabolomics. MS of metabolites extracted from mesothelial cells (HPMCs) treated with metformin (1 mM, 48 h)were analyzed using a two-sided t test with a FDR of 0.25 and an S 0 value of 0.125. Left: volcano plot showing significant metabolites by red dots (n = 2 patients in duplicate). Right: principal-component analysis (PCA) assessing metabolite profiles of HPMCs treated with metformin compared to control. Open circles represent each group (n = 2 patients in duplicate). (B) Proteomics. MS of proteins extracted from HPMCs treated with metformin were analyzed using a two-sided t test with an FDR of 0.05 and an S 0 value of 0.1. Left: volcano plot showing significant proteins by red dots (n = 4 patients in triplicate). Right: PCA assessing protein profiles of HPMCs treated with metformin compared to control. Open circles represent each group (n = 4 patients in triplicate). (C) Relative MS intensity of TCA metabolites in HPMCs treated with metformin from (A) was analyzed using a paired t test. (D) Relative protein expression of the TCA enzyme SUCLG2 in HPMCs treated with metformin from (B) was analyzed using a paired t test. (E) Western blot of HIF1α expression. HPMCs treated with metformin (250 μM, 72 h) followed by the addition of TYKnu OvCa CM ± succinate (suc, 5 mM) (representative blot from n = 2 patients in duplicate). (F) Prolyl hydroxylase (PHD) activity using a GFP reporter construct. A GFP-tagged HIF1α oxygen-dependent degradation domain construct (ODD-GFP) was expressed in HPMCs; cells were then treated with TYKnu OvCa CM ± metformin (1 mM) ± succinate (suc, 5 mM) for 16 h. An increased GFP signal indicates depleted PHD activity (n = 2 patients in duplicate). (G) Western blot of HIF1α expression. HPMCs were transfected with SUCLG2 and then treated with TYKnu OvCa CM ± metformin (1 mM, 48 h) (representative blot from n = 3 patients in duplicate). (H) SUCLG2 immunohistochemical staining in primary ovarian and omental metastasis from OvCa patients with diabetes using metformin compared to non-diabetic patients with OvCa not taking metformin. Quantification of primary tumor (n = 15 and 17 for control and metformin, respectively) or omental metastasis (n = 12 and 8 for control and metformin, respectively) was performed using ImageScope software. Images are representative and were taken at 20× magnification; scale bar represents 100 μm. Data represent mean values ± SDs. #p

    Journal: Cell reports

    Article Title: Mesothelial Cell HIF1α Expression Is Metabolically Downregulated by Metformin to Prevent Oncogenic Tumor-Stromal Crosstalk

    doi: 10.1016/j.celrep.2019.11.079

    Figure Lengend Snippet: Metformin Represses SUCLG2 and TCA Intermediates to Inhibit HIF1α in Mesothelial Cells (A) Metabolomics. MS of metabolites extracted from mesothelial cells (HPMCs) treated with metformin (1 mM, 48 h)were analyzed using a two-sided t test with a FDR of 0.25 and an S 0 value of 0.125. Left: volcano plot showing significant metabolites by red dots (n = 2 patients in duplicate). Right: principal-component analysis (PCA) assessing metabolite profiles of HPMCs treated with metformin compared to control. Open circles represent each group (n = 2 patients in duplicate). (B) Proteomics. MS of proteins extracted from HPMCs treated with metformin were analyzed using a two-sided t test with an FDR of 0.05 and an S 0 value of 0.1. Left: volcano plot showing significant proteins by red dots (n = 4 patients in triplicate). Right: PCA assessing protein profiles of HPMCs treated with metformin compared to control. Open circles represent each group (n = 4 patients in triplicate). (C) Relative MS intensity of TCA metabolites in HPMCs treated with metformin from (A) was analyzed using a paired t test. (D) Relative protein expression of the TCA enzyme SUCLG2 in HPMCs treated with metformin from (B) was analyzed using a paired t test. (E) Western blot of HIF1α expression. HPMCs treated with metformin (250 μM, 72 h) followed by the addition of TYKnu OvCa CM ± succinate (suc, 5 mM) (representative blot from n = 2 patients in duplicate). (F) Prolyl hydroxylase (PHD) activity using a GFP reporter construct. A GFP-tagged HIF1α oxygen-dependent degradation domain construct (ODD-GFP) was expressed in HPMCs; cells were then treated with TYKnu OvCa CM ± metformin (1 mM) ± succinate (suc, 5 mM) for 16 h. An increased GFP signal indicates depleted PHD activity (n = 2 patients in duplicate). (G) Western blot of HIF1α expression. HPMCs were transfected with SUCLG2 and then treated with TYKnu OvCa CM ± metformin (1 mM, 48 h) (representative blot from n = 3 patients in duplicate). (H) SUCLG2 immunohistochemical staining in primary ovarian and omental metastasis from OvCa patients with diabetes using metformin compared to non-diabetic patients with OvCa not taking metformin. Quantification of primary tumor (n = 15 and 17 for control and metformin, respectively) or omental metastasis (n = 12 and 8 for control and metformin, respectively) was performed using ImageScope software. Images are representative and were taken at 20× magnification; scale bar represents 100 μm. Data represent mean values ± SDs. #p

    Article Snippet: For proteomic analysis of mesothelial cells co-cultured with OvCa cells, HPMCs were plated at 150,000 cells per well on a 6-well plate and OvCa cells were plated at 100,000 cells per transwell polyester membrane insert (0.4μm pore size, for use with 6-well plate; Corning) on a separate plate.

    Techniques: Expressing, Western Blot, Activity Assay, Construct, Transfection, Immunohistochemistry, Staining, Software

    Metformin Targets OvCa TGF-β1 Signaling to Inhibit HIF1α Stabilization in HPMCs (A) Proteomics. MS of proteins extracted from DOV13 OvCa cells treated with metformin (1 mM, 72 h) were analyzed using a two-sided t test. Color gradient indicates the density of the distribution, with green dots as outer 5%. Proteins related to the TGF-β pathway are annotated for comparison. Top pathways regulated by metformin were determined by one-dimensional pathway enrichment analysis using FDR

    Journal: Cell reports

    Article Title: Mesothelial Cell HIF1α Expression Is Metabolically Downregulated by Metformin to Prevent Oncogenic Tumor-Stromal Crosstalk

    doi: 10.1016/j.celrep.2019.11.079

    Figure Lengend Snippet: Metformin Targets OvCa TGF-β1 Signaling to Inhibit HIF1α Stabilization in HPMCs (A) Proteomics. MS of proteins extracted from DOV13 OvCa cells treated with metformin (1 mM, 72 h) were analyzed using a two-sided t test. Color gradient indicates the density of the distribution, with green dots as outer 5%. Proteins related to the TGF-β pathway are annotated for comparison. Top pathways regulated by metformin were determined by one-dimensional pathway enrichment analysis using FDR

    Article Snippet: For proteomic analysis of mesothelial cells co-cultured with OvCa cells, HPMCs were plated at 150,000 cells per well on a 6-well plate and OvCa cells were plated at 100,000 cells per transwell polyester membrane insert (0.4μm pore size, for use with 6-well plate; Corning) on a separate plate.

    Techniques:

    Metformin Prevents Tumor-Cell-Induced Stabilization of HIF1α in Mesothelial Cells (A) Mass spectrometry (MS) analysis of mesothelial cells (HPMCs) co-cultured with OvCa cells and treated with metformin (1 mM, 48 h). Cell lysates were collected and measured on a Q Exactive HF mass spectrometer, and the quantification of proteins significantly altered by metformin is shown by volcano plot (Perseus), with the significantly altered proteins highlighted in red. Significance was defined by a false discovery rate (FDR) of 0.05 and an S 0 value of 0.05 (n = 4 patients in triplicate). (B) Unsupervised hierarchical clustering (Perseus) was performed on the dataset from (A), assessing hypoxia-related proteins in HPMCs following exposure to TYKnu OvCa co-culture in the presence or absence of metformin (1 mM, 48 h) (n = 4 patients per group in triplicate). (C) Western blot of HIF1α and HIF2α protein expression in HPMCs following exposure to TYKnu OvCa co-culture in the presence of metformin (1 mM, 48 h) (representative blot from n = 2 patients in duplicate). (D) Western blot of HIF1α protein expression in HPMCs expressing constitutively stable HIF1α (P402A/P564A substitutions) ± metformin (1 mM, 48 h) (representative blot from n = 2 patients in duplicate). (E) qRT-PCR of IL-8 (left) and CCL2 (right) mRNA expression in HPMCs transfected with constitutively stable HIF1α (P402A/P564A) with or without metformin (1 mM) (n = 2 patients in duplicate). (F) Invasion of TYKnu OvCa cells (36 h) in 3D culture with metformin (pretreatment of HPMCs with 250 μM for 72 h) in the presence of N -oxalylglycine (OG, 1 mM) and 2-oxoglutarate (2-oxo, 5 mM) (n = 2 patients per group in triplicate). (G) Invasion of TYKnu OvCa cells (36 h) in 3D culture containing control or HPMCs expressing constitutively stable HIF1α (P402A/P564A) treated with metformin (pretreatment of HPMCs with 250 μM for 72 h), with concurrent exposure to IL-8RA/CXCR1 neutralizing antibody (150 ng/mL) during the invasion assay (n = 2 patients in duplicate). (H) Western blot of HIF1α and CAIX expression in normal/benign omental explants from patients using metformin for diabetes compared to control patients without diabetes not using metformin. Rightside, densitometry (area under the curve [AUC]) of western immunoblot bands of HIF1α and CAIX was quantified and normalized to vinculin (n = 3 patients per group in duplicate). (I) CAIX immunohistochemical staining in primary ovarian and omental tumors from patients with OvCa who were using metformin for diabetes compared to non-diabetic patients with OvCa not taking metformin. Quantification of primary tumor (n = 15 and 18 for control and metformin, respectively) or omental metastasis (n = 15 and 12 for control and metformin, respectively) was performed using ImageScope software. Images are representative and were taken at 20× magnification; scale bar represents 100 μm. Data represent mean values ± SDs. *p

    Journal: Cell reports

    Article Title: Mesothelial Cell HIF1α Expression Is Metabolically Downregulated by Metformin to Prevent Oncogenic Tumor-Stromal Crosstalk

    doi: 10.1016/j.celrep.2019.11.079

    Figure Lengend Snippet: Metformin Prevents Tumor-Cell-Induced Stabilization of HIF1α in Mesothelial Cells (A) Mass spectrometry (MS) analysis of mesothelial cells (HPMCs) co-cultured with OvCa cells and treated with metformin (1 mM, 48 h). Cell lysates were collected and measured on a Q Exactive HF mass spectrometer, and the quantification of proteins significantly altered by metformin is shown by volcano plot (Perseus), with the significantly altered proteins highlighted in red. Significance was defined by a false discovery rate (FDR) of 0.05 and an S 0 value of 0.05 (n = 4 patients in triplicate). (B) Unsupervised hierarchical clustering (Perseus) was performed on the dataset from (A), assessing hypoxia-related proteins in HPMCs following exposure to TYKnu OvCa co-culture in the presence or absence of metformin (1 mM, 48 h) (n = 4 patients per group in triplicate). (C) Western blot of HIF1α and HIF2α protein expression in HPMCs following exposure to TYKnu OvCa co-culture in the presence of metformin (1 mM, 48 h) (representative blot from n = 2 patients in duplicate). (D) Western blot of HIF1α protein expression in HPMCs expressing constitutively stable HIF1α (P402A/P564A substitutions) ± metformin (1 mM, 48 h) (representative blot from n = 2 patients in duplicate). (E) qRT-PCR of IL-8 (left) and CCL2 (right) mRNA expression in HPMCs transfected with constitutively stable HIF1α (P402A/P564A) with or without metformin (1 mM) (n = 2 patients in duplicate). (F) Invasion of TYKnu OvCa cells (36 h) in 3D culture with metformin (pretreatment of HPMCs with 250 μM for 72 h) in the presence of N -oxalylglycine (OG, 1 mM) and 2-oxoglutarate (2-oxo, 5 mM) (n = 2 patients per group in triplicate). (G) Invasion of TYKnu OvCa cells (36 h) in 3D culture containing control or HPMCs expressing constitutively stable HIF1α (P402A/P564A) treated with metformin (pretreatment of HPMCs with 250 μM for 72 h), with concurrent exposure to IL-8RA/CXCR1 neutralizing antibody (150 ng/mL) during the invasion assay (n = 2 patients in duplicate). (H) Western blot of HIF1α and CAIX expression in normal/benign omental explants from patients using metformin for diabetes compared to control patients without diabetes not using metformin. Rightside, densitometry (area under the curve [AUC]) of western immunoblot bands of HIF1α and CAIX was quantified and normalized to vinculin (n = 3 patients per group in duplicate). (I) CAIX immunohistochemical staining in primary ovarian and omental tumors from patients with OvCa who were using metformin for diabetes compared to non-diabetic patients with OvCa not taking metformin. Quantification of primary tumor (n = 15 and 18 for control and metformin, respectively) or omental metastasis (n = 15 and 12 for control and metformin, respectively) was performed using ImageScope software. Images are representative and were taken at 20× magnification; scale bar represents 100 μm. Data represent mean values ± SDs. *p

    Article Snippet: For proteomic analysis of mesothelial cells co-cultured with OvCa cells, HPMCs were plated at 150,000 cells per well on a 6-well plate and OvCa cells were plated at 100,000 cells per transwell polyester membrane insert (0.4μm pore size, for use with 6-well plate; Corning) on a separate plate.

    Techniques: Mass Spectrometry, Cell Culture, Co-Culture Assay, Western Blot, Expressing, Quantitative RT-PCR, Transfection, Invasion Assay, Immunohistochemistry, Staining, Software

    Suppression of Mesothelial Cell-Derived CCL2 and IL-8 Is Required for the Inhibition of Invasion In Vitro and the Colonization Ex Vivo by Metformin for details) (n = 2 mice per group). (B) Validation of candidate cytokines CXCL1 and CCL2 from (A) using ELISA (n = 8 mice per group). (C) qRT-PCR of IL-8 mRNA expression in HPMCs exposed to TYKnu OvCa CM (36 h). HPMCs were pretreated with metformin (250 μM) for 72 h before CM exposure (n = 4 patients in duplicate). (D) Invasion of TYKnu OvCa cells (36 h) through the 3D model with or without metformin pretreatment of HPMCs (250 μM for 72 h), or simultaneous treatment ± recombinant human IL-8 protein (150 ng/mL) (n = 2 patients in triplicate). (E) qRT-PCR of CCL2 mRNA expression in HPMCs subjected to TYKnu OvCa CM (36 h). HPMCs were pretreated with metformin (250 μM) for 72 h before CM exposure (n = 4 patients in duplicate). (F) Invasion of TYKnu OvCa cells (36 h) through the 3D model with or without metformin pretreatment of HPMCs (250 μM for 72 h), or simultaneous treatment ± recombinant human CCL2 protein (150 ng/mL) (n = 2 patients in triplicate). (G and H) 3D invasion (G) or omental explant colonization (H): TYKnu OvCa cells expressing constitutively active IL-8 receptor (CXCR1-V247N) or CCL2 receptor (CCR2-T94K) were added to the 3D model constructed with HPMCs pretreated with metformin (G) or to a human omental explant pretreated with metformin (H). Data represent mean values ± SDs. #p

    Journal: Cell reports

    Article Title: Mesothelial Cell HIF1α Expression Is Metabolically Downregulated by Metformin to Prevent Oncogenic Tumor-Stromal Crosstalk

    doi: 10.1016/j.celrep.2019.11.079

    Figure Lengend Snippet: Suppression of Mesothelial Cell-Derived CCL2 and IL-8 Is Required for the Inhibition of Invasion In Vitro and the Colonization Ex Vivo by Metformin for details) (n = 2 mice per group). (B) Validation of candidate cytokines CXCL1 and CCL2 from (A) using ELISA (n = 8 mice per group). (C) qRT-PCR of IL-8 mRNA expression in HPMCs exposed to TYKnu OvCa CM (36 h). HPMCs were pretreated with metformin (250 μM) for 72 h before CM exposure (n = 4 patients in duplicate). (D) Invasion of TYKnu OvCa cells (36 h) through the 3D model with or without metformin pretreatment of HPMCs (250 μM for 72 h), or simultaneous treatment ± recombinant human IL-8 protein (150 ng/mL) (n = 2 patients in triplicate). (E) qRT-PCR of CCL2 mRNA expression in HPMCs subjected to TYKnu OvCa CM (36 h). HPMCs were pretreated with metformin (250 μM) for 72 h before CM exposure (n = 4 patients in duplicate). (F) Invasion of TYKnu OvCa cells (36 h) through the 3D model with or without metformin pretreatment of HPMCs (250 μM for 72 h), or simultaneous treatment ± recombinant human CCL2 protein (150 ng/mL) (n = 2 patients in triplicate). (G and H) 3D invasion (G) or omental explant colonization (H): TYKnu OvCa cells expressing constitutively active IL-8 receptor (CXCR1-V247N) or CCL2 receptor (CCR2-T94K) were added to the 3D model constructed with HPMCs pretreated with metformin (G) or to a human omental explant pretreated with metformin (H). Data represent mean values ± SDs. #p

    Article Snippet: For proteomic analysis of mesothelial cells co-cultured with OvCa cells, HPMCs were plated at 150,000 cells per well on a 6-well plate and OvCa cells were plated at 100,000 cells per transwell polyester membrane insert (0.4μm pore size, for use with 6-well plate; Corning) on a separate plate.

    Techniques: Derivative Assay, Inhibition, In Vitro, Ex Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Recombinant, Construct

    Metformin Inhibits Colonization of the Omentum Ex Vivo and Invasion of the 3D TME In Vitro (A) Schematic: ex vivo adhesion of GFP-tagged HeyA8 OvCa cells to fresh human omental biopsies from patients without cancer taking metformin for type 2 diabetes or control patients not taking metformin. (B) Omental explants from control or metformin patients were seeded with GFP-tagged HeyA8 OvCa cells. Tumor cells were allowed to adhere to omental explants for 1.5 h before imaging and quantification (n = 3 patients per group in triplicate). Images are representative and were taken at 10× magnification; scale bar represents 200 μm. (C and D) Ex vivo adhesion and colonization: human omental explants were pretreated with metformin (250 μM) for 72 h before seeding of GFP-tagged HeyA8 OvCa cells, and the number of tumor cells that adhered to (1.5 h, C) or colonized (72 h, D) per omentum were quantified (n = 2 patients in triplicate for both experiments). (E) Migration of HeyA8 OvCa cells through transwell chamber (15 h) toward omental explants that had been pretreated with metformin (250 μM, 72 h). During the assay, metformin was removed, and all media were replaced with serum-free media containing 0.1% BSA (n = 2 patients in triplicate). (F) Invasion through 3D organotypic model (36 h) after seeding of TYKnu OvCa cells. Each cell type was individually treated with metformin (250 μM, 72 h) before the 3D model was constructed. HPMC, human primary mesothelial cells; NOF, normal omental fibroblast; n = 3 patients in triplicate. (G) Invasion of TYKnu cells through 3D model (36 h) treated with the indicated compounds: metformin (1 mM), AMPK inhibitor compound C (CompC, 1 μM), and AMPK activator AICAR (1 mM) (n = 3 patients in triplicate). Data represent mean values ± SDs. *p

    Journal: Cell reports

    Article Title: Mesothelial Cell HIF1α Expression Is Metabolically Downregulated by Metformin to Prevent Oncogenic Tumor-Stromal Crosstalk

    doi: 10.1016/j.celrep.2019.11.079

    Figure Lengend Snippet: Metformin Inhibits Colonization of the Omentum Ex Vivo and Invasion of the 3D TME In Vitro (A) Schematic: ex vivo adhesion of GFP-tagged HeyA8 OvCa cells to fresh human omental biopsies from patients without cancer taking metformin for type 2 diabetes or control patients not taking metformin. (B) Omental explants from control or metformin patients were seeded with GFP-tagged HeyA8 OvCa cells. Tumor cells were allowed to adhere to omental explants for 1.5 h before imaging and quantification (n = 3 patients per group in triplicate). Images are representative and were taken at 10× magnification; scale bar represents 200 μm. (C and D) Ex vivo adhesion and colonization: human omental explants were pretreated with metformin (250 μM) for 72 h before seeding of GFP-tagged HeyA8 OvCa cells, and the number of tumor cells that adhered to (1.5 h, C) or colonized (72 h, D) per omentum were quantified (n = 2 patients in triplicate for both experiments). (E) Migration of HeyA8 OvCa cells through transwell chamber (15 h) toward omental explants that had been pretreated with metformin (250 μM, 72 h). During the assay, metformin was removed, and all media were replaced with serum-free media containing 0.1% BSA (n = 2 patients in triplicate). (F) Invasion through 3D organotypic model (36 h) after seeding of TYKnu OvCa cells. Each cell type was individually treated with metformin (250 μM, 72 h) before the 3D model was constructed. HPMC, human primary mesothelial cells; NOF, normal omental fibroblast; n = 3 patients in triplicate. (G) Invasion of TYKnu cells through 3D model (36 h) treated with the indicated compounds: metformin (1 mM), AMPK inhibitor compound C (CompC, 1 μM), and AMPK activator AICAR (1 mM) (n = 3 patients in triplicate). Data represent mean values ± SDs. *p

    Article Snippet: For proteomic analysis of mesothelial cells co-cultured with OvCa cells, HPMCs were plated at 150,000 cells per well on a 6-well plate and OvCa cells were plated at 100,000 cells per transwell polyester membrane insert (0.4μm pore size, for use with 6-well plate; Corning) on a separate plate.

    Techniques: Ex Vivo, In Vitro, Imaging, Migration, Construct

    C3larvin and C3 toxin cell entry experiments. Cell morphology assays were performed with J774A.1 mouse macrophage cells that were grown to confluence in 25-cm 2 culture flasks, the cells were resuspended, and 100 μl was transferred to 6- or 96-well

    Journal: The Journal of Biological Chemistry

    Article Title: C3larvin Toxin, an ADP-ribosyltransferase from Paenibacillus larvae *

    doi: 10.1074/jbc.M114.589846

    Figure Lengend Snippet: C3larvin and C3 toxin cell entry experiments. Cell morphology assays were performed with J774A.1 mouse macrophage cells that were grown to confluence in 25-cm 2 culture flasks, the cells were resuspended, and 100 μl was transferred to 6- or 96-well

    Article Snippet: J774A.1 or RAW 264.7 cells were grown to confluence in 25-cm2 culture flasks, the cells were resuspended, and 100 μl was transferred to 6-well or 96-well culture plates (Corning) containing 4 ml of supplemented medium (200 μl for the 96-well plates).

    Techniques:

    Inhibition of Akt signaling in AML by ISC-4. (A) Western blot analysis of AML cells exposed to increasing concentrations of ISC-4 or GDC-0941 (1 μM) for 24 h. GAPDH was used as a loading control. (B) Densitometric quantification of western blot bands. (C) Detection of PI3K activation by flow cytometry in bulk primary human AML cells ( n = 3). (D,E) Flow cytometric detection of phospho-Akt (p-Akt) in CD34 + primary human AML cells ( n = 5) after treatment with DMSO or ISC-4 (1–10 μM). Results are mean ± standard error of the mean (SEM). * P

    Journal: Frontiers in Oncology

    Article Title: The PI3K/AKT Pathway Inhibitor ISC-4 Induces Apoptosis and Inhibits Growth of Leukemia in Preclinical Models of Acute Myeloid Leukemia

    doi: 10.3389/fonc.2020.00393

    Figure Lengend Snippet: Inhibition of Akt signaling in AML by ISC-4. (A) Western blot analysis of AML cells exposed to increasing concentrations of ISC-4 or GDC-0941 (1 μM) for 24 h. GAPDH was used as a loading control. (B) Densitometric quantification of western blot bands. (C) Detection of PI3K activation by flow cytometry in bulk primary human AML cells ( n = 3). (D,E) Flow cytometric detection of phospho-Akt (p-Akt) in CD34 + primary human AML cells ( n = 5) after treatment with DMSO or ISC-4 (1–10 μM). Results are mean ± standard error of the mean (SEM). * P

    Article Snippet: Cell Cycle Analysis Cells were plated in 6-well plate and treated with increasing concentrations of ISC-4 (0.5–3 μM) for 24 h. Post-treatment 1 × 106 cells were washed with phosphate-buffered saline (PBS) (Corning) and fixed in chilled 70% ethanol for at least 3 h. Cells were washed with PBS and resuspended in 200 μL of Muse Cell Cycle reagent (MilliporeSigma), incubated for 30 min at room temperature, and analyzed on Muse Cell Analyzer (MilliporeSigma).

    Techniques: Inhibition, Western Blot, Activation Assay, Flow Cytometry

    ISC-4 inhibits leukemia progression in vivo and extends overall survival. (A–D) Albino B6 mice ( n = 3) transplanted with Luciferase and dsRed-expressing C1498 (C1498-dsRed-Luc) were treated either with vehicle control (DMSO) or ISC-4 (7 mg/kg). (A) Experimental scheme of C1498 animal study. (B,C) The decrease in the leukemic burden of ISC-4-treated mice monitored by bioluminescence imaging over the time course of the study. Data are mean ± SEM, * P

    Journal: Frontiers in Oncology

    Article Title: The PI3K/AKT Pathway Inhibitor ISC-4 Induces Apoptosis and Inhibits Growth of Leukemia in Preclinical Models of Acute Myeloid Leukemia

    doi: 10.3389/fonc.2020.00393

    Figure Lengend Snippet: ISC-4 inhibits leukemia progression in vivo and extends overall survival. (A–D) Albino B6 mice ( n = 3) transplanted with Luciferase and dsRed-expressing C1498 (C1498-dsRed-Luc) were treated either with vehicle control (DMSO) or ISC-4 (7 mg/kg). (A) Experimental scheme of C1498 animal study. (B,C) The decrease in the leukemic burden of ISC-4-treated mice monitored by bioluminescence imaging over the time course of the study. Data are mean ± SEM, * P

    Article Snippet: Cell Cycle Analysis Cells were plated in 6-well plate and treated with increasing concentrations of ISC-4 (0.5–3 μM) for 24 h. Post-treatment 1 × 106 cells were washed with phosphate-buffered saline (PBS) (Corning) and fixed in chilled 70% ethanol for at least 3 h. Cells were washed with PBS and resuspended in 200 μL of Muse Cell Cycle reagent (MilliporeSigma), incubated for 30 min at room temperature, and analyzed on Muse Cell Analyzer (MilliporeSigma).

    Techniques: In Vivo, Mouse Assay, Luciferase, Expressing, Imaging

    Effect of ISC-4 on AML cell proliferation. (A) Sensitivity of AML cell lines ( n = 7) to ISC-4 (0.75–24 μM) after 24 h of treatment. (B) Inhibition of cell growth in MV4-11 and OCI-AML3 cells with ISC-4 treatment. (C) Effect of ISC-4 and cytarabine (AraC) combination treatment on U937 cell viability at 72 h (D) ISC-4-mediated reduction in clonogenicity of human AML cell lines in colony growth medium. (E) Sensitivity of primary human AML cells or cord blood mononuclear cells clonogenicity to ISC-4 treatment. Data are the mean ± standard deviation (SD) **** P

    Journal: Frontiers in Oncology

    Article Title: The PI3K/AKT Pathway Inhibitor ISC-4 Induces Apoptosis and Inhibits Growth of Leukemia in Preclinical Models of Acute Myeloid Leukemia

    doi: 10.3389/fonc.2020.00393

    Figure Lengend Snippet: Effect of ISC-4 on AML cell proliferation. (A) Sensitivity of AML cell lines ( n = 7) to ISC-4 (0.75–24 μM) after 24 h of treatment. (B) Inhibition of cell growth in MV4-11 and OCI-AML3 cells with ISC-4 treatment. (C) Effect of ISC-4 and cytarabine (AraC) combination treatment on U937 cell viability at 72 h (D) ISC-4-mediated reduction in clonogenicity of human AML cell lines in colony growth medium. (E) Sensitivity of primary human AML cells or cord blood mononuclear cells clonogenicity to ISC-4 treatment. Data are the mean ± standard deviation (SD) **** P

    Article Snippet: Cell Cycle Analysis Cells were plated in 6-well plate and treated with increasing concentrations of ISC-4 (0.5–3 μM) for 24 h. Post-treatment 1 × 106 cells were washed with phosphate-buffered saline (PBS) (Corning) and fixed in chilled 70% ethanol for at least 3 h. Cells were washed with PBS and resuspended in 200 μL of Muse Cell Cycle reagent (MilliporeSigma), incubated for 30 min at room temperature, and analyzed on Muse Cell Analyzer (MilliporeSigma).

    Techniques: Inhibition, Standard Deviation

    ISC-4 induces apoptosis in AML. (A,B) ISC-4-mediated apoptosis in human AML cell lines as the percentage of Annexin V + or Caspase-3/7 activity. (C) Increase in Annexin V + primary human AML cells after treatment (24 h) with ISC-4 ( n = 11) or cytarabine (AraC, n = 8). Data are the mean ± SEM, * P

    Journal: Frontiers in Oncology

    Article Title: The PI3K/AKT Pathway Inhibitor ISC-4 Induces Apoptosis and Inhibits Growth of Leukemia in Preclinical Models of Acute Myeloid Leukemia

    doi: 10.3389/fonc.2020.00393

    Figure Lengend Snippet: ISC-4 induces apoptosis in AML. (A,B) ISC-4-mediated apoptosis in human AML cell lines as the percentage of Annexin V + or Caspase-3/7 activity. (C) Increase in Annexin V + primary human AML cells after treatment (24 h) with ISC-4 ( n = 11) or cytarabine (AraC, n = 8). Data are the mean ± SEM, * P

    Article Snippet: Cell Cycle Analysis Cells were plated in 6-well plate and treated with increasing concentrations of ISC-4 (0.5–3 μM) for 24 h. Post-treatment 1 × 106 cells were washed with phosphate-buffered saline (PBS) (Corning) and fixed in chilled 70% ethanol for at least 3 h. Cells were washed with PBS and resuspended in 200 μL of Muse Cell Cycle reagent (MilliporeSigma), incubated for 30 min at room temperature, and analyzed on Muse Cell Analyzer (MilliporeSigma).

    Techniques: Activity Assay

    ISC-4 induces apoptosis in primary human leukemic stem cells. (A) Dose-dependent apoptotic response of bulk primary human cells (CD45 + ), or leukemic stem cells (CD34 + or CD123 + cells) to ISC-4. Error bars are mean ± SEM. (B) Reduction in leukemic stem cells after ISC-4 treatment in AML Pt. 1172. (C) Apoptosis in CD45 + , CD34 + , or TIM-3 + cells after ISC-4 and cytarabine (AraC) combination treatment. Error bars are mean ± SEM. * P

    Journal: Frontiers in Oncology

    Article Title: The PI3K/AKT Pathway Inhibitor ISC-4 Induces Apoptosis and Inhibits Growth of Leukemia in Preclinical Models of Acute Myeloid Leukemia

    doi: 10.3389/fonc.2020.00393

    Figure Lengend Snippet: ISC-4 induces apoptosis in primary human leukemic stem cells. (A) Dose-dependent apoptotic response of bulk primary human cells (CD45 + ), or leukemic stem cells (CD34 + or CD123 + cells) to ISC-4. Error bars are mean ± SEM. (B) Reduction in leukemic stem cells after ISC-4 treatment in AML Pt. 1172. (C) Apoptosis in CD45 + , CD34 + , or TIM-3 + cells after ISC-4 and cytarabine (AraC) combination treatment. Error bars are mean ± SEM. * P

    Article Snippet: Cell Cycle Analysis Cells were plated in 6-well plate and treated with increasing concentrations of ISC-4 (0.5–3 μM) for 24 h. Post-treatment 1 × 106 cells were washed with phosphate-buffered saline (PBS) (Corning) and fixed in chilled 70% ethanol for at least 3 h. Cells were washed with PBS and resuspended in 200 μL of Muse Cell Cycle reagent (MilliporeSigma), incubated for 30 min at room temperature, and analyzed on Muse Cell Analyzer (MilliporeSigma).

    Techniques:

    IL-1 but not IL-33 affects fibroblast collagen I gel contraction and fiber formation. Primary airway fibroblasts (PAFs) from non-asthmatics and asthmatics were grown to confluence and seeded on 3D collagen I gels in the presence or absence of 1 ng/ml IL-1α, IL-1β or IL-33 and allowed to migrated into and contract the gel for 24 hours. ( a ) Representative gel contraction images after the 24 hour stimulation of non-asthmatic and asthmatic PAF-seeded gels with 1 ng/ml IL-1α, IL-1β or IL-33, ( b ) Percentage gel contraction comparing control non-stimulated rates of non-asthmatic and asthmatic -derived PAFs, ( c ) Percentage gel contraction after stimulation with IL-1α, IL-1β and IL-33, ( d ) semi-dry weight of contracted gels after stimulation with IL-1α, IL-1β and IL-33. ( e ) Representative images of fibrillar collagen taken with second harmonic generation (SHG) microscopy in gels, ( f ) SHG peak intensity of fibrillar collagen. ( g ) Entropy of collagen fibers, calculated using texturel analysis of SHG images. Representative images are 20X magnification and scale bars are provided. Exact P values indicated. SHG images are pseudo-coloured, showing fibrillar collagen in blue (un-labeled).

    Journal: Scientific Reports

    Article Title: Epithelial-interleukin-1 inhibits collagen formation by airway fibroblasts: Implications for asthma

    doi: 10.1038/s41598-020-65567-z

    Figure Lengend Snippet: IL-1 but not IL-33 affects fibroblast collagen I gel contraction and fiber formation. Primary airway fibroblasts (PAFs) from non-asthmatics and asthmatics were grown to confluence and seeded on 3D collagen I gels in the presence or absence of 1 ng/ml IL-1α, IL-1β or IL-33 and allowed to migrated into and contract the gel for 24 hours. ( a ) Representative gel contraction images after the 24 hour stimulation of non-asthmatic and asthmatic PAF-seeded gels with 1 ng/ml IL-1α, IL-1β or IL-33, ( b ) Percentage gel contraction comparing control non-stimulated rates of non-asthmatic and asthmatic -derived PAFs, ( c ) Percentage gel contraction after stimulation with IL-1α, IL-1β and IL-33, ( d ) semi-dry weight of contracted gels after stimulation with IL-1α, IL-1β and IL-33. ( e ) Representative images of fibrillar collagen taken with second harmonic generation (SHG) microscopy in gels, ( f ) SHG peak intensity of fibrillar collagen. ( g ) Entropy of collagen fibers, calculated using texturel analysis of SHG images. Representative images are 20X magnification and scale bars are provided. Exact P values indicated. SHG images are pseudo-coloured, showing fibrillar collagen in blue (un-labeled).

    Article Snippet: Fibroblast stimulations and collagen I gel contraction assayAt passage 3–5, PAFs were grown to confluence in DMEM10% fetal calf serum (FCS) (Invitrogen, Burlington, ON, Canada) on collagen I (Corning, New York, NY, USA) coated 6-well plates.

    Techniques: Derivative Assay, Microscopy, Labeling

    IL-1 but not IL-33 stimulates the release of inflammatory mediators from primary airway fibroblasts (PAFs). Primary airway fibroblasts from non-asthmatics (n = 5–9) and asthmatics (n = 5–9) were grown to confluence on collagen I coated plates and stimulated with or without 1 ng/ml recombinant human IL-1α, IL-1β or IL-33 for 24 hours. Concentration of ( a ) IL-6, ( b ) CXCL8/IL-8, ( c ) granulocyte-monocyte colony stimulating factor (GM-CSF) ( d ) thymic stromal lymphopoietin (TSLP) released from primary airway fibroblasts after 24 hours. Exact P values indicated.

    Journal: Scientific Reports

    Article Title: Epithelial-interleukin-1 inhibits collagen formation by airway fibroblasts: Implications for asthma

    doi: 10.1038/s41598-020-65567-z

    Figure Lengend Snippet: IL-1 but not IL-33 stimulates the release of inflammatory mediators from primary airway fibroblasts (PAFs). Primary airway fibroblasts from non-asthmatics (n = 5–9) and asthmatics (n = 5–9) were grown to confluence on collagen I coated plates and stimulated with or without 1 ng/ml recombinant human IL-1α, IL-1β or IL-33 for 24 hours. Concentration of ( a ) IL-6, ( b ) CXCL8/IL-8, ( c ) granulocyte-monocyte colony stimulating factor (GM-CSF) ( d ) thymic stromal lymphopoietin (TSLP) released from primary airway fibroblasts after 24 hours. Exact P values indicated.

    Article Snippet: Fibroblast stimulations and collagen I gel contraction assayAt passage 3–5, PAFs were grown to confluence in DMEM10% fetal calf serum (FCS) (Invitrogen, Burlington, ON, Canada) on collagen I (Corning, New York, NY, USA) coated 6-well plates.

    Techniques: Recombinant, Concentration Assay

    Lysyl oxidase activity is essential for fibroblast contraction of collagen I gels. Primary airway fibroblasts from non-asthmatics and asthmatics (n = 4–6) were grown to confluence and seeded on collagen I gels and allowed to contract and migrate through the gel for 24 hours in the presence or absence of 10 mg/ml β-aminopropionitrile which inhibits lysyl oxidase activity. Collagen I gels were then stained with DAPI and Phalloidin 488 for F-actin in the seeded fibroblasts. ( a ) Representative composite images of co-localized un-labeled second harmonic generation images (SHG, shown in blue) and two-photon excited fluorescence labeled with DAPI and phalloidin staining (TPEF, shown in green) of fibrillar collagen and fibroblasts respectively, ( b ) Cell area of fibroblasts seeded in collagen I gels after contraction. ( c ) Second harmoni generation (SHG) peak intensity of fibrillar collagen and ( d ) entropy score for collagen fibers calculated using texturel analysis of second harmonic generation images ( e ) Percentage gel contraction of collagen I gels. ( f ) Semi-dry weight of contracted gels. ( g ) Primary airway fibroblasts (PAFs) were grown to confluence on collagen I coated plates and stimulated with or without 10 mg/ml BAPN. Percentage lactate dehydrogenase (LDH) released from cells after 24 hours. Representative images are 20X magnification and scale bars are provided. Exact P values indicated.

    Journal: Scientific Reports

    Article Title: Epithelial-interleukin-1 inhibits collagen formation by airway fibroblasts: Implications for asthma

    doi: 10.1038/s41598-020-65567-z

    Figure Lengend Snippet: Lysyl oxidase activity is essential for fibroblast contraction of collagen I gels. Primary airway fibroblasts from non-asthmatics and asthmatics (n = 4–6) were grown to confluence and seeded on collagen I gels and allowed to contract and migrate through the gel for 24 hours in the presence or absence of 10 mg/ml β-aminopropionitrile which inhibits lysyl oxidase activity. Collagen I gels were then stained with DAPI and Phalloidin 488 for F-actin in the seeded fibroblasts. ( a ) Representative composite images of co-localized un-labeled second harmonic generation images (SHG, shown in blue) and two-photon excited fluorescence labeled with DAPI and phalloidin staining (TPEF, shown in green) of fibrillar collagen and fibroblasts respectively, ( b ) Cell area of fibroblasts seeded in collagen I gels after contraction. ( c ) Second harmoni generation (SHG) peak intensity of fibrillar collagen and ( d ) entropy score for collagen fibers calculated using texturel analysis of second harmonic generation images ( e ) Percentage gel contraction of collagen I gels. ( f ) Semi-dry weight of contracted gels. ( g ) Primary airway fibroblasts (PAFs) were grown to confluence on collagen I coated plates and stimulated with or without 10 mg/ml BAPN. Percentage lactate dehydrogenase (LDH) released from cells after 24 hours. Representative images are 20X magnification and scale bars are provided. Exact P values indicated.

    Article Snippet: Fibroblast stimulations and collagen I gel contraction assayAt passage 3–5, PAFs were grown to confluence in DMEM10% fetal calf serum (FCS) (Invitrogen, Burlington, ON, Canada) on collagen I (Corning, New York, NY, USA) coated 6-well plates.

    Techniques: Activity Assay, Staining, Labeling, Fluorescence

    IL-1α and IL-1β induce decreased extracellular matrix protein expression in airway fibroblasts. Primary airway fibroblasts from non-asthmatics and asthmatics were grown to confluence on collagen I coated plates and stimulated with or without 1 ng/ml recombinant human IL-1α, IL-1β or IL-33 for 24 hours. The mRNA expression of Collagen Iα1 ( a–c ), and glioma-associated oncogene homolog 1 (GLI-1) ( d–f ) was assessed at 24 hours. Exact P values indicated.

    Journal: Scientific Reports

    Article Title: Epithelial-interleukin-1 inhibits collagen formation by airway fibroblasts: Implications for asthma

    doi: 10.1038/s41598-020-65567-z

    Figure Lengend Snippet: IL-1α and IL-1β induce decreased extracellular matrix protein expression in airway fibroblasts. Primary airway fibroblasts from non-asthmatics and asthmatics were grown to confluence on collagen I coated plates and stimulated with or without 1 ng/ml recombinant human IL-1α, IL-1β or IL-33 for 24 hours. The mRNA expression of Collagen Iα1 ( a–c ), and glioma-associated oncogene homolog 1 (GLI-1) ( d–f ) was assessed at 24 hours. Exact P values indicated.

    Article Snippet: Fibroblast stimulations and collagen I gel contraction assayAt passage 3–5, PAFs were grown to confluence in DMEM10% fetal calf serum (FCS) (Invitrogen, Burlington, ON, Canada) on collagen I (Corning, New York, NY, USA) coated 6-well plates.

    Techniques: Expressing, Recombinant

    Interleukin-1 alters fibroblast interaction with collagen I. Primary airway fibroblasts from non-asthmatics and asthmatics were grown to confluence and seeded on collagen I gels in the presence or absence of 1 ng/ml IL-1α, IL-1β or IL-33 and allowed to migrate through and contract for 24 hours. Collagen I gels were then stained with DAPI to localize cell nuclei and Phalloidin 488 for F-actin in the seeded fibroblasts. ( a ) Representative composite images of co-localized un-labeled second harmonic generation images (SHG, shown in blue) and two-photon excited fluorescence labeled with DAPI and phalloidin (TPEF, shown in green) of fibrillar collagen and fibroblasts respectively in gels from non-asthmatic and asthmatic PAF’s ( b ) Average number of cells per region of interest, ( c ) Cell area of fibroblasts seeded in collagen I gels after contraction. ( d ) Correlation of fibroblast cell area with SHG peak intensity of fibrillar collagen, (e ) Correlation of fibroblast cell area with collagen fiber entropy. Representative images are 20X magnification and scale bars are provided. Exact P values indicated.

    Journal: Scientific Reports

    Article Title: Epithelial-interleukin-1 inhibits collagen formation by airway fibroblasts: Implications for asthma

    doi: 10.1038/s41598-020-65567-z

    Figure Lengend Snippet: Interleukin-1 alters fibroblast interaction with collagen I. Primary airway fibroblasts from non-asthmatics and asthmatics were grown to confluence and seeded on collagen I gels in the presence or absence of 1 ng/ml IL-1α, IL-1β or IL-33 and allowed to migrate through and contract for 24 hours. Collagen I gels were then stained with DAPI to localize cell nuclei and Phalloidin 488 for F-actin in the seeded fibroblasts. ( a ) Representative composite images of co-localized un-labeled second harmonic generation images (SHG, shown in blue) and two-photon excited fluorescence labeled with DAPI and phalloidin (TPEF, shown in green) of fibrillar collagen and fibroblasts respectively in gels from non-asthmatic and asthmatic PAF’s ( b ) Average number of cells per region of interest, ( c ) Cell area of fibroblasts seeded in collagen I gels after contraction. ( d ) Correlation of fibroblast cell area with SHG peak intensity of fibrillar collagen, (e ) Correlation of fibroblast cell area with collagen fiber entropy. Representative images are 20X magnification and scale bars are provided. Exact P values indicated.

    Article Snippet: Fibroblast stimulations and collagen I gel contraction assayAt passage 3–5, PAFs were grown to confluence in DMEM10% fetal calf serum (FCS) (Invitrogen, Burlington, ON, Canada) on collagen I (Corning, New York, NY, USA) coated 6-well plates.

    Techniques: Staining, Labeling, Fluorescence

    IL-1 down-regulates the expression of lysyl oxidase (LOX) in airway fibroblasts. Primary airway fibroblasts (PAFs) from non-asthmatics and asthmatics were seeded on 96 well plates in the presence of 1 ng/ml IL-1α, ( a ) Optical magnetic twisting cytometry was then used to measure cell stiffness presented as G 1 (Pa/nm) (n = 6). PAFs were grown to confluence on collagen I coated 6-well plates and stimulated with or without 1 ng/ml recombinant IL-1α/β or IL-33 for 24 hours. Relative protein expression and representative blot images of ( b ) non muscle myosin IIB and ( c ) β-tubulin. mRNA expression of LOX after stimulation with ( d ) IL-1α, ( e ) IL-1β and (f ) IL-33. Exact P values indicated.

    Journal: Scientific Reports

    Article Title: Epithelial-interleukin-1 inhibits collagen formation by airway fibroblasts: Implications for asthma

    doi: 10.1038/s41598-020-65567-z

    Figure Lengend Snippet: IL-1 down-regulates the expression of lysyl oxidase (LOX) in airway fibroblasts. Primary airway fibroblasts (PAFs) from non-asthmatics and asthmatics were seeded on 96 well plates in the presence of 1 ng/ml IL-1α, ( a ) Optical magnetic twisting cytometry was then used to measure cell stiffness presented as G 1 (Pa/nm) (n = 6). PAFs were grown to confluence on collagen I coated 6-well plates and stimulated with or without 1 ng/ml recombinant IL-1α/β or IL-33 for 24 hours. Relative protein expression and representative blot images of ( b ) non muscle myosin IIB and ( c ) β-tubulin. mRNA expression of LOX after stimulation with ( d ) IL-1α, ( e ) IL-1β and (f ) IL-33. Exact P values indicated.

    Article Snippet: Fibroblast stimulations and collagen I gel contraction assayAt passage 3–5, PAFs were grown to confluence in DMEM10% fetal calf serum (FCS) (Invitrogen, Burlington, ON, Canada) on collagen I (Corning, New York, NY, USA) coated 6-well plates.

    Techniques: Expressing, Cytometry, Recombinant