Structured Review

Horizon Discovery individual sirna duplexes
Confirmation that SF3A1 and GOLGA4 regulate LPS-induced IL-6 production using a second mouse macrophage cell line. A and B, indicated pools of <t>siRNA</t> duplexes were <t>transfected</t> into the RAW264.7 mouse macrophage cell line; cells were simulated with LPS
Individual Sirna Duplexes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/individual sirna duplexes/product/Horizon Discovery
Average 88 stars, based on 6 article reviews
Price from $9.99 to $1999.99
individual sirna duplexes - by Bioz Stars, 2020-09
88/100 stars

Images

1) Product Images from "An Evolutionarily Conserved Innate Immunity Protein Interaction Network *"

Article Title: An Evolutionarily Conserved Innate Immunity Protein Interaction Network *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.407205

Confirmation that SF3A1 and GOLGA4 regulate LPS-induced IL-6 production using a second mouse macrophage cell line. A and B, indicated pools of siRNA duplexes were transfected into the RAW264.7 mouse macrophage cell line; cells were simulated with LPS
Figure Legend Snippet: Confirmation that SF3A1 and GOLGA4 regulate LPS-induced IL-6 production using a second mouse macrophage cell line. A and B, indicated pools of siRNA duplexes were transfected into the RAW264.7 mouse macrophage cell line; cells were simulated with LPS

Techniques Used: Transfection

Most genes in the innate immunity protein interaction network affect LPS-induced IL-6 production in the J774A.1 mouse macrophage cell line. A, pools of four siRNA duplexes per gene were transfected into the mouse macrophage cell line J774A.1; cells were
Figure Legend Snippet: Most genes in the innate immunity protein interaction network affect LPS-induced IL-6 production in the J774A.1 mouse macrophage cell line. A, pools of four siRNA duplexes per gene were transfected into the mouse macrophage cell line J774A.1; cells were

Techniques Used: Transfection

MACF1 inhibits PAMP-induced cytokine production in RAW264.7 cells and in vivo . A, pool of four siRNA duplexes targeting either Macf1 or a nontargeting control siRNA duplex pool were transfected into the mouse macrophage cell line RAW264.7; cells were
Figure Legend Snippet: MACF1 inhibits PAMP-induced cytokine production in RAW264.7 cells and in vivo . A, pool of four siRNA duplexes targeting either Macf1 or a nontargeting control siRNA duplex pool were transfected into the mouse macrophage cell line RAW264.7; cells were

Techniques Used: In Vivo, Transfection

2) Product Images from "A Unique Role for Nonmuscle Myosin Heavy Chain IIA in Regulation of Epithelial Apical Junctions"

Article Title: A Unique Role for Nonmuscle Myosin Heavy Chain IIA in Regulation of Epithelial Apical Junctions

Journal: PLoS ONE

doi: 10.1371/journal.pone.0000658

Downregulation of either NMMIIB or NMMIIC has no effect on reformation of epithelial TJs. Control, NMMIIB or NMMIIC siRNA-transfected SK-CO15 monolayers were subjected to overnight calcium depletion to disrupt cell-cell adhesion. Reformation of TJs was triggered by transferring cells for 5 h into the HCM. Similarly to control cells, NMMIIB and NMMIIC-deficient cell monolayers rapidly restore normal junctional labeling pattern for ZO-1 (arrows). Bar, 10 µm.
Figure Legend Snippet: Downregulation of either NMMIIB or NMMIIC has no effect on reformation of epithelial TJs. Control, NMMIIB or NMMIIC siRNA-transfected SK-CO15 monolayers were subjected to overnight calcium depletion to disrupt cell-cell adhesion. Reformation of TJs was triggered by transferring cells for 5 h into the HCM. Similarly to control cells, NMMIIB and NMMIIC-deficient cell monolayers rapidly restore normal junctional labeling pattern for ZO-1 (arrows). Bar, 10 µm.

Techniques Used: Transfection, Transferring, Labeling

siRNA-mediated knock-down of NMMIIA inhibits cell contractility triggered by calcium-depletion. Control, NMMIIA, NMMIIB, and NMMIIC-deficient SK-CO15 monolayers were subjected to 1 h calcium depletion, and their overall cell shape was analyzed by F-actin labeling. Calcium depletion causes rapid rounding of control, NMMIIB, and NMMIIC-deficient cells (arrows). In contrast, cell rounding was significantly attenuated in cells monolayers subjected to NMMIIA knock-down. Bar, 20 µm.
Figure Legend Snippet: siRNA-mediated knock-down of NMMIIA inhibits cell contractility triggered by calcium-depletion. Control, NMMIIA, NMMIIB, and NMMIIC-deficient SK-CO15 monolayers were subjected to 1 h calcium depletion, and their overall cell shape was analyzed by F-actin labeling. Calcium depletion causes rapid rounding of control, NMMIIB, and NMMIIC-deficient cells (arrows). In contrast, cell rounding was significantly attenuated in cells monolayers subjected to NMMIIA knock-down. Bar, 20 µm.

Techniques Used: Labeling

siRNA-mediated depletion of NMMIIA attenuates the development of tight junctions. SK-CO15 cells were transfected with either control (cyclophilin) or NMMII isoform specific siRNAs and on day 3 post-transfection were subjected to overnight calcium depletion in order to disrupt cell-cell adhesion. Reassembly of TJs in control and NMMIIA-deficient cells was investigated after 5 h of calcium repletion by monitoring the formation of characteristic ‘chicken wire’ labeling pattern of the TJ proteins occludin and ZO-1 (red). Control SK-CO15 cell monolayers show almost complete restoration of normal localization of occludin and ZO-1 at TJs (arrows). In contrast, occludin and ZO-1 labeling demonstrates abnormal discontinuous pattern at TJs in NMMIIA-deficient cells (arrowheads). Bar, 10 µm.
Figure Legend Snippet: siRNA-mediated depletion of NMMIIA attenuates the development of tight junctions. SK-CO15 cells were transfected with either control (cyclophilin) or NMMII isoform specific siRNAs and on day 3 post-transfection were subjected to overnight calcium depletion in order to disrupt cell-cell adhesion. Reassembly of TJs in control and NMMIIA-deficient cells was investigated after 5 h of calcium repletion by monitoring the formation of characteristic ‘chicken wire’ labeling pattern of the TJ proteins occludin and ZO-1 (red). Control SK-CO15 cell monolayers show almost complete restoration of normal localization of occludin and ZO-1 at TJs (arrows). In contrast, occludin and ZO-1 labeling demonstrates abnormal discontinuous pattern at TJs in NMMIIA-deficient cells (arrowheads). Bar, 10 µm.

Techniques Used: Transfection, Labeling

siRNA-mediated knock-down of NMMIIA selectively attenuates disassembly of the AJC in calcium-depleted cells. Control, NMMIIA, NMMIIB, and NMMIIC-deficient SK-CO15 monolayers were incubated for 1 h in the LCM-EGTA, and the integrity of their AJC was analyzed by immunolabeling for occludin (red). Calcium depletion causes rapid disruption of the AJC and accumulation of occludin in cytosolic ring-like structures in control, NMMIIB and NMMIIC-deficient cells (arrows). In contrast, the majority of occludin-labeled TJs remained intact in cells monolayers subjected to NMMIIA knock-down (arrowheads). Bar, 20 µm.
Figure Legend Snippet: siRNA-mediated knock-down of NMMIIA selectively attenuates disassembly of the AJC in calcium-depleted cells. Control, NMMIIA, NMMIIB, and NMMIIC-deficient SK-CO15 monolayers were incubated for 1 h in the LCM-EGTA, and the integrity of their AJC was analyzed by immunolabeling for occludin (red). Calcium depletion causes rapid disruption of the AJC and accumulation of occludin in cytosolic ring-like structures in control, NMMIIB and NMMIIC-deficient cells (arrows). In contrast, the majority of occludin-labeled TJs remained intact in cells monolayers subjected to NMMIIA knock-down (arrowheads). Bar, 20 µm.

Techniques Used: Incubation, Laser Capture Microdissection, Immunolabeling, Labeling

Downregulation of the NMMIIA expression alters epithelial cell shape and attenuates development of the paracellular barrier. (A) Western blots of SK-CO15 cell lysates prepared 3 days after transfection show selective downregulation of protein expression of NMMIIA. NMMIIB, and NMMIIC by two different siRNA duplexes, each specific for the NMMII isoform. (B) siRNA-mediated knock-down of NMMIIA but not NMMIIB or NMMIIC causes dramatic changes from an orthogonal epithelial to a protrusive fibroblast-like shape in low-density colonies of SK-CO15 cells. (C) siRNA -mediated knock-down of NMMIIA but not NMMIIB or NMMIIC significantly attenuates the increase in TEER in confluent SK-CO15 cell monolayers when compared to the control (cyclophilin) siRNA-transfected cells (*p
Figure Legend Snippet: Downregulation of the NMMIIA expression alters epithelial cell shape and attenuates development of the paracellular barrier. (A) Western blots of SK-CO15 cell lysates prepared 3 days after transfection show selective downregulation of protein expression of NMMIIA. NMMIIB, and NMMIIC by two different siRNA duplexes, each specific for the NMMII isoform. (B) siRNA-mediated knock-down of NMMIIA but not NMMIIB or NMMIIC causes dramatic changes from an orthogonal epithelial to a protrusive fibroblast-like shape in low-density colonies of SK-CO15 cells. (C) siRNA -mediated knock-down of NMMIIA but not NMMIIB or NMMIIC significantly attenuates the increase in TEER in confluent SK-CO15 cell monolayers when compared to the control (cyclophilin) siRNA-transfected cells (*p

Techniques Used: Expressing, Western Blot, Transfection

siRNA-mediated knock-down of either NMMIIB or NMMIIC has no effect on reformation of initial adherens-like junctions. Control, NMMIIB or NMMIIC siRNA-transfected SK-CO15 monolayers were subjected to overnight calcium depletion to disrupt cell-cell adhesion. Reformation of the initial adherens-like junctions was triggered by transferring cells for 1 h into the HCM. Similarly to control cells, NMMIIB and NMMIIC knock-down rapidly translocate E-cadherin (red) to areas of cell-cell contacts (arrows). Bar, 10 µm.
Figure Legend Snippet: siRNA-mediated knock-down of either NMMIIB or NMMIIC has no effect on reformation of initial adherens-like junctions. Control, NMMIIB or NMMIIC siRNA-transfected SK-CO15 monolayers were subjected to overnight calcium depletion to disrupt cell-cell adhesion. Reformation of the initial adherens-like junctions was triggered by transferring cells for 1 h into the HCM. Similarly to control cells, NMMIIB and NMMIIC knock-down rapidly translocate E-cadherin (red) to areas of cell-cell contacts (arrows). Bar, 10 µm.

Techniques Used: Transfection, Transferring

3) Product Images from "Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation"

Article Title: Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation

Journal: Nature Communications

doi: 10.1038/s41467-018-08154-1

SGK1 signals downstream of active Src in non-small cell lung cancer. a Src inhibition decreases SGK1 expression levels. Lysates from A549 cells treated for 16 h with AZD0530 (AZD) or PP2 were western blotted as indicated. Representative blots are shown from n = 2 biological replicates. b Src knockdown decreases SGK1 expression levels. A549 cells were transfected with a Src siRNA SMARTpool or non-targeting control (NTP) (20 nM) and cell lysates harvested at 72 h for western blot analysis. c SGK1 knockdown by tetracycline-inducible shRNAs. A549 cells stably programmed to express tetracycline-inducible SGK1 shRNAs or empty vector (Ctrl) were treated with tetracyclin (1 μg/ml) for 48 h and lysates were collected for western blot. d Effect on A549 colony formation of combining SGK1 knockdown with Src inhibitor (AZD) treatment. A549 cells stably transfected with tetracyclin-inducible SGK1 shRNA#1 were treated +/−tetracyclin and +/−AZD. Data are expressed relative to the DMSO control which was arbitrarily set at 100. e Effect on colony formation of combining SGK1 inhibitor (SGKi) with Src inhibitor (AZD) treatment. A549 cells were treated +/−SGKi and +/−AZD. Data are expressed relative to the DMSO control which was arbitrarily set at 100. Coefficient of drug interaction (CDI) is 0.68 (SGKi and PP2) and 0.34 (SGKi and AZD), respectively, indicating synergy. d , e Results were quantified from n = 3 biological replicates. Error bars represent s.e.m., * p
Figure Legend Snippet: SGK1 signals downstream of active Src in non-small cell lung cancer. a Src inhibition decreases SGK1 expression levels. Lysates from A549 cells treated for 16 h with AZD0530 (AZD) or PP2 were western blotted as indicated. Representative blots are shown from n = 2 biological replicates. b Src knockdown decreases SGK1 expression levels. A549 cells were transfected with a Src siRNA SMARTpool or non-targeting control (NTP) (20 nM) and cell lysates harvested at 72 h for western blot analysis. c SGK1 knockdown by tetracycline-inducible shRNAs. A549 cells stably programmed to express tetracycline-inducible SGK1 shRNAs or empty vector (Ctrl) were treated with tetracyclin (1 μg/ml) for 48 h and lysates were collected for western blot. d Effect on A549 colony formation of combining SGK1 knockdown with Src inhibitor (AZD) treatment. A549 cells stably transfected with tetracyclin-inducible SGK1 shRNA#1 were treated +/−tetracyclin and +/−AZD. Data are expressed relative to the DMSO control which was arbitrarily set at 100. e Effect on colony formation of combining SGK1 inhibitor (SGKi) with Src inhibitor (AZD) treatment. A549 cells were treated +/−SGKi and +/−AZD. Data are expressed relative to the DMSO control which was arbitrarily set at 100. Coefficient of drug interaction (CDI) is 0.68 (SGKi and PP2) and 0.34 (SGKi and AZD), respectively, indicating synergy. d , e Results were quantified from n = 3 biological replicates. Error bars represent s.e.m., * p

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Stable Transfection, Plasmid Preparation, shRNA

Functional characterization of SGK1 in MCF-10A_Src cells. a SGK1 knockdown or inhibition increases apoptosis in MCF-10A_Src acini. MCF-10A_Src cells were transfected with a SGK1 siRNA SMARTpool or non-targeting control (NTP) (40 nM) (left) or treated with a selective SGK1 inhibitor (SGKi) (10 μM) (right), and grown in 3D culture for 3 days or 5 days, respectively. Immunofluorescent imaging was performed using Ki67 and cleaved Caspase-3 antibodies (green) and DAPI counterstain (blue). Representative images are shown. Arrows indicate cleaved Caspase-3 positive acini. Scale bar represents 100 μm. b Percentage of cleaved Caspase-3 or Ki67 positive acini from n = 3 biological replicates. At least 50 acini were analyzed in each replicate. ** p
Figure Legend Snippet: Functional characterization of SGK1 in MCF-10A_Src cells. a SGK1 knockdown or inhibition increases apoptosis in MCF-10A_Src acini. MCF-10A_Src cells were transfected with a SGK1 siRNA SMARTpool or non-targeting control (NTP) (40 nM) (left) or treated with a selective SGK1 inhibitor (SGKi) (10 μM) (right), and grown in 3D culture for 3 days or 5 days, respectively. Immunofluorescent imaging was performed using Ki67 and cleaved Caspase-3 antibodies (green) and DAPI counterstain (blue). Representative images are shown. Arrows indicate cleaved Caspase-3 positive acini. Scale bar represents 100 μm. b Percentage of cleaved Caspase-3 or Ki67 positive acini from n = 3 biological replicates. At least 50 acini were analyzed in each replicate. ** p

Techniques Used: Functional Assay, Inhibition, Transfection, Imaging

SGK1 signals downstream of active Src in TNBC. a SGK1 expression and activity in breast cancer cell lines. Cell lysates were western blotted as indicated. The blots for Src and Src-pY416 were run in a different order but have been re-assembled to match the others, for ease of comparison. ‘MM’ indicates MDA-MB-. b , c Src inhibition decreases SGK1 expression levels in TNBC cell lines. Lysates were prepared from cells treated overnight with AZD0530 (AZD) ( b ) or PP2 ( c ). DMSO was the vehicle control. Representative blots are shown from n = 2 biological replicates. d Src knockdown reduces SGK1 expression levels in TNBC. Cell lysates from Hs578T cells transfected with a Src siRNA SMARTpool or non-targeting control (NTP) (20 nM) for 72 h were western blotted as indicated. Representative blots are shown from n = 2 biological replicates. e SGK1 knockdown by tetracycline-inducible shRNAs. MDA-MB-231 cells were programmed to express tetracycline-inducible SGK1 shRNAs or empty vector (Ctrl) and treated with tetracyclin (1 μg/ml) for 48 h. Cell lysates were prepared and Western blotted as indicated. f SGK1 knockdown reduces colony formation by MDA-MB-231 TNBC cells. Cells from ( e ) were used in a colony formation assay. g SGK1 inhibition reduces colony formation. MDA-MB-231 cells treated with SGK1 inhibitor (SGKi) or vehicle control (DMSO) were assessed in a colony formation assay. h Effect on colony formation of combining SGK1 knockdown with Src inhibitor (AZD) treatment. MDA-MB-231 cells stably transfected with tetracyclin-inducible SGK1 shRNA#1 were treated +/−tetracyclin and +/−AZD. i Effect on colony formation of combining the SGK1 inhibitor (SGKi) with Src inhibitor (AZD) treatment. MDA-MB-231 cells were treated +/−SGKi and +/−AZD. Combination index (CI) was calculated by the Chou–Talalay method using the CompuSyn program. The CI value for 10 μM SGKi with 1 μM AZD is 0.8, indicating weak synergy. f – i In the column graph, data are expressed relative to the empty vector or DMSO control which was arbitrarily set at 100. Results were quantified from n = 3 biological replicates. Error bars represent s.e.m., * p
Figure Legend Snippet: SGK1 signals downstream of active Src in TNBC. a SGK1 expression and activity in breast cancer cell lines. Cell lysates were western blotted as indicated. The blots for Src and Src-pY416 were run in a different order but have been re-assembled to match the others, for ease of comparison. ‘MM’ indicates MDA-MB-. b , c Src inhibition decreases SGK1 expression levels in TNBC cell lines. Lysates were prepared from cells treated overnight with AZD0530 (AZD) ( b ) or PP2 ( c ). DMSO was the vehicle control. Representative blots are shown from n = 2 biological replicates. d Src knockdown reduces SGK1 expression levels in TNBC. Cell lysates from Hs578T cells transfected with a Src siRNA SMARTpool or non-targeting control (NTP) (20 nM) for 72 h were western blotted as indicated. Representative blots are shown from n = 2 biological replicates. e SGK1 knockdown by tetracycline-inducible shRNAs. MDA-MB-231 cells were programmed to express tetracycline-inducible SGK1 shRNAs or empty vector (Ctrl) and treated with tetracyclin (1 μg/ml) for 48 h. Cell lysates were prepared and Western blotted as indicated. f SGK1 knockdown reduces colony formation by MDA-MB-231 TNBC cells. Cells from ( e ) were used in a colony formation assay. g SGK1 inhibition reduces colony formation. MDA-MB-231 cells treated with SGK1 inhibitor (SGKi) or vehicle control (DMSO) were assessed in a colony formation assay. h Effect on colony formation of combining SGK1 knockdown with Src inhibitor (AZD) treatment. MDA-MB-231 cells stably transfected with tetracyclin-inducible SGK1 shRNA#1 were treated +/−tetracyclin and +/−AZD. i Effect on colony formation of combining the SGK1 inhibitor (SGKi) with Src inhibitor (AZD) treatment. MDA-MB-231 cells were treated +/−SGKi and +/−AZD. Combination index (CI) was calculated by the Chou–Talalay method using the CompuSyn program. The CI value for 10 μM SGKi with 1 μM AZD is 0.8, indicating weak synergy. f – i In the column graph, data are expressed relative to the empty vector or DMSO control which was arbitrarily set at 100. Results were quantified from n = 3 biological replicates. Error bars represent s.e.m., * p

Techniques Used: Expressing, Activity Assay, Western Blot, Multiple Displacement Amplification, Inhibition, Transfection, Plasmid Preparation, Colony Assay, Stable Transfection, shRNA

4) Product Images from "Recruitment of factors linking transcription and processing of pre-rRNA to NOR chromatin is UBF-dependent and occurs independent of transcription in human cells"

Article Title: Recruitment of factors linking transcription and processing of pre-rRNA to NOR chromatin is UBF-dependent and occurs independent of transcription in human cells

Journal:

doi: 10.1101/gad.436707

SiRNA depletion of t-UTPs impairs pre-rRNA processing. ( A ); © 1993 Blackwell Publishing. The position of
Figure Legend Snippet: SiRNA depletion of t-UTPs impairs pre-rRNA processing. ( A ); © 1993 Blackwell Publishing. The position of

Techniques Used:

UBF is required for recruitment of pol I, t-UTPs, and TCOF1 to pseudo-NORs. ( A ) 3D-1 cells were repeatedly transfected with either control or UBF siRNA duplexes. Depletion of UBF was monitored by Western blot ( right panel) loaded with equal amounts of
Figure Legend Snippet: UBF is required for recruitment of pol I, t-UTPs, and TCOF1 to pseudo-NORs. ( A ) 3D-1 cells were repeatedly transfected with either control or UBF siRNA duplexes. Depletion of UBF was monitored by Western blot ( right panel) loaded with equal amounts of

Techniques Used: Transfection, Western Blot

5) Product Images from "RNA interference screen for RGS protein specificity at muscarinic and protease-activated receptors reveals bidirectional modulation of signaling"

Article Title: RNA interference screen for RGS protein specificity at muscarinic and protease-activated receptors reveals bidirectional modulation of signaling

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00441.2009

Changes in mAChR and PAR-1 expression levels in cells treated with RGS -specific siRNAs. A : evaluation of endogenous levels of total mAChR in cells treated with indicated siRNA was performed with a radioligand binding assay using 3-quinuclydinyl benzylate
Figure Legend Snippet: Changes in mAChR and PAR-1 expression levels in cells treated with RGS -specific siRNAs. A : evaluation of endogenous levels of total mAChR in cells treated with indicated siRNA was performed with a radioligand binding assay using 3-quinuclydinyl benzylate

Techniques Used: Expressing, Radio Ligand Binding Assay

Specific RGS protein “hits” identified as modulators of carbachol and/or PAR-1 responses following the siRNA SMARTpool screen. A and B each depict examples of three members of R4 and R7 subfamilies. A : dose-response curves following carbachol
Figure Legend Snippet: Specific RGS protein “hits” identified as modulators of carbachol and/or PAR-1 responses following the siRNA SMARTpool screen. A and B each depict examples of three members of R4 and R7 subfamilies. A : dose-response curves following carbachol

Techniques Used:

Validation of primary siRNA pool screen data using individual siRNA oligonucleotide duplexes. Fluorescence calcium transient assays were performed with each individual siRNA duplex from previously identified SMARTpool hits. Dose-response curves showing
Figure Legend Snippet: Validation of primary siRNA pool screen data using individual siRNA oligonucleotide duplexes. Fluorescence calcium transient assays were performed with each individual siRNA duplex from previously identified SMARTpool hits. Dose-response curves showing

Techniques Used: Fluorescence

RGS protein-mediated regulation of total inositol phosphate production after mAChR activation. HEK293 cells were transfected with indicated siRNA duplexes and then labeled with myo -[ 3 H]inositol for 12 h. Cells were then stimulated with 25 μM carbachol
Figure Legend Snippet: RGS protein-mediated regulation of total inositol phosphate production after mAChR activation. HEK293 cells were transfected with indicated siRNA duplexes and then labeled with myo -[ 3 H]inositol for 12 h. Cells were then stimulated with 25 μM carbachol

Techniques Used: Activation Assay, Transfection, Labeling

6) Product Images from "LKB1 tumor suppressor protein regulates actin filament assembly through Rho and its exchange factor Dbl independently of kinase activity"

Article Title: LKB1 tumor suppressor protein regulates actin filament assembly through Rho and its exchange factor Dbl independently of kinase activity

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-11-77

Stress fiber formation is mediated by RhoA and Dbl . HeLa-S3 cells were transfected with siRNAs and then re-transfected 2 days later with myc-tagged wild type LKB1. A . HeLa-S3 cells transfected with control siRNA followed 2 days later by empty vector. B, C . Cells transfected with control siRNA followed 2 days later by a myc-tagged LKB1 construct. D, E . Cells transfected with RhoA siRNA SMARTpool followed 2 days later by a myc-tagged LKB1 construct. F, G . Cells transfected with Dbl SMARTpool siRNA followed 2 days later by a myc-tagged LKB1 construct. H . Quantification of stress fiber formation. Four individual siRNAs (1-4) and the SMARTpool (sp) are shown for RhoA and two individual siRNAs (1,2) and SMARTpool (sp) are shown for Dbl. I . Western blot showing effectiveness of individual siRNAs in depleting endogenous RhoA and Dbl proteins. Cell lysates were harvested 3 days after siRNA transfection. Scale bar = 20 μm.
Figure Legend Snippet: Stress fiber formation is mediated by RhoA and Dbl . HeLa-S3 cells were transfected with siRNAs and then re-transfected 2 days later with myc-tagged wild type LKB1. A . HeLa-S3 cells transfected with control siRNA followed 2 days later by empty vector. B, C . Cells transfected with control siRNA followed 2 days later by a myc-tagged LKB1 construct. D, E . Cells transfected with RhoA siRNA SMARTpool followed 2 days later by a myc-tagged LKB1 construct. F, G . Cells transfected with Dbl SMARTpool siRNA followed 2 days later by a myc-tagged LKB1 construct. H . Quantification of stress fiber formation. Four individual siRNAs (1-4) and the SMARTpool (sp) are shown for RhoA and two individual siRNAs (1,2) and SMARTpool (sp) are shown for Dbl. I . Western blot showing effectiveness of individual siRNAs in depleting endogenous RhoA and Dbl proteins. Cell lysates were harvested 3 days after siRNA transfection. Scale bar = 20 μm.

Techniques Used: Transfection, Plasmid Preparation, Construct, Western Blot

7) Product Images from "Inhibition of BCL2 expression and activity increases H460 sensitivity to the growth inhibitory effects of polyphenon E"

Article Title: Inhibition of BCL2 expression and activity increases H460 sensitivity to the growth inhibitory effects of polyphenon E

Journal: Journal of experimental therapeutics & oncology

doi:

Treatment of H460 cells with Poly E, bcl-2 -specific siRNA, and/or HA14-1 decreases MMP and increases PARP cleavage. A, H460 cells were treated under the following conditions: no treatment, 10 nM siControl RNA (negative control), 10 nM D1 siRNA, 10 nM D2 siRNA, 10 nM D2 plus Poly E (25 μg/mL), 10 nM D2 plus Poly E (25 μg/mL) plus HA14-1, CCCP (positive control). MMP was measured using the JC-1 staining assay and flow cytometry. Values were normalized to untreated cells (value 1.0).The results are the means +/− standard deviations from duplicate experiments (*, p ≤ .01). B, H460 cells were treated under the following conditions: 10 nM D1 siRNA, 10 nM D2 siRNA, 10 nM D2 plus Poly E (25 μg/mL, 24 h), and 10 nM D2 plus Poly E (25 μg/mL, 48 h). Cell lysates were collected and evaluated for cleaved PARP by western blotting. Immunoblotting for actin was performed to verify equivalent amounts of loaded protein ( data not shown ).
Figure Legend Snippet: Treatment of H460 cells with Poly E, bcl-2 -specific siRNA, and/or HA14-1 decreases MMP and increases PARP cleavage. A, H460 cells were treated under the following conditions: no treatment, 10 nM siControl RNA (negative control), 10 nM D1 siRNA, 10 nM D2 siRNA, 10 nM D2 plus Poly E (25 μg/mL), 10 nM D2 plus Poly E (25 μg/mL) plus HA14-1, CCCP (positive control). MMP was measured using the JC-1 staining assay and flow cytometry. Values were normalized to untreated cells (value 1.0).The results are the means +/− standard deviations from duplicate experiments (*, p ≤ .01). B, H460 cells were treated under the following conditions: 10 nM D1 siRNA, 10 nM D2 siRNA, 10 nM D2 plus Poly E (25 μg/mL, 24 h), and 10 nM D2 plus Poly E (25 μg/mL, 48 h). Cell lysates were collected and evaluated for cleaved PARP by western blotting. Immunoblotting for actin was performed to verify equivalent amounts of loaded protein ( data not shown ).

Techniques Used: Negative Control, Positive Control, Staining, Flow Cytometry, Cytometry, Western Blot

Inhibition of BCL2 expression and activity attenuates H460 cell resistance to the growth inhibitory effects of Poly E. A, H460 cells were transfected with 5 nM and 10 nM of SMARTpool bcl-2 -specific siRNA for 24 h, 48 h, and 72 h ( 5 nM sample not shown ). Cell lysates from untreated (UT) and transfected cells were collected after each time point and evaluated for BCL2 protein expression by western blotting. Immunoblotting for actin was performed to verify equivalent amounts of loaded protein. B, H460 cells were transfected with 10 nM of two individual bcl-2 -specific siRNA duplexes (D1 – 96 h; D2 – 24 h, 48 h, and 96 h). Cell lysates from UT and transfected cells were collected after each time point and evaluated for BCL2 protein expression by western blotting. C, exponentially dividing cells were transected with 10 nM of Duplex 1 or Duplex 2 bcl-2 -specific siRNA or (negative) non-targeting siControl RNA for 24 h and then treated with increasing concentrations of Poly E for 72 h. “No siRNA” cells were not transfected with any siRNA. Cell viability was determined using the MTT assay. The percentage of growth was calculated, with 100% representing control cells treated with 0.1% DMSO alone. The results are the means ± SDs from triplicate experiments (*, p
Figure Legend Snippet: Inhibition of BCL2 expression and activity attenuates H460 cell resistance to the growth inhibitory effects of Poly E. A, H460 cells were transfected with 5 nM and 10 nM of SMARTpool bcl-2 -specific siRNA for 24 h, 48 h, and 72 h ( 5 nM sample not shown ). Cell lysates from untreated (UT) and transfected cells were collected after each time point and evaluated for BCL2 protein expression by western blotting. Immunoblotting for actin was performed to verify equivalent amounts of loaded protein. B, H460 cells were transfected with 10 nM of two individual bcl-2 -specific siRNA duplexes (D1 – 96 h; D2 – 24 h, 48 h, and 96 h). Cell lysates from UT and transfected cells were collected after each time point and evaluated for BCL2 protein expression by western blotting. C, exponentially dividing cells were transected with 10 nM of Duplex 1 or Duplex 2 bcl-2 -specific siRNA or (negative) non-targeting siControl RNA for 24 h and then treated with increasing concentrations of Poly E for 72 h. “No siRNA” cells were not transfected with any siRNA. Cell viability was determined using the MTT assay. The percentage of growth was calculated, with 100% representing control cells treated with 0.1% DMSO alone. The results are the means ± SDs from triplicate experiments (*, p

Techniques Used: Inhibition, Expressing, Activity Assay, Transfection, Western Blot, MTT Assay

8) Product Images from "Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation"

Article Title: Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation

Journal: Nature Communications

doi: 10.1038/s41467-018-08154-1

SGK1 signals downstream of active Src in non-small cell lung cancer. a Src inhibition decreases SGK1 expression levels. Lysates from A549 cells treated for 16 h with AZD0530 (AZD) or PP2 were western blotted as indicated. Representative blots are shown from n = 2 biological replicates. b Src knockdown decreases SGK1 expression levels. A549 cells were transfected with a Src siRNA SMARTpool or non-targeting control (NTP) (20 nM) and cell lysates harvested at 72 h for western blot analysis. c SGK1 knockdown by tetracycline-inducible shRNAs. A549 cells stably programmed to express tetracycline-inducible SGK1 shRNAs or empty vector (Ctrl) were treated with tetracyclin (1 μg/ml) for 48 h and lysates were collected for western blot. d Effect on A549 colony formation of combining SGK1 knockdown with Src inhibitor (AZD) treatment. A549 cells stably transfected with tetracyclin-inducible SGK1 shRNA#1 were treated +/−tetracyclin and +/−AZD. Data are expressed relative to the DMSO control which was arbitrarily set at 100. e Effect on colony formation of combining SGK1 inhibitor (SGKi) with Src inhibitor (AZD) treatment. A549 cells were treated +/−SGKi and +/−AZD. Data are expressed relative to the DMSO control which was arbitrarily set at 100. Coefficient of drug interaction (CDI) is 0.68 (SGKi and PP2) and 0.34 (SGKi and AZD), respectively, indicating synergy. d , e Results were quantified from n = 3 biological replicates. Error bars represent s.e.m., * p
Figure Legend Snippet: SGK1 signals downstream of active Src in non-small cell lung cancer. a Src inhibition decreases SGK1 expression levels. Lysates from A549 cells treated for 16 h with AZD0530 (AZD) or PP2 were western blotted as indicated. Representative blots are shown from n = 2 biological replicates. b Src knockdown decreases SGK1 expression levels. A549 cells were transfected with a Src siRNA SMARTpool or non-targeting control (NTP) (20 nM) and cell lysates harvested at 72 h for western blot analysis. c SGK1 knockdown by tetracycline-inducible shRNAs. A549 cells stably programmed to express tetracycline-inducible SGK1 shRNAs or empty vector (Ctrl) were treated with tetracyclin (1 μg/ml) for 48 h and lysates were collected for western blot. d Effect on A549 colony formation of combining SGK1 knockdown with Src inhibitor (AZD) treatment. A549 cells stably transfected with tetracyclin-inducible SGK1 shRNA#1 were treated +/−tetracyclin and +/−AZD. Data are expressed relative to the DMSO control which was arbitrarily set at 100. e Effect on colony formation of combining SGK1 inhibitor (SGKi) with Src inhibitor (AZD) treatment. A549 cells were treated +/−SGKi and +/−AZD. Data are expressed relative to the DMSO control which was arbitrarily set at 100. Coefficient of drug interaction (CDI) is 0.68 (SGKi and PP2) and 0.34 (SGKi and AZD), respectively, indicating synergy. d , e Results were quantified from n = 3 biological replicates. Error bars represent s.e.m., * p

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Stable Transfection, Plasmid Preparation, shRNA

Functional characterization of SGK1 in MCF-10A_Src cells. a SGK1 knockdown or inhibition increases apoptosis in MCF-10A_Src acini. MCF-10A_Src cells were transfected with a SGK1 siRNA SMARTpool or non-targeting control (NTP) (40 nM) (left) or treated with a selective SGK1 inhibitor (SGKi) (10 μM) (right), and grown in 3D culture for 3 days or 5 days, respectively. Immunofluorescent imaging was performed using Ki67 and cleaved Caspase-3 antibodies (green) and DAPI counterstain (blue). Representative images are shown. Arrows indicate cleaved Caspase-3 positive acini. Scale bar represents 100 μm. b Percentage of cleaved Caspase-3 or Ki67 positive acini from n = 3 biological replicates. At least 50 acini were analyzed in each replicate. ** p
Figure Legend Snippet: Functional characterization of SGK1 in MCF-10A_Src cells. a SGK1 knockdown or inhibition increases apoptosis in MCF-10A_Src acini. MCF-10A_Src cells were transfected with a SGK1 siRNA SMARTpool or non-targeting control (NTP) (40 nM) (left) or treated with a selective SGK1 inhibitor (SGKi) (10 μM) (right), and grown in 3D culture for 3 days or 5 days, respectively. Immunofluorescent imaging was performed using Ki67 and cleaved Caspase-3 antibodies (green) and DAPI counterstain (blue). Representative images are shown. Arrows indicate cleaved Caspase-3 positive acini. Scale bar represents 100 μm. b Percentage of cleaved Caspase-3 or Ki67 positive acini from n = 3 biological replicates. At least 50 acini were analyzed in each replicate. ** p

Techniques Used: Functional Assay, Inhibition, Transfection, Imaging

SGK1 signals downstream of active Src in TNBC. a SGK1 expression and activity in breast cancer cell lines. Cell lysates were western blotted as indicated. The blots for Src and Src-pY416 were run in a different order but have been re-assembled to match the others, for ease of comparison. ‘MM’ indicates MDA-MB-. b , c Src inhibition decreases SGK1 expression levels in TNBC cell lines. Lysates were prepared from cells treated overnight with AZD0530 (AZD) ( b ) or PP2 ( c ). DMSO was the vehicle control. Representative blots are shown from n = 2 biological replicates. d Src knockdown reduces SGK1 expression levels in TNBC. Cell lysates from Hs578T cells transfected with a Src siRNA SMARTpool or non-targeting control (NTP) (20 nM) for 72 h were western blotted as indicated. Representative blots are shown from n = 2 biological replicates. e SGK1 knockdown by tetracycline-inducible shRNAs. MDA-MB-231 cells were programmed to express tetracycline-inducible SGK1 shRNAs or empty vector (Ctrl) and treated with tetracyclin (1 μg/ml) for 48 h. Cell lysates were prepared and Western blotted as indicated. f SGK1 knockdown reduces colony formation by MDA-MB-231 TNBC cells. Cells from ( e ) were used in a colony formation assay. g SGK1 inhibition reduces colony formation. MDA-MB-231 cells treated with SGK1 inhibitor (SGKi) or vehicle control (DMSO) were assessed in a colony formation assay. h Effect on colony formation of combining SGK1 knockdown with Src inhibitor (AZD) treatment. MDA-MB-231 cells stably transfected with tetracyclin-inducible SGK1 shRNA#1 were treated +/−tetracyclin and +/−AZD. i Effect on colony formation of combining the SGK1 inhibitor (SGKi) with Src inhibitor (AZD) treatment. MDA-MB-231 cells were treated +/−SGKi and +/−AZD. Combination index (CI) was calculated by the Chou–Talalay method using the CompuSyn program. The CI value for 10 μM SGKi with 1 μM AZD is 0.8, indicating weak synergy. f – i In the column graph, data are expressed relative to the empty vector or DMSO control which was arbitrarily set at 100. Results were quantified from n = 3 biological replicates. Error bars represent s.e.m., * p
Figure Legend Snippet: SGK1 signals downstream of active Src in TNBC. a SGK1 expression and activity in breast cancer cell lines. Cell lysates were western blotted as indicated. The blots for Src and Src-pY416 were run in a different order but have been re-assembled to match the others, for ease of comparison. ‘MM’ indicates MDA-MB-. b , c Src inhibition decreases SGK1 expression levels in TNBC cell lines. Lysates were prepared from cells treated overnight with AZD0530 (AZD) ( b ) or PP2 ( c ). DMSO was the vehicle control. Representative blots are shown from n = 2 biological replicates. d Src knockdown reduces SGK1 expression levels in TNBC. Cell lysates from Hs578T cells transfected with a Src siRNA SMARTpool or non-targeting control (NTP) (20 nM) for 72 h were western blotted as indicated. Representative blots are shown from n = 2 biological replicates. e SGK1 knockdown by tetracycline-inducible shRNAs. MDA-MB-231 cells were programmed to express tetracycline-inducible SGK1 shRNAs or empty vector (Ctrl) and treated with tetracyclin (1 μg/ml) for 48 h. Cell lysates were prepared and Western blotted as indicated. f SGK1 knockdown reduces colony formation by MDA-MB-231 TNBC cells. Cells from ( e ) were used in a colony formation assay. g SGK1 inhibition reduces colony formation. MDA-MB-231 cells treated with SGK1 inhibitor (SGKi) or vehicle control (DMSO) were assessed in a colony formation assay. h Effect on colony formation of combining SGK1 knockdown with Src inhibitor (AZD) treatment. MDA-MB-231 cells stably transfected with tetracyclin-inducible SGK1 shRNA#1 were treated +/−tetracyclin and +/−AZD. i Effect on colony formation of combining the SGK1 inhibitor (SGKi) with Src inhibitor (AZD) treatment. MDA-MB-231 cells were treated +/−SGKi and +/−AZD. Combination index (CI) was calculated by the Chou–Talalay method using the CompuSyn program. The CI value for 10 μM SGKi with 1 μM AZD is 0.8, indicating weak synergy. f – i In the column graph, data are expressed relative to the empty vector or DMSO control which was arbitrarily set at 100. Results were quantified from n = 3 biological replicates. Error bars represent s.e.m., * p

Techniques Used: Expressing, Activity Assay, Western Blot, Multiple Displacement Amplification, Inhibition, Transfection, Plasmid Preparation, Colony Assay, Stable Transfection, shRNA

9) Product Images from "Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity"

Article Title: Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

Journal: Genetics

doi: 10.1534/genetics.113.160499

Use of siRNA pools to identify genes that regulate LPS-induced IL-6 production in murine macrophages. Pools of siRNA duplexes targeting the indicated genes (or nontargeting control pools CT1 or CT2) were transfected into the mouse macrophage cell line
Figure Legend Snippet: Use of siRNA pools to identify genes that regulate LPS-induced IL-6 production in murine macrophages. Pools of siRNA duplexes targeting the indicated genes (or nontargeting control pools CT1 or CT2) were transfected into the mouse macrophage cell line

Techniques Used: Transfection

10) Product Images from "Cyclin A/Cdk2 regulates Cdh1 and claspin during late S/G2 phase of the cell cycle"

Article Title: Cyclin A/Cdk2 regulates Cdh1 and claspin during late S/G2 phase of the cell cycle

Journal: Cell Cycle

doi: 10.4161/15384101.2014.949111

Chk1 depletion attenuates the Cyclin A dependent G2 phase delay. ( A ) HeLa cells were transfected with nonsense control (NS), cyclin A siRNAs (#1, #3) or Cdh1 siRNA, then synchronised with overnight thymidine arrest. Cells were harvested 7 h after
Figure Legend Snippet: Chk1 depletion attenuates the Cyclin A dependent G2 phase delay. ( A ) HeLa cells were transfected with nonsense control (NS), cyclin A siRNAs (#1, #3) or Cdh1 siRNA, then synchronised with overnight thymidine arrest. Cells were harvested 7 h after

Techniques Used: Transfection

Cyclin A regulates S/G2 phase Cdh1 and Claspin levels.( A ) HeLa cells were transfected with ether nonsense (NS), Cyclin A (#1) or Cdh1 siRNA for 24 h then fixed, stained for their DNA content with propidium iodide and sorted into four fractions,
Figure Legend Snippet: Cyclin A regulates S/G2 phase Cdh1 and Claspin levels.( A ) HeLa cells were transfected with ether nonsense (NS), Cyclin A (#1) or Cdh1 siRNA for 24 h then fixed, stained for their DNA content with propidium iodide and sorted into four fractions,

Techniques Used: Transfection, Staining

Cdh1 levels are reduced with Cyclin A depletion. ( A ) HeLa cells were transfected with nonsense control (NS), cyclin A siRNA #1 and #3 or Cdh1 siRNA, then synchronised with overnight thymidine arrest. Untransfected cells were treated with inhibitors of
Figure Legend Snippet: Cdh1 levels are reduced with Cyclin A depletion. ( A ) HeLa cells were transfected with nonsense control (NS), cyclin A siRNA #1 and #3 or Cdh1 siRNA, then synchronised with overnight thymidine arrest. Untransfected cells were treated with inhibitors of

Techniques Used: Transfection

Cyclin A/cdk2 regulates a subset of Cdh1 targets. HeLa cells were transfected with either Lipofectamine alone (Con), nonsense (NS), or Cyclin A siRNAs (#1, #3) without and with co-transfection with Cdh1 siRNA. Cells were synchronised with thymidine and
Figure Legend Snippet: Cyclin A/cdk2 regulates a subset of Cdh1 targets. HeLa cells were transfected with either Lipofectamine alone (Con), nonsense (NS), or Cyclin A siRNAs (#1, #3) without and with co-transfection with Cdh1 siRNA. Cells were synchronised with thymidine and

Techniques Used: Transfection, Cotransfection

11) Product Images from "The THAP-Zinc Finger Protein THAP1 Associates with Coactivator HCF-1 and O-GlcNAc Transferase"

Article Title: The THAP-Zinc Finger Protein THAP1 Associates with Coactivator HCF-1 and O-GlcNAc Transferase

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.072579

Endogenous HCF-1 regulates RRM1 expression in primary human endothelial cells. A , knockdown of endogenous HCF-1 with a pool of specific siRNAs reduces RRM1 mRNA levels. RNA was isolated from cells transfected with ON-TARGET-plus SMARTpool HCF-1 siRNAs, 48 h after siRNA transfection, and used for qPCR analysis with the indicated human gene primers ( RRM1, HCF-1 , and control gene GAPDH ). Actin was used as a control gene for normalization. Results are shown as means with S.D. from three separate data points. B and C , knockdown of endogenous HCF-1 with 4 individual ON-TARGET-plus HCF-1 siRNAs. B , HCF-1, THAP1 and Tubα (loading control) expression levels were analyzed by Western blot. C , knockdown of HCF-1 with individual HCF-1 siRNAs results in down-regulation of RRM1 mRNA levels. qPCR analyses were performed as described in A .
Figure Legend Snippet: Endogenous HCF-1 regulates RRM1 expression in primary human endothelial cells. A , knockdown of endogenous HCF-1 with a pool of specific siRNAs reduces RRM1 mRNA levels. RNA was isolated from cells transfected with ON-TARGET-plus SMARTpool HCF-1 siRNAs, 48 h after siRNA transfection, and used for qPCR analysis with the indicated human gene primers ( RRM1, HCF-1 , and control gene GAPDH ). Actin was used as a control gene for normalization. Results are shown as means with S.D. from three separate data points. B and C , knockdown of endogenous HCF-1 with 4 individual ON-TARGET-plus HCF-1 siRNAs. B , HCF-1, THAP1 and Tubα (loading control) expression levels were analyzed by Western blot. C , knockdown of HCF-1 with individual HCF-1 siRNAs results in down-regulation of RRM1 mRNA levels. qPCR analyses were performed as described in A .

Techniques Used: Expressing, Isolation, Transfection, Real-time Polymerase Chain Reaction, Western Blot

12) Product Images from "CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer"

Article Title: CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer

Journal: Nature Communications

doi: 10.1038/s41467-020-16393-4

Sensitivity to CX-5461 is associated with induced MYC target genes, BRCA1-mutated and HRD gene expression signatures. a GSEA of microarray expression data of 12 CX-5461-sensitive and 11 CX-5461-resistant cell lines (Fig. 1a ). Enrichment plots of gene sets identified to be enriched in the CX-5461-sensitive cell lines are shown. b Single sample GSEA (ssGSEA) was utilized to obtain the level of activity of a HRD gene expression signature 24 in individual samples. Genes in each sample were ranked according to their expression levels, and a score for each pathway was generated based on the empirical cumulative distribution function, reflecting how highly or lowly genes were found in the ranked list. n = 32 cell lines in Fig. 1a , box plot—median, upper and lower hinges correspond to the first and third quartiles (25th and 75th percentiles). Upper whisker extends to the largest value no further than 1.5*IQR (IQR = inter-quartile range). Lower whisker extends to the smallest value at 1.5*IQR. Data beyond the end of the whiskers are plotted as individual points. Statistical significance was obtained using two-sided Wilcoxon tests. c The level of expression of MYC target genes (MYC_UP-sig) and BRCA1 mutated (BRCAm-sig) gene signatures were calculated in RNA expression data from the Broad Institute CCLE using ssGSEA. The MYC_UP-sig and BRCAm-sig gene expression signatures are more highly expressed in the CX-5461-sensitive group compared with the resistant group (one-sided Wilcoxon tests p -value 0.004762 and 0.009524, respectively). d The combination of CX-5461 and siRNAs targeting HR genes in OVCAR4 cells synergistically inhibits proliferation. Four individual siRNA duplexes per gene were reversed transfected for 24 h, followed by treatment with CX-5461 (80 nM) or vehicle for 48 h. Cell counts were measured using DAPI staining and imaged using Cellomics. n = 4, each data point represents individual siRNA duplexes. Error bars represent mean ± SEM (standard error of the mean). Statistical analysis was performed using a two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p -values are shown). Accompanying graph (right) is a bliss plot with each dot representing an individual siRNA duplex. A combination index of CI
Figure Legend Snippet: Sensitivity to CX-5461 is associated with induced MYC target genes, BRCA1-mutated and HRD gene expression signatures. a GSEA of microarray expression data of 12 CX-5461-sensitive and 11 CX-5461-resistant cell lines (Fig. 1a ). Enrichment plots of gene sets identified to be enriched in the CX-5461-sensitive cell lines are shown. b Single sample GSEA (ssGSEA) was utilized to obtain the level of activity of a HRD gene expression signature 24 in individual samples. Genes in each sample were ranked according to their expression levels, and a score for each pathway was generated based on the empirical cumulative distribution function, reflecting how highly or lowly genes were found in the ranked list. n = 32 cell lines in Fig. 1a , box plot—median, upper and lower hinges correspond to the first and third quartiles (25th and 75th percentiles). Upper whisker extends to the largest value no further than 1.5*IQR (IQR = inter-quartile range). Lower whisker extends to the smallest value at 1.5*IQR. Data beyond the end of the whiskers are plotted as individual points. Statistical significance was obtained using two-sided Wilcoxon tests. c The level of expression of MYC target genes (MYC_UP-sig) and BRCA1 mutated (BRCAm-sig) gene signatures were calculated in RNA expression data from the Broad Institute CCLE using ssGSEA. The MYC_UP-sig and BRCAm-sig gene expression signatures are more highly expressed in the CX-5461-sensitive group compared with the resistant group (one-sided Wilcoxon tests p -value 0.004762 and 0.009524, respectively). d The combination of CX-5461 and siRNAs targeting HR genes in OVCAR4 cells synergistically inhibits proliferation. Four individual siRNA duplexes per gene were reversed transfected for 24 h, followed by treatment with CX-5461 (80 nM) or vehicle for 48 h. Cell counts were measured using DAPI staining and imaged using Cellomics. n = 4, each data point represents individual siRNA duplexes. Error bars represent mean ± SEM (standard error of the mean). Statistical analysis was performed using a two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p -values are shown). Accompanying graph (right) is a bliss plot with each dot representing an individual siRNA duplex. A combination index of CI

Techniques Used: Expressing, Microarray, Activity Assay, Generated, Whisker Assay, RNA Expression, Transfection, Staining

13) Product Images from "ABCC Multidrug Transporters in Childhood Neuroblastoma: Clinical and Biological Effects Independent of Cytotoxic Drug Efflux"

Article Title: ABCC Multidrug Transporters in Childhood Neuroblastoma: Clinical and Biological Effects Independent of Cytotoxic Drug Efflux

Journal: JNCI Journal of the National Cancer Institute

doi: 10.1093/jnci/djr256

Impact of ABCC4 suppression in SH-SY5Y neuroblastoma cells. A ) Western blot analysis of ABCC4 protein expression following exposure of SH-SY5Y cells to ABCC4 -specific SMARTpool siRNA, or to control siRNA. B ) Enhanced neurite extension in SH-SY5Y cells upon depletion of ABCC4. C ) 3 H-thymidine incorporation in SH-SY5Y cells depleted of ABCC4. D ) Clonogenic capacity upon depletion of ABCC4 was assayed after 12 days. In panel ( B ) and ( D ), P values were derived from two-sided Student t test vs control, whereas one-sample t test was used in panel ( C ) (H 0 , μ = 100%). Means are derived from three replicate experiments and error bars represent 95% confidence intervals. ABCC4 = ATP-binding cassette, subfamily C, member 4; siRNA = short interfering RNA.
Figure Legend Snippet: Impact of ABCC4 suppression in SH-SY5Y neuroblastoma cells. A ) Western blot analysis of ABCC4 protein expression following exposure of SH-SY5Y cells to ABCC4 -specific SMARTpool siRNA, or to control siRNA. B ) Enhanced neurite extension in SH-SY5Y cells upon depletion of ABCC4. C ) 3 H-thymidine incorporation in SH-SY5Y cells depleted of ABCC4. D ) Clonogenic capacity upon depletion of ABCC4 was assayed after 12 days. In panel ( B ) and ( D ), P values were derived from two-sided Student t test vs control, whereas one-sample t test was used in panel ( C ) (H 0 , μ = 100%). Means are derived from three replicate experiments and error bars represent 95% confidence intervals. ABCC4 = ATP-binding cassette, subfamily C, member 4; siRNA = short interfering RNA.

Techniques Used: Western Blot, Expressing, Derivative Assay, Binding Assay, Small Interfering RNA

Impact of ABCC1 and ABCC4 suppression in BE(2)-C human neuroblastoma cells. A ) Western blot analysis of ABCC1 and ABCC4 protein expression following exposure of BE(2)-C cells to ABCC1 - and ABCC4 -specific siRNA molecules, alone or in combination, or to control siRNA (lane 1). B ) Representative images of wound closure assay; Scale bar = 125 μm. C ) Quantification demonstrating impaired motility of BE(2)-C cells depleted of ABCC1, ABCC4, or both, as measured by wound closure assay for 48 hours. D ) 3 H-thymidine incorporation assay in BE(2)-C cells depleted of ABCC1, ABCC4, or both. E ) Clonogenic capacity of BE(2)-C cells upon depletion of ABCC1 and ABCC4 was assayed after 10 days. F ) Neurite extension in BE(2)-C cells upon depletion of ABCC1 and ABCC4. In panels ( C ), ( E ), and ( F ), P values were derived from two-sided Student t test vs control, whereas one-sample t test (H 0 , μ = 100%) was used in panel ( D ). Means are derived from at least three independent experiments. Error bars represent 95% confidence intervals. ABCC = ATP-binding cassette, sub family C ; siRNA = short interfering RNA.
Figure Legend Snippet: Impact of ABCC1 and ABCC4 suppression in BE(2)-C human neuroblastoma cells. A ) Western blot analysis of ABCC1 and ABCC4 protein expression following exposure of BE(2)-C cells to ABCC1 - and ABCC4 -specific siRNA molecules, alone or in combination, or to control siRNA (lane 1). B ) Representative images of wound closure assay; Scale bar = 125 μm. C ) Quantification demonstrating impaired motility of BE(2)-C cells depleted of ABCC1, ABCC4, or both, as measured by wound closure assay for 48 hours. D ) 3 H-thymidine incorporation assay in BE(2)-C cells depleted of ABCC1, ABCC4, or both. E ) Clonogenic capacity of BE(2)-C cells upon depletion of ABCC1 and ABCC4 was assayed after 10 days. F ) Neurite extension in BE(2)-C cells upon depletion of ABCC1 and ABCC4. In panels ( C ), ( E ), and ( F ), P values were derived from two-sided Student t test vs control, whereas one-sample t test (H 0 , μ = 100%) was used in panel ( D ). Means are derived from at least three independent experiments. Error bars represent 95% confidence intervals. ABCC = ATP-binding cassette, sub family C ; siRNA = short interfering RNA.

Techniques Used: Western Blot, Expressing, Wound Closure Assay, Thymidine Incorporation Assay, Derivative Assay, Binding Assay, Small Interfering RNA

Related Articles

Transfection:

Article Title: Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation
Article Snippet: .. Transfections SMARTpool siRNA or individual siRNA duplexes were applied to cells using DharmaFECT (Dharmacon, Lafayette, CO) transfection reagent according to the manufacturer instructions. ..

Article Title: Recruitment of factors linking transcription and processing of pre-rRNA to NOR chromatin is UBF-dependent and occurs independent of transcription in human cells
Article Snippet: .. HeLa cells were transfected with pools or individual siRNA duplexes (Dharmacon) directed against t-UTPs, the pol I subunit RPA43, the U3 snoRNP protein hU3–55K, or a control duplex. ..

Article Title: An Evolutionarily Conserved Innate Immunity Protein Interaction Network *
Article Snippet: .. In brief, pools of four siRNA duplexes or individual siRNA duplexes (Dharmacon) were transfected into either of two mouse macrophage cell lines (J774A.1 or RAW264.7) using the Amaxa nucleofector 96-well shuttle according to the manufacturer's instructions. .. Negative control siRNAs used were either a pool of four siRNA duplexes that do not target any mouse gene (Dharmacon nontargeting pool 1) or a single siRNA duplex not targeting any gene (Dharmacon nontargeting siRNA 1).

Cotransfection:

Article Title: Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation
Article Snippet: .. Transfections SMARTpool siRNA or individual siRNA duplexes were applied to cells using DharmaFECT (Dharmacon, Lafayette, CO) transfection reagent according to the manufacturer instructions. ..

Article Title: Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation
Article Snippet: .. SMARTpool siRNA or individual siRNA duplexes were applied to cells using DharmaFECT (Dharmacon, Lafayette, CO) transfection reagent according to the manufacturer instructions. ..

Synthesized:

Article Title: RNA interference screen for RGS protein specificity at muscarinic and protease-activated receptors reveals bidirectional modulation of signaling
Article Snippet: .. All siRNA used in this study were chemically synthesized by Dharmacon RNAi Technologies (Lafayette, CO). siRNA directed toward human RGS transcripts ( RGS1 to -22 ) and M 1 , M 3 , and M 4 mAChR mRNAs were purchased as siGENOME SMARTpools containing four distinct siRNA oligonucleotide duplexes; for those RGS transcripts examined after the primary SMARTpool screen, the four individual siRNA duplexes of the pool were obtained separately from Dharmacon. .. The siRNA duplex targeting a highly conserved region of sequence shared between human Gαq and Gα11 mRNAs (5′-AAGATGTTCGTGGACCTGAAC-3′) was previously described ( ).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Horizon Discovery hct116 cell line
    Analyses of VPS 4A and VPS 4B mRNA and protein abundance in HCT 116 cells and the dependencies between both genes in different cell lines mRNA levels of VPS4A (upper panel) and VPS4B (lower panel) in <t>HCT116</t> cells 72 h upon siRNA transfection. For VPS4A or VPS4B depletion, two different duplexes (#1 or #2) of siVPS4A or siVPS4B were used. For simultaneous VPS4A+B depletion, various combinations of siVPS4A and siVPS4B duplexes were used. Two different duplexes of siCTRL (#1 or #2) were used as non‐targeting controls. NT—non‐transfected. Values represent normalized counts after including variance normalized transformation performed by the DESeq2 package for RNA‐Seq data analysis. Data are means of four independent experiments ± SEM. Two‐tailed unpaired t ‐test; ns—non‐significant ( P ≥ 0.05). Upper panel, representative immunoblotting analysis of VPS4A and VPS4B abundance in lysates of HCT116 cells collected 72 h after transfection as in (A). GAPDH was used as a loading control. Lower panel, densitometry analysis of VPS4A and VPS4B abundance based on immunoblotting analysis as shown in the upper panel. NT—non‐transfected. Data are means of four independent experiments ± SEM. Wilcoxon signed rank test; ns—non‐significant ( P ≥ 0.05). Correlation between dependency scores and VPS4B copy number for selected cancer cell lines from the DepMap portal dataset ( https://depmap.org/portal/ ). According to the portal, a lower score (below −0.5) means that a gene is more likely to be dependent in a given cell line. A score of 0 is equivalent to a gene that is not essential, whereas a score of −1 corresponds to the median of all common essential genes. Immunoblotting analysis of VPS4A silencing efficiency in HOP62 and SNU410 cell lines. Lysates were prepared 6 days after siRNA transfection with non‐targeting (siCTRL#1) or VPS4A ‐targeting (siVPS4A#2 or #5) duplexes. Vinculin was used as a loading control. Data information: The exact P ‐values can be found in Appendix Table S3 .
    Hct116 Cell Line, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hct116 cell line/product/Horizon Discovery
    Average 90 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    hct116 cell line - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    92
    Horizon Discovery non targeting sirna
    Analysis of Pathogen- and Apoptotic Cell-Associated Phagocytosis (A) FACS analysis to measure uptake of pHrodo E. coli particles in variant <t>TREM2</t> iPSC-MGLCs compared with controls. n = 3, four control iPSC lines and two iPSC clones per TREM2 variant iPSC line were analyzed. (B) FACS analysis to measure uptake of pHrodo E. coli particles following <t>siRNA</t> knockdown of TREM2 in iPSC-MGLCs (T2 siRNA) or non-targeting siRNA (non-targeting [NT] siRNA) (n = 3). (C) FACS analysis of phagocytosis of fluorescent dye-labeled apoptotic SH-SY5Y cells by control and TREM2 variant iPSC-MGLCs. Quantification of phagocytosis shown. (D) 21%, 45%, and 45% reduction in T66Mhet, T66Mhom, W50Chom, respectively, compared with controls (n = 4, ∗ p
    Non Targeting Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/non targeting sirna/product/Horizon Discovery
    Average 92 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    non targeting sirna - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    84
    Horizon Discovery sirna targeting sec22a
    <t>SEC22A</t> associates with RAB18 and influences LD morphology. (A) Confocal micrograph to show overlapping localization of exogenous mEmerald-SEC22A (Green) and mCherry-ER (Red) in HeLa cells. (B) RAB18 LFQ intensities from a reciprocal BioID experiment showing a reduced association between BioID2(Gly40Ser)-SEC22A and endogenous RAB18 in RAB3GAP-null compared to wild-type HeLa cells. Data were adjusted to account for non-specific binding of RAB18 to beads and normalized by SEC22A LFQ intensities in each replicate experiment. Error bars represent s.e.m. Data for other BioID2(Gly40Ser)-SEC22A-associated proteins are provided in table S5. (C) Bar graphs to show effects of ZW10, NBAS and SEC22A knockdowns on lipid droplet number and diameter. siRNA-treated IHH cells were loaded with 200nM BSA-conjugated oleate, fixed and stained with BODIPY and DAPI, and imaged. Images were analysed using ImageJ. Data are derived from measurements from > 100 cells/condition and are representative of three independent experiments. Error bars represent SD. *p
    Sirna Targeting Sec22a, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna targeting sec22a/product/Horizon Discovery
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sirna targeting sec22a - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    Image Search Results


    Analyses of VPS 4A and VPS 4B mRNA and protein abundance in HCT 116 cells and the dependencies between both genes in different cell lines mRNA levels of VPS4A (upper panel) and VPS4B (lower panel) in HCT116 cells 72 h upon siRNA transfection. For VPS4A or VPS4B depletion, two different duplexes (#1 or #2) of siVPS4A or siVPS4B were used. For simultaneous VPS4A+B depletion, various combinations of siVPS4A and siVPS4B duplexes were used. Two different duplexes of siCTRL (#1 or #2) were used as non‐targeting controls. NT—non‐transfected. Values represent normalized counts after including variance normalized transformation performed by the DESeq2 package for RNA‐Seq data analysis. Data are means of four independent experiments ± SEM. Two‐tailed unpaired t ‐test; ns—non‐significant ( P ≥ 0.05). Upper panel, representative immunoblotting analysis of VPS4A and VPS4B abundance in lysates of HCT116 cells collected 72 h after transfection as in (A). GAPDH was used as a loading control. Lower panel, densitometry analysis of VPS4A and VPS4B abundance based on immunoblotting analysis as shown in the upper panel. NT—non‐transfected. Data are means of four independent experiments ± SEM. Wilcoxon signed rank test; ns—non‐significant ( P ≥ 0.05). Correlation between dependency scores and VPS4B copy number for selected cancer cell lines from the DepMap portal dataset ( https://depmap.org/portal/ ). According to the portal, a lower score (below −0.5) means that a gene is more likely to be dependent in a given cell line. A score of 0 is equivalent to a gene that is not essential, whereas a score of −1 corresponds to the median of all common essential genes. Immunoblotting analysis of VPS4A silencing efficiency in HOP62 and SNU410 cell lines. Lysates were prepared 6 days after siRNA transfection with non‐targeting (siCTRL#1) or VPS4A ‐targeting (siVPS4A#2 or #5) duplexes. Vinculin was used as a loading control. Data information: The exact P ‐values can be found in Appendix Table S3 .

    Journal: EMBO Molecular Medicine

    Article Title: Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer

    doi: 10.15252/emmm.201910812

    Figure Lengend Snippet: Analyses of VPS 4A and VPS 4B mRNA and protein abundance in HCT 116 cells and the dependencies between both genes in different cell lines mRNA levels of VPS4A (upper panel) and VPS4B (lower panel) in HCT116 cells 72 h upon siRNA transfection. For VPS4A or VPS4B depletion, two different duplexes (#1 or #2) of siVPS4A or siVPS4B were used. For simultaneous VPS4A+B depletion, various combinations of siVPS4A and siVPS4B duplexes were used. Two different duplexes of siCTRL (#1 or #2) were used as non‐targeting controls. NT—non‐transfected. Values represent normalized counts after including variance normalized transformation performed by the DESeq2 package for RNA‐Seq data analysis. Data are means of four independent experiments ± SEM. Two‐tailed unpaired t ‐test; ns—non‐significant ( P ≥ 0.05). Upper panel, representative immunoblotting analysis of VPS4A and VPS4B abundance in lysates of HCT116 cells collected 72 h after transfection as in (A). GAPDH was used as a loading control. Lower panel, densitometry analysis of VPS4A and VPS4B abundance based on immunoblotting analysis as shown in the upper panel. NT—non‐transfected. Data are means of four independent experiments ± SEM. Wilcoxon signed rank test; ns—non‐significant ( P ≥ 0.05). Correlation between dependency scores and VPS4B copy number for selected cancer cell lines from the DepMap portal dataset ( https://depmap.org/portal/ ). According to the portal, a lower score (below −0.5) means that a gene is more likely to be dependent in a given cell line. A score of 0 is equivalent to a gene that is not essential, whereas a score of −1 corresponds to the median of all common essential genes. Immunoblotting analysis of VPS4A silencing efficiency in HOP62 and SNU410 cell lines. Lysates were prepared 6 days after siRNA transfection with non‐targeting (siCTRL#1) or VPS4A ‐targeting (siVPS4A#2 or #5) duplexes. Vinculin was used as a loading control. Data information: The exact P ‐values can be found in Appendix Table S3 .

    Article Snippet: Generation of VPS4B knockout cell line VPS4B knockout in HCT116 cell line (HCT116 VPS4B −/− ) was generated using the CRISPR/Cas9 genome editing technology by Horizon Discovery Ltd. One sgRNA targeting exon 3 was used (5′‐ > 3′ TGATA GAGCAGAAAAAACTAA).

    Techniques: Transfection, Transformation Assay, RNA Sequencing Assay, Two Tailed Test

    Synthetic lethality between VPS 4A and VPS 4B inhibits growth of CRC cells in a mouse xenograft model Schematic illustration of xenograft experiments with HCT116 VPS4B −/− cells having doxycycline (Dox)‐inducible expression of shRNA targeting VPS4A . Left panel, growth of HCT116 VPS4B −/− shVPS4A cells as xenografts in mice in the presence or absence of doxycycline. Day 1 indicates the first day of doxycycline administration. n = 9 for each group, each mouse bearing one tumor, ± SEM. Two‐tailed unpaired t ‐test; ns—non‐significant ( P ≥ 0.05), ** P

    Journal: EMBO Molecular Medicine

    Article Title: Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer

    doi: 10.15252/emmm.201910812

    Figure Lengend Snippet: Synthetic lethality between VPS 4A and VPS 4B inhibits growth of CRC cells in a mouse xenograft model Schematic illustration of xenograft experiments with HCT116 VPS4B −/− cells having doxycycline (Dox)‐inducible expression of shRNA targeting VPS4A . Left panel, growth of HCT116 VPS4B −/− shVPS4A cells as xenografts in mice in the presence or absence of doxycycline. Day 1 indicates the first day of doxycycline administration. n = 9 for each group, each mouse bearing one tumor, ± SEM. Two‐tailed unpaired t ‐test; ns—non‐significant ( P ≥ 0.05), ** P

    Article Snippet: Generation of VPS4B knockout cell line VPS4B knockout in HCT116 cell line (HCT116 VPS4B −/− ) was generated using the CRISPR/Cas9 genome editing technology by Horizon Discovery Ltd. One sgRNA targeting exon 3 was used (5′‐ > 3′ TGATA GAGCAGAAAAAACTAA).

    Techniques: Expressing, shRNA, Mouse Assay, Two Tailed Test

    Combined VPS 4A+B depletion induces NF ‐κB signaling and caspase‐dependent and caspase‐independent cell death pathways Immunoblotting analysis of the canonical and noncanonical branches of the NF‐κB pathway and mediators of caspase‐dependent cell death. Lysates of HCT116 VPS4B −/− cells were collected 66 h after transfection with siRNA (siCTRL#1 or different siVPS4A duplexes: #2, #4, or #5). Lysates of HCT116 VPS4B +/+ and non‐transfected HCT116 VPS4B −/− cells were used to monitor the basal pathway activity. Representative blot from 10 experiments is shown. NT—non‐transfected; p‐RelA—phospho‐RelA; p‐IκBα—phospho‐IκBα; cl—cleaved caspases or PARP‐1. GAPDH or vinculin served as loading controls. Densitometry analysis of the abundance of the indicated proteins based on immunoblot images as shown in (A). Data are means of 10 (phospho‐Rel and cleaved caspase 7), nine (p52, caspase 8 and cleaved caspase 9), seven (phosphorylated and total IκB), or five (cleaved caspase 3) independent experiments. Error bars are SEM. Statistical significance was assessed using the following tests: one‐sample t ‐test (caspase 8, cleaved caspases 9, 7, and 3, total and phospho‐IκBα) or Wilcoxon signed rank test (phospho‐RelA and p52); * P

    Journal: EMBO Molecular Medicine

    Article Title: Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer

    doi: 10.15252/emmm.201910812

    Figure Lengend Snippet: Combined VPS 4A+B depletion induces NF ‐κB signaling and caspase‐dependent and caspase‐independent cell death pathways Immunoblotting analysis of the canonical and noncanonical branches of the NF‐κB pathway and mediators of caspase‐dependent cell death. Lysates of HCT116 VPS4B −/− cells were collected 66 h after transfection with siRNA (siCTRL#1 or different siVPS4A duplexes: #2, #4, or #5). Lysates of HCT116 VPS4B +/+ and non‐transfected HCT116 VPS4B −/− cells were used to monitor the basal pathway activity. Representative blot from 10 experiments is shown. NT—non‐transfected; p‐RelA—phospho‐RelA; p‐IκBα—phospho‐IκBα; cl—cleaved caspases or PARP‐1. GAPDH or vinculin served as loading controls. Densitometry analysis of the abundance of the indicated proteins based on immunoblot images as shown in (A). Data are means of 10 (phospho‐Rel and cleaved caspase 7), nine (p52, caspase 8 and cleaved caspase 9), seven (phosphorylated and total IκB), or five (cleaved caspase 3) independent experiments. Error bars are SEM. Statistical significance was assessed using the following tests: one‐sample t ‐test (caspase 8, cleaved caspases 9, 7, and 3, total and phospho‐IκBα) or Wilcoxon signed rank test (phospho‐RelA and p52); * P

    Article Snippet: Generation of VPS4B knockout cell line VPS4B knockout in HCT116 cell line (HCT116 VPS4B −/− ) was generated using the CRISPR/Cas9 genome editing technology by Horizon Discovery Ltd. One sgRNA targeting exon 3 was used (5′‐ > 3′ TGATA GAGCAGAAAAAACTAA).

    Techniques: Transfection, Activity Assay

    Inhibition of endocytosis and cell cycle progression upon simultaneous depletion of VPS 4A and VPS 4B Upper panel, microscopy images of HCT116 cells collected 48 h after transfection with control (siCTRL#1) or VPS4A‐ and/or VPS4B ‐targeting siRNA (siVPS4A#2 or #5 and siVPS4B#1 or #2). EEA1, Rab7, and LAMP1 were used as markers of early endosomes (EE), late endosomes (LE), and lysosomes, respectively, and were visualized in green. Nuclei were stained with Hoechst 33342 (blue). NT—non‐transfected cells. Scale bar, 10 μm. Lower panel, quantified fluorescence signals from microscopy images. The boxes denote the 25 th to 75 th percentile range, the center lines mark the 50 th percentile (median) and the whiskers reflect the largest and smallest observed values in at least four z‐stacks from three independent experiments. The Welch t ‐test (for EEA1 and Rab7) and the Mann–Whitney U ‐test (for LAMP1); * P

    Journal: EMBO Molecular Medicine

    Article Title: Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer

    doi: 10.15252/emmm.201910812

    Figure Lengend Snippet: Inhibition of endocytosis and cell cycle progression upon simultaneous depletion of VPS 4A and VPS 4B Upper panel, microscopy images of HCT116 cells collected 48 h after transfection with control (siCTRL#1) or VPS4A‐ and/or VPS4B ‐targeting siRNA (siVPS4A#2 or #5 and siVPS4B#1 or #2). EEA1, Rab7, and LAMP1 were used as markers of early endosomes (EE), late endosomes (LE), and lysosomes, respectively, and were visualized in green. Nuclei were stained with Hoechst 33342 (blue). NT—non‐transfected cells. Scale bar, 10 μm. Lower panel, quantified fluorescence signals from microscopy images. The boxes denote the 25 th to 75 th percentile range, the center lines mark the 50 th percentile (median) and the whiskers reflect the largest and smallest observed values in at least four z‐stacks from three independent experiments. The Welch t ‐test (for EEA1 and Rab7) and the Mann–Whitney U ‐test (for LAMP1); * P

    Article Snippet: Generation of VPS4B knockout cell line VPS4B knockout in HCT116 cell line (HCT116 VPS4B −/− ) was generated using the CRISPR/Cas9 genome editing technology by Horizon Discovery Ltd. One sgRNA targeting exon 3 was used (5′‐ > 3′ TGATA GAGCAGAAAAAACTAA).

    Techniques: Inhibition, Microscopy, Transfection, Staining, Fluorescence, MANN-WHITNEY

    Synthetic lethality between VPS 4A and VPS 4B inhibits growth of CRC lines in vitro Analysis of viability of HCT116 cells assessed 96 h after transfection with independent non‐targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #1 or #2), VPS4B (duplexes #1 or #2), or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of three independent experiments ± SEM. All values were normalized, averaged (avrg) viability of siCTRL#1‐ and #2‐transfected cells was set as 100%, the Kruskal–Wallis test followed by Dunn's multiple comparison post hoc test; ns—non‐significant ( P ≥ 0.05), ** P

    Journal: EMBO Molecular Medicine

    Article Title: Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer

    doi: 10.15252/emmm.201910812

    Figure Lengend Snippet: Synthetic lethality between VPS 4A and VPS 4B inhibits growth of CRC lines in vitro Analysis of viability of HCT116 cells assessed 96 h after transfection with independent non‐targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #1 or #2), VPS4B (duplexes #1 or #2), or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of three independent experiments ± SEM. All values were normalized, averaged (avrg) viability of siCTRL#1‐ and #2‐transfected cells was set as 100%, the Kruskal–Wallis test followed by Dunn's multiple comparison post hoc test; ns—non‐significant ( P ≥ 0.05), ** P

    Article Snippet: Generation of VPS4B knockout cell line VPS4B knockout in HCT116 cell line (HCT116 VPS4B −/− ) was generated using the CRISPR/Cas9 genome editing technology by Horizon Discovery Ltd. One sgRNA targeting exon 3 was used (5′‐ > 3′ TGATA GAGCAGAAAAAACTAA).

    Techniques: In Vitro, Transfection

    Synthetic lethality between VPS 4A and VPS 4B induces release of immunogenic DAMP s and promotes M1 macrophage polarization Measurement of ATP (A) and HMGB1 (B) released to the cell medium by HCT116 VPS4B −/− cells non‐transfected (NT) or transfected with siRNA (non‐targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4, or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52‐58 h. For non‐transfected cells (NT) , the same treatment protocol was used but without the transfection mixture. Data are means of five (A) or four (B) independent experiments ± SEM. Two‐tailed unpaired t ‐test; * *P

    Journal: EMBO Molecular Medicine

    Article Title: Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer

    doi: 10.15252/emmm.201910812

    Figure Lengend Snippet: Synthetic lethality between VPS 4A and VPS 4B induces release of immunogenic DAMP s and promotes M1 macrophage polarization Measurement of ATP (A) and HMGB1 (B) released to the cell medium by HCT116 VPS4B −/− cells non‐transfected (NT) or transfected with siRNA (non‐targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4, or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52‐58 h. For non‐transfected cells (NT) , the same treatment protocol was used but without the transfection mixture. Data are means of five (A) or four (B) independent experiments ± SEM. Two‐tailed unpaired t ‐test; * *P

    Article Snippet: Generation of VPS4B knockout cell line VPS4B knockout in HCT116 cell line (HCT116 VPS4B −/− ) was generated using the CRISPR/Cas9 genome editing technology by Horizon Discovery Ltd. One sgRNA targeting exon 3 was used (5′‐ > 3′ TGATA GAGCAGAAAAAACTAA).

    Techniques: Transfection, Cell Culture, Two Tailed Test

    Characterization of HCT 116 VPS 4B −/− cell line and engineering of HCT 116 VPS 4B −/− cells with doxycycline (Dox)‐inducible VPS 4A ‐targeting sh RNA expression ( HCT 116 VPS 4B −/− sh VPS 4A) for in vivo studies Left panel, PCR sequencing analysis verifying bi‐allelic VPS4B knockout in two CRISPR/Cas9 engineered clones derived from the HCT116 VPS4B +/+ parental line. Right panel, immunoblotting analysis of VPS4B in cell lysates of these clones. Comparison of clonal growth of isogenic HCT116 lines: parental VPS4B +/+ and VPS4B −/− . Upper panel, analysis of colony area of parental HCT116 VPS4B +/+ cells and VPS4B −/− clones assessed in the colony formation assay. Data are means of five independent experiments. Values were normalized to the colony area of parental HCT116 VPS4B +/+ cells that was set as 1. Error bars are SEM. One‐sample t ‐test; ns—non‐significant ( P ≥ 0.05). Lower panel, representative images of clonal growth of parental and VPS4B −/− HCT116 cells that were used for the quantification presented on the top. Analysis of the tumor growth in mice bearing parental HCT116 VPS4B +/+ or VPS4B −/− xenografts. n = 3 mice for each group, each mouse bearing two tumors, ± SEM. Confirmation of cell death of HCT116 VPS4B −/− cells upon VPS4A depletion. Upper panel, viability of HCT116 VPS4B −/− assessed 72 h after siVPS4A transfection (three independent siVPS4A duplexes #2, #4, and #5 were used). Non‐transfected (NT) or siCTRL#1‐transfected cells served as viability controls. Data are means of four independent experiments. Values were normalized to the viability of siCTRL#1‐transfected cells that was set as 100%. Error bars are SEM. One‐sample t ‐test; **** P

    Journal: EMBO Molecular Medicine

    Article Title: Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer

    doi: 10.15252/emmm.201910812

    Figure Lengend Snippet: Characterization of HCT 116 VPS 4B −/− cell line and engineering of HCT 116 VPS 4B −/− cells with doxycycline (Dox)‐inducible VPS 4A ‐targeting sh RNA expression ( HCT 116 VPS 4B −/− sh VPS 4A) for in vivo studies Left panel, PCR sequencing analysis verifying bi‐allelic VPS4B knockout in two CRISPR/Cas9 engineered clones derived from the HCT116 VPS4B +/+ parental line. Right panel, immunoblotting analysis of VPS4B in cell lysates of these clones. Comparison of clonal growth of isogenic HCT116 lines: parental VPS4B +/+ and VPS4B −/− . Upper panel, analysis of colony area of parental HCT116 VPS4B +/+ cells and VPS4B −/− clones assessed in the colony formation assay. Data are means of five independent experiments. Values were normalized to the colony area of parental HCT116 VPS4B +/+ cells that was set as 1. Error bars are SEM. One‐sample t ‐test; ns—non‐significant ( P ≥ 0.05). Lower panel, representative images of clonal growth of parental and VPS4B −/− HCT116 cells that were used for the quantification presented on the top. Analysis of the tumor growth in mice bearing parental HCT116 VPS4B +/+ or VPS4B −/− xenografts. n = 3 mice for each group, each mouse bearing two tumors, ± SEM. Confirmation of cell death of HCT116 VPS4B −/− cells upon VPS4A depletion. Upper panel, viability of HCT116 VPS4B −/− assessed 72 h after siVPS4A transfection (three independent siVPS4A duplexes #2, #4, and #5 were used). Non‐transfected (NT) or siCTRL#1‐transfected cells served as viability controls. Data are means of four independent experiments. Values were normalized to the viability of siCTRL#1‐transfected cells that was set as 100%. Error bars are SEM. One‐sample t ‐test; **** P

    Article Snippet: Generation of VPS4B knockout cell line VPS4B knockout in HCT116 cell line (HCT116 VPS4B −/− ) was generated using the CRISPR/Cas9 genome editing technology by Horizon Discovery Ltd. One sgRNA targeting exon 3 was used (5′‐ > 3′ TGATA GAGCAGAAAAAACTAA).

    Techniques: RNA Expression, In Vivo, Polymerase Chain Reaction, Sequencing, Knock-Out, CRISPR, Clone Assay, Derivative Assay, Colony Assay, Mouse Assay, Transfection

    Evaluation of VPS 4A and VPS 4B protein abundance in human tissues and in CRC qPCR analysis of VPS4A mRNA abundance in normal colon, adenoma, and CRC samples. Adenocarcinoma ( n = 26); adenoma ( n = 42); normal colon ( n = 24). Green horizontal bars indicate means, and red whiskers indicate SD. Differences were analyzed using the Kruskal–Wallis test followed by Dunn's multiple comparison post hoc test; ns—non‐significant ( P ≥ 0.05). Immunohistochemistry (IHC) evaluation of VPS4A abundance in pairs of normal colon versus matched CRC samples (2 representative pairs out of 100 analyzed). 3+—very intensive staining. Scale bar, 20 μm. Specificity tests of VPS4B IHC staining using human tissues and mouse xenografts. C1—strong granular cytoplasmic staining in the mucosa of the appendix. C2, C3—negative staining in the appendix (C2) and CRC (C3) when primary antibody (anti‐VPS4B) was omitted. C4—granular cytoplasmic staining in the xenograft from wild‐type ( VPS4B +/+ ) HCT116 cells. C5—negative staining in the xenograft from HCT116 VPS4B −/− cells. Scale bars: C1, C4, and C5—20 μm; C2, C3—50 μm. Specificity tests of VPS4A IHC staining in various human tissues with high and low expression of VPS4A . D1—strong granular cytoplasmic staining in the mucosa of the appendix (arrowhead, and at a higher magnification in D2) and weak staining in the muscle (arrow, and at a higher magnification in D3). Scale bars: D1—200 μm; D2 and D3—20 μm. Data information: The exact P ‐values can be found in Appendix Table S3 .

    Journal: EMBO Molecular Medicine

    Article Title: Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer

    doi: 10.15252/emmm.201910812

    Figure Lengend Snippet: Evaluation of VPS 4A and VPS 4B protein abundance in human tissues and in CRC qPCR analysis of VPS4A mRNA abundance in normal colon, adenoma, and CRC samples. Adenocarcinoma ( n = 26); adenoma ( n = 42); normal colon ( n = 24). Green horizontal bars indicate means, and red whiskers indicate SD. Differences were analyzed using the Kruskal–Wallis test followed by Dunn's multiple comparison post hoc test; ns—non‐significant ( P ≥ 0.05). Immunohistochemistry (IHC) evaluation of VPS4A abundance in pairs of normal colon versus matched CRC samples (2 representative pairs out of 100 analyzed). 3+—very intensive staining. Scale bar, 20 μm. Specificity tests of VPS4B IHC staining using human tissues and mouse xenografts. C1—strong granular cytoplasmic staining in the mucosa of the appendix. C2, C3—negative staining in the appendix (C2) and CRC (C3) when primary antibody (anti‐VPS4B) was omitted. C4—granular cytoplasmic staining in the xenograft from wild‐type ( VPS4B +/+ ) HCT116 cells. C5—negative staining in the xenograft from HCT116 VPS4B −/− cells. Scale bars: C1, C4, and C5—20 μm; C2, C3—50 μm. Specificity tests of VPS4A IHC staining in various human tissues with high and low expression of VPS4A . D1—strong granular cytoplasmic staining in the mucosa of the appendix (arrowhead, and at a higher magnification in D2) and weak staining in the muscle (arrow, and at a higher magnification in D3). Scale bars: D1—200 μm; D2 and D3—20 μm. Data information: The exact P ‐values can be found in Appendix Table S3 .

    Article Snippet: Generation of VPS4B knockout cell line VPS4B knockout in HCT116 cell line (HCT116 VPS4B −/− ) was generated using the CRISPR/Cas9 genome editing technology by Horizon Discovery Ltd. One sgRNA targeting exon 3 was used (5′‐ > 3′ TGATA GAGCAGAAAAAACTAA).

    Techniques: Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining, Negative Staining, Expressing

    Analysis of Pathogen- and Apoptotic Cell-Associated Phagocytosis (A) FACS analysis to measure uptake of pHrodo E. coli particles in variant TREM2 iPSC-MGLCs compared with controls. n = 3, four control iPSC lines and two iPSC clones per TREM2 variant iPSC line were analyzed. (B) FACS analysis to measure uptake of pHrodo E. coli particles following siRNA knockdown of TREM2 in iPSC-MGLCs (T2 siRNA) or non-targeting siRNA (non-targeting [NT] siRNA) (n = 3). (C) FACS analysis of phagocytosis of fluorescent dye-labeled apoptotic SH-SY5Y cells by control and TREM2 variant iPSC-MGLCs. Quantification of phagocytosis shown. (D) 21%, 45%, and 45% reduction in T66Mhet, T66Mhom, W50Chom, respectively, compared with controls (n = 4, ∗ p

    Journal: Cell Reports

    Article Title: Human Induced Pluripotent Stem Cell-Derived Microglia-Like Cells Harboring TREM2 Missense Mutations Show Specific Deficits in Phagocytosis

    doi: 10.1016/j.celrep.2018.07.094

    Figure Lengend Snippet: Analysis of Pathogen- and Apoptotic Cell-Associated Phagocytosis (A) FACS analysis to measure uptake of pHrodo E. coli particles in variant TREM2 iPSC-MGLCs compared with controls. n = 3, four control iPSC lines and two iPSC clones per TREM2 variant iPSC line were analyzed. (B) FACS analysis to measure uptake of pHrodo E. coli particles following siRNA knockdown of TREM2 in iPSC-MGLCs (T2 siRNA) or non-targeting siRNA (non-targeting [NT] siRNA) (n = 3). (C) FACS analysis of phagocytosis of fluorescent dye-labeled apoptotic SH-SY5Y cells by control and TREM2 variant iPSC-MGLCs. Quantification of phagocytosis shown. (D) 21%, 45%, and 45% reduction in T66Mhet, T66Mhom, W50Chom, respectively, compared with controls (n = 4, ∗ p

    Article Snippet: Cells were transfected using SMARTpool ON-TARGETplus TREM2 (Dharmacon, USA) or non-targeting siRNA (Horizon Discovery, UK) and Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions.

    Techniques: FACS, Variant Assay, Clone Assay, Labeling

    SEC22A associates with RAB18 and influences LD morphology. (A) Confocal micrograph to show overlapping localization of exogenous mEmerald-SEC22A (Green) and mCherry-ER (Red) in HeLa cells. (B) RAB18 LFQ intensities from a reciprocal BioID experiment showing a reduced association between BioID2(Gly40Ser)-SEC22A and endogenous RAB18 in RAB3GAP-null compared to wild-type HeLa cells. Data were adjusted to account for non-specific binding of RAB18 to beads and normalized by SEC22A LFQ intensities in each replicate experiment. Error bars represent s.e.m. Data for other BioID2(Gly40Ser)-SEC22A-associated proteins are provided in table S5. (C) Bar graphs to show effects of ZW10, NBAS and SEC22A knockdowns on lipid droplet number and diameter. siRNA-treated IHH cells were loaded with 200nM BSA-conjugated oleate, fixed and stained with BODIPY and DAPI, and imaged. Images were analysed using ImageJ. Data are derived from measurements from > 100 cells/condition and are representative of three independent experiments. Error bars represent SD. *p

    Journal: bioRxiv

    Article Title: Comparative proximity biotinylation implicates RAB18 in cholesterol mobilization and biosynthesis

    doi: 10.1101/871517

    Figure Lengend Snippet: SEC22A associates with RAB18 and influences LD morphology. (A) Confocal micrograph to show overlapping localization of exogenous mEmerald-SEC22A (Green) and mCherry-ER (Red) in HeLa cells. (B) RAB18 LFQ intensities from a reciprocal BioID experiment showing a reduced association between BioID2(Gly40Ser)-SEC22A and endogenous RAB18 in RAB3GAP-null compared to wild-type HeLa cells. Data were adjusted to account for non-specific binding of RAB18 to beads and normalized by SEC22A LFQ intensities in each replicate experiment. Error bars represent s.e.m. Data for other BioID2(Gly40Ser)-SEC22A-associated proteins are provided in table S5. (C) Bar graphs to show effects of ZW10, NBAS and SEC22A knockdowns on lipid droplet number and diameter. siRNA-treated IHH cells were loaded with 200nM BSA-conjugated oleate, fixed and stained with BODIPY and DAPI, and imaged. Images were analysed using ImageJ. Data are derived from measurements from > 100 cells/condition and are representative of three independent experiments. Error bars represent SD. *p

    Article Snippet: Lipid loading experimentsFor LD number and diameter measurements, IHH cells were seeded onto glass coverslips. siRNA transfections were carried out using FuGene reagent (Promega) according to manufacturer’s instructions. siRNAs targeting ZW10 and NBAS were obtained from IDT, Coralville, IA; siRNA targeting SEC22A was obtained from Horizon Discovery, Cambridge, UK.

    Techniques: Binding Assay, Staining, Derivative Assay