anti-gapdh Search Results


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  • 99
    Thermo Fisher anti gapdh
    RP4010 inhibited calcium-regulated Akt/mTOR and NFAT signaling. ( A and B ) MiaPaCa-2 cells were grown overnight in 100-mm petri dishes to nearly 50% confluence. The cells were then treated on the following day with RP4010 (10 µM) for 72 h. Protein extraction, determination of protein concentration, SDS-PAGE, and Western blot were performed as described in the Methods ( n = 1). <t>β-actin</t> and <t>GAPDH</t> were used as loading controls. The expression of marker proteins was indicated as fold change relative to the control, and the quantitative analysis of mean pixel density of the blots was performed using ImageJ software. ( C ) MiaPaCa-2 cells were exposed to RP4010 (10 µM) for 48 h. At the end of the treatment period, RNA was isolated and RT-qPCR was performed as described in Methods (* p
    Anti Gapdh, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1766 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti gapdh
    Expression of <t>Cav3‐WT,</t> ‐F97C or ‐S141R with Ca v 1.2 channels in HEK293 cells HEK293 cells were transfected with Cav3‐WT, Cav3‐F97C or Cav3‐S141R with Ca v 1.2 + Ca v β 2cN4 subunits. Cells were immunolabelled with anti‐Cav‐3 (red) and anti‐HA (green) antibodies and imaged using confocal microscopy ( A – I ). J , representative western blot from HEK293 cells expressing Ca V 1.2 and Ca v β 2CN4 subunits with Cav3‐WT, F97C or S141R plasmids probed for Cav3 protein expression with <t>GAPDH</t> loading control. Lysates from HEK293 cells with no plasmid transfected served as a negative control. K , normalized Cav3 protein signals (Cav3/GAPDH) were analysed using ANOVA and were not statistically different between transfected groups.
    Anti Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Santa Cruz Biotechnology gapdh
    <t>OXPHOS</t> protein expression in breast cancer cell lines. A) Cells were maintained in normal growth medium and 40 μg of whole cell extract proteins were separated on NuPAGE 4-12% Bis-Tris gels. A representative western blot is shown. For A and C, the membranes were stripped and re-probed for <t>GAPDH</t> for normalization. B) Values are the ratio of target protein/ GAPDH normalized to each value for MCF-7, set to one. Values are the average of 5 separate experiments ± SEM. C) Western blot for UQCRC2. D) Complex III activity was measured in triplicate in three biological replicate samples for each cell line. Thus values are the avg. of 9 separate determinations ± SEM. For B, C, and D: * p
    Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 33546 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc gapdh
    Knockdown of CPSF4 inhibits <t>PI3K/AKT,</t> MAPK signaling and activates caspase-dependent apoptotic pathway in H1299 and A549 cells. ( A ) At 72 hours after siRNA treatment, the expression of CPSF4 protein and the total and phosphorylated Akt, PI3K, ERK1/2, JNK and p38 proteins in H1299 and A549 was detected by Western blot. <t>GAPDH</t> served as the loading control. ( B ) Apoptosis in H1299 and A549 was determined by flow cytometry 72 h after siRNA transfection using an Annexin V-FITC/PI-staining kit. The representative data from three independent experiments are shown. ( C ) Apoptosis was calculated in terms of the FITC-positive in cells. Results are shown as the mean ± SD of three independent experiments (*, P
    Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 30846 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gapdh  (Abcam)
    99
    Abcam gapdh
    AA6 prevents metastasization targeting the <t>TET–miR200–Zeb1/CtBP1–MMP3</t> axis. a Relative enrichment of 5mC in selected CCpGG sites of miR-200 family promoter regions for cluster 1 (left panel) and cluster 2 (right panel) in 4T1-injected mice DNA treated with AA6 (50 mg/kg; grey bars) versus control mice DNA (black bars); n = 5. b Pri-miR-200 cluster 1 (left panel) and cluster 2 (right panel) level of AA6 (50 mg/kg; grey bars) treated 4T1-injected mice expressed as fold-induction compared to untreated mice (black bars); n = 3. c Cluster 1 (miR-200b, miR-200a and miR-429; left panel) and cluster 2 (miR-200c and miR-141; right panel) expression in 4T1-injected mice treated with AA6 (50 mg/kg; grey bars), the graph represents average fold changes versus controls (black bars); n = 4. d Cluster 1 (miR-200b, miR-200a and miR-429; left panel) and cluster 2 (miR-200c and miR-141; right panel) expression in 4T1 cells treated with AA6 (50 µM; grey bars) for 6, 16, and 24 h, bar graphs represent average fold changes versus vehicle-treated cells (black bars); n = 4. e Representative WB (left panel) and relative densitometry (right panel; n = 5) of ZEB1 protein level in AA6 (50 mg/kg; grey bar) treated mice compared to controls (black bar). <t>GAPDH</t> and Red Ponceau were used as loading controls. f , g Zeb1 mRNA expression levels ( f ) and representative western blotting analysis of ZEB1 protein expression ( g ) in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); the right panel shows the relative densitometry as fold-change versus vehicle. α-tubulin was used as loading control; n = 4. h Representative WB (left panel) and relative densitometry (right panel) of ZEB1 protein expression level in AA6 treated 4T1 cells compared to vehicle-treated cells after transfection either with scramble-LNA (vehicle: black bar; AA6 50 µM: light grey bar) or anti-miR-200c-LNA (vehicle: dark grey bar; AA6 50 µM: medium grey bar). α-tubulin was used as loading control; n = 4. Data are presented as mean ± SE; * p
    Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 18820 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti gapdh
    Inhibition of Ep3 suppresses the growth of A549 cells via inhibition of TGF-β/Smad signaling. The protein expression of (A) TGF-β, (B) <t>p-Smad2</t> and Smad2, and (C) p-Smad3 and Smad3 were detected by western blotting and normalized to <t>GAPDH</t> levels. (D) Cell viability was assessed by MTT assay. (E) Cell apoptosis was assessed by flow cytometry assay. The data are presented as the mean ± the standard error of the mean of three independent experiments *P
    Anti Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8049 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore gapdh
    Investigation of mechanisms underlying NMR-detected changes in the levels of choline metabolites and lactate following treatment of SF188 pediatric glioblastoma cells with PI-103 (5×GI 50 ). (A) Representative Western blots showing changes in protein expression levels of enzymes involved in choline metabolism (CHKA) and glucose metabolism including: GLUT1, <t>HK2</t> and LDHA, at selected time points post treatment with PI-103. <t>GAPDH</t> was used as a loading control. (B) Quantitative measurement of 1 H-NMR detected percentage changes in the levels of lactate (Lac, internal external) and glucose (external) at selected time points post treatment with PI-103 relative to controls, 8 hours n = 2. Results are expressed as percentage of treated to control and presented as the mean ± SD (error bars). Statistically significant different from the control *p≤0.05, **p
    Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15716 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mouse monoclonal antibody
    Chromatin architecture of the Reg locus in adult cortex and pancreas. ( A ) Levels of cohesin were assayed by immunoblot with <t>SA1</t> and Rad21 antibodies in pancreas from wild-type and SA1 heterozygous mice (two individuals per genotype). <t>GAPDH</t> serves as loading control. ( B ) Table showing the four genes differentially expressed (FDR
    Mouse Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc rabbit anti gapdh
    K nockdown of <t>FTO</t> does not significantly change mRNA levels of genes involved in miRNA biogenesis. The steady-state mRNA levels of DICER , DROSHA , DGCR8 and ADAR were analyzed by qRT-PCR in cells treated with scrambled (scr) and FTO- specific siRNAs, respectively. <t>GAPDH</t> was used as a reference gene. The observed changes were not significant. Merged values of mean ± SD from triplicates per assay for the three independent cell lines FTO1C1, FTO2D4 and FTO3C3 are depicted. FTO kd, FTO -specific siRNA treated cells, scr siRNA, scrambled siRNA treated cells.
    Rabbit Anti Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 3270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology mouse anti gapdh
    YY1 and rs398206 affect <t>MX2</t> expression in melanoma cells. a YY1 was knocked down using four different siRNAs in UACC647 cells, and MX2 levels were measured. <t>GAPDH</t> -normalized MX2 mRNA levels are shown as fold change over those from non-targeting siRNA. Four biological replicates of n = 6 were combined (total n = 24). b CRISPRi using dCAS9-KRAB-MeCP2 and four different gRNAs targeting rs398206 in UACC903 cells. MX2 mRNA levels ( GAPDH -normalized) are shown as fold change over those from non-targeting gRNA. Three biological replicates of n = 6 were combined (total n = 18, except gRNA-3, n = 17). gRNA 1, 3, and 4 directly overlap rs398206. gRNA 2 targets ~25 bp upstream of rs398206. AAVS1 (gRNA targeting adeno-associated virus integration site on Chr19). Box: Median and 25th to 75th percentile. Whisker: 10th to 90th percentile. P -values are shown from one-sample Wilcoxon test (two-sided) for difference from non-targeting siRNA/gRNA. Dotted line denotes the MX2 levels in non-targeting siRNA/gRNA control. Source data are provided as a Source Data file.
    Mouse Anti Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1920 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam anti gapdh antibody epr16891
    YY1 and rs398206 affect <t>MX2</t> expression in melanoma cells. a YY1 was knocked down using four different siRNAs in UACC647 cells, and MX2 levels were measured. <t>GAPDH</t> -normalized MX2 mRNA levels are shown as fold change over those from non-targeting siRNA. Four biological replicates of n = 6 were combined (total n = 24). b CRISPRi using dCAS9-KRAB-MeCP2 and four different gRNAs targeting rs398206 in UACC903 cells. MX2 mRNA levels ( GAPDH -normalized) are shown as fold change over those from non-targeting gRNA. Three biological replicates of n = 6 were combined (total n = 18, except gRNA-3, n = 17). gRNA 1, 3, and 4 directly overlap rs398206. gRNA 2 targets ~25 bp upstream of rs398206. AAVS1 (gRNA targeting adeno-associated virus integration site on Chr19). Box: Median and 25th to 75th percentile. Whisker: 10th to 90th percentile. P -values are shown from one-sample Wilcoxon test (two-sided) for difference from non-targeting siRNA/gRNA. Dotted line denotes the MX2 levels in non-targeting siRNA/gRNA control. Source data are provided as a Source Data file.
    Anti Gapdh Antibody Epr16891, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 737 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology rabbit anti gapdh
    The αN4 is an integral part of OASL. ( A ) The crystal structure of OLD with a zoom-in on the αN4 helix. The anchoring residues Val67, Phe70 and Asn72 are indicated and their side chains are shown in sticks. ( B ) An alignment of porcine OAS1 (pOAS1), human OAS1 (hOAS1), human cGAS (hcGAS), human OASL (hOASL), Archaeoglobus fulgidus CCA (AfCCA) and Saccharomyces cerevisiae PAP (yPAP). The residues spanning the αN4 or the αN4-equivalent helices in cGAS, CCA and PAP are indicated. The asterisks indicate the three carboxyl acid residues in the catalytic triad of OAS1, cGAS, CCA and PAP. The residues are colored according to conservation. ( C ) HEK293T OASL neg cells were co-transfected with <t>V5-tagged</t> empty pcDNA3.1 vector (mock), OASL, OASL V67G or OASL N72K in combination with an IFN-β promoter firefly luciferase reporter construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. Error bars indicate standard deviation from three independent experiments. The inset shows an immunoblot against V5 and <t>GAPDH</t> to confirm the expression of the transfected constructs and to ensure that similar amounts were loaded on the gel, respectively. ( D ) HeLa cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL V67G or OASL N72K in combination with an IFN-β promoter firefly luciferase reporter construct and a β-actin promoter Renilla luciferase construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. The measurements are shown as a ratio of firefly to Renilla luciferase activities. Error bars indicate standard deviation from three independent experiments. **: P -value ≤ 0.01. ***: P -value ≤ 0.001.
    Rabbit Anti Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1789 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    RP4010 inhibited calcium-regulated Akt/mTOR and NFAT signaling. ( A and B ) MiaPaCa-2 cells were grown overnight in 100-mm petri dishes to nearly 50% confluence. The cells were then treated on the following day with RP4010 (10 µM) for 72 h. Protein extraction, determination of protein concentration, SDS-PAGE, and Western blot were performed as described in the Methods ( n = 1). β-actin and GAPDH were used as loading controls. The expression of marker proteins was indicated as fold change relative to the control, and the quantitative analysis of mean pixel density of the blots was performed using ImageJ software. ( C ) MiaPaCa-2 cells were exposed to RP4010 (10 µM) for 48 h. At the end of the treatment period, RNA was isolated and RT-qPCR was performed as described in Methods (* p

    Journal: Cancers

    Article Title: Calcium Release-Activated Calcium (CRAC) Channel Inhibition Suppresses Pancreatic Ductal Adenocarcinoma Cell Proliferation and Patient-Derived Tumor Growth

    doi: 10.3390/cancers12030750

    Figure Lengend Snippet: RP4010 inhibited calcium-regulated Akt/mTOR and NFAT signaling. ( A and B ) MiaPaCa-2 cells were grown overnight in 100-mm petri dishes to nearly 50% confluence. The cells were then treated on the following day with RP4010 (10 µM) for 72 h. Protein extraction, determination of protein concentration, SDS-PAGE, and Western blot were performed as described in the Methods ( n = 1). β-actin and GAPDH were used as loading controls. The expression of marker proteins was indicated as fold change relative to the control, and the quantitative analysis of mean pixel density of the blots was performed using ImageJ software. ( C ) MiaPaCa-2 cells were exposed to RP4010 (10 µM) for 48 h. At the end of the treatment period, RNA was isolated and RT-qPCR was performed as described in Methods (* p

    Article Snippet: Anti-NFAT1 (Cell Signaling, Danvers, MA, USA), anti-p-Akt (Cell Signaling), anti-p-4EBP1 (Cell Signaling), anti-S6K (Cell Signaling), anti-p-S6K (Cell Signaling), anti-NF-κB (MilliporeSigma, Burlington, MA, USA), anti-β-actin (Cell Signaling), and anti-GAPDH (Thermo Fisher Scientific, Waltham, MA, USA) primary antibodies were used for Western Blot analysis.

    Techniques: Protein Extraction, Protein Concentration, SDS Page, Western Blot, Expressing, Marker, Software, Isolation, Quantitative RT-PCR

    Expression of Cav3‐WT, ‐F97C or ‐S141R with Ca v 1.2 channels in HEK293 cells HEK293 cells were transfected with Cav3‐WT, Cav3‐F97C or Cav3‐S141R with Ca v 1.2 + Ca v β 2cN4 subunits. Cells were immunolabelled with anti‐Cav‐3 (red) and anti‐HA (green) antibodies and imaged using confocal microscopy ( A – I ). J , representative western blot from HEK293 cells expressing Ca V 1.2 and Ca v β 2CN4 subunits with Cav3‐WT, F97C or S141R plasmids probed for Cav3 protein expression with GAPDH loading control. Lysates from HEK293 cells with no plasmid transfected served as a negative control. K , normalized Cav3 protein signals (Cav3/GAPDH) were analysed using ANOVA and were not statistically different between transfected groups.

    Journal: The Journal of Physiology

    Article Title: Long QT syndrome caveolin‐3 mutations differentially modulate Kv4 and Cav1.2 channels to contribute to action potential prolongation

    doi: 10.1113/JP276014

    Figure Lengend Snippet: Expression of Cav3‐WT, ‐F97C or ‐S141R with Ca v 1.2 channels in HEK293 cells HEK293 cells were transfected with Cav3‐WT, Cav3‐F97C or Cav3‐S141R with Ca v 1.2 + Ca v β 2cN4 subunits. Cells were immunolabelled with anti‐Cav‐3 (red) and anti‐HA (green) antibodies and imaged using confocal microscopy ( A – I ). J , representative western blot from HEK293 cells expressing Ca V 1.2 and Ca v β 2CN4 subunits with Cav3‐WT, F97C or S141R plasmids probed for Cav3 protein expression with GAPDH loading control. Lysates from HEK293 cells with no plasmid transfected served as a negative control. K , normalized Cav3 protein signals (Cav3/GAPDH) were analysed using ANOVA and were not statistically different between transfected groups.

    Article Snippet: To detect multiple proteins, blots were cut and incubated with anti‐Cav3 (catalogue number 610421; Becton‐Dickinson Biosciences, Franklin Lakes, NJ, USA) or anti‐GAPDH (MAB374; Millipore Sigma, Burlington, MA, USA) and then incubated with HRP‐conjugated secondary antibodies.

    Techniques: Expressing, Transfection, Confocal Microscopy, Western Blot, Plasmid Preparation, Negative Control

    A short isoform of Sup35 appears during fermentative growth in glucose. A, GT17 [ psi − pin − ] cells grown in the mid log-phase ( lane 5 ) were starved in YPA medium for 12 h ( lane 6 ), and cultured in YPDA. Cells were harvested at the specified time points ( lanes 7–14 ) and analyzed by Western blotting ( WB ) using anti-Sup35 and anti-GAPDH. The four leftmost lanes indicate 2-fold dilutions of the protein to confirm that the condition used was semi-quantitative. B, the protein levels of Sup35 ( blue ) and Sup35s ( red ) were measured by MultiGauge and normalized to that of GAPDH, and the normalized Sup35 level (0 h) was defined as 100%. Dotted line indicates the A 600 of the yeast culture.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteolysis suppresses spontaneous prion generation in yeast

    doi: 10.1074/jbc.M117.811323

    Figure Lengend Snippet: A short isoform of Sup35 appears during fermentative growth in glucose. A, GT17 [ psi − pin − ] cells grown in the mid log-phase ( lane 5 ) were starved in YPA medium for 12 h ( lane 6 ), and cultured in YPDA. Cells were harvested at the specified time points ( lanes 7–14 ) and analyzed by Western blotting ( WB ) using anti-Sup35 and anti-GAPDH. The four leftmost lanes indicate 2-fold dilutions of the protein to confirm that the condition used was semi-quantitative. B, the protein levels of Sup35 ( blue ) and Sup35s ( red ) were measured by MultiGauge and normalized to that of GAPDH, and the normalized Sup35 level (0 h) was defined as 100%. Dotted line indicates the A 600 of the yeast culture.

    Article Snippet: The extracts were analyzed by Western blotting using the following antibodies; anti-Sup35 antibody raised against His-tagged Sup35 (136–483), anti-Prb1 antibody raised against His-tagged mature Prb1 (281–573), anti-GAPDH antibody raised against His-tagged human GAPDH (1–335), and anti-FLAG M2 antibody (Sigma).

    Techniques: Cell Culture, Western Blot

    Senescence of P8 cells requires p53. (A) Left: Representative immunoblots for p16, p19, and p53 in P2 neurospheres from 2-m R1 and P8 mice. (B) Densitometric quantification of p16, p19, and p53 relative to Gapdh levels (R1 n = 7, P8 n = 7). (C) Percentage of cells with γ-H2AX + foci. Positive control (C+) is a doxorubicin-treated (0.5 μg/ml, 6 h) neurosphere culture. (D) Left : Representative immunoblot for phospho-p53 in P2 neurospheres from 2-m R1 and P8 mice. Right : Densitometric quantification of pp53 relative to Gapdh levels (R1 n = 3, P8 n = 3). (E) Treatment with 20 μ m p53 inhibitor PFTα or 10 μ m ATM inhibitor KU55933 prevents the P8 senescent phenotype. (F) SA β-gal labeling of P8 cells infected with a control or with a p53 shRNA-carrying retrovirus (R1 n = 4, P8 n = 4). (G) Representative images of p53 shRNA and control-infected cultures. Upper panels : phase contrast. Lower panels : SA β-gal staining. Data are shown as mean ± SEM of the indicated number of cultures ( n ) from each strain (* P

    Journal: Aging Cell

    Article Title: Regulation of the p19Arf/p53 pathway by histone acetylation underlies neural stem cell behavior in senescence-prone SAMP8 mice

    doi: 10.1111/acel.12328

    Figure Lengend Snippet: Senescence of P8 cells requires p53. (A) Left: Representative immunoblots for p16, p19, and p53 in P2 neurospheres from 2-m R1 and P8 mice. (B) Densitometric quantification of p16, p19, and p53 relative to Gapdh levels (R1 n = 7, P8 n = 7). (C) Percentage of cells with γ-H2AX + foci. Positive control (C+) is a doxorubicin-treated (0.5 μg/ml, 6 h) neurosphere culture. (D) Left : Representative immunoblot for phospho-p53 in P2 neurospheres from 2-m R1 and P8 mice. Right : Densitometric quantification of pp53 relative to Gapdh levels (R1 n = 3, P8 n = 3). (E) Treatment with 20 μ m p53 inhibitor PFTα or 10 μ m ATM inhibitor KU55933 prevents the P8 senescent phenotype. (F) SA β-gal labeling of P8 cells infected with a control or with a p53 shRNA-carrying retrovirus (R1 n = 4, P8 n = 4). (G) Representative images of p53 shRNA and control-infected cultures. Upper panels : phase contrast. Lower panels : SA β-gal staining. Data are shown as mean ± SEM of the indicated number of cultures ( n ) from each strain (* P

    Article Snippet: The membranes were blocked 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline supplied with 0.1% Tween 20 (TBS-T) and incubated with rabbit anti-p53 (1:500, Novocastra), ac-p53 (1:1000, Cell Signaling), ac-H3 (1:10 000, Sigma), p16 (1:200, Santa Cruz) or pp53 (S18) (1:500, RyD), rat anti-p19 (1:1000, Abcam), or mouse anti-Gapdh (1:1500, Millipore) antibodies in blocking buffer, o/n at 4 °C.

    Techniques: Western Blot, Mouse Assay, Positive Control, Labeling, Infection, shRNA, Staining

    Inhibition of HDACs in neurospheres induces senescence. (A) Left: Representative immunoblot for Ac-p53 and p19 in R1 neurospheres treated with TSA or VPA. Right : Densitometric quantification reveals a TSA and VPA dose-dependent increase in Ac-p53 and p19 protein levels, relative to Gapdh (in arbitrary units, a.u.). (B) Percentage of BrdU-positive cells in P8, R1, and R1 TSA- or VPA-treated neurospheres. (C) Secondary spheres from R1 mice treated with vehicle (DMSO), 50 n m TSA, or 4 m m VPA. Insets : TSA- and VPA-treated R1 neurospheres exhibit SA β-gal staining. (D) SA β-gal labeling of P8 cells infected with control or p53 shRNA-carrying retroviruses and treated with DMSO or 50 n m TSA (R1 n = 5, P8 n = 5). (E) Fold changes in the number of neurospheres, of SA β-gal + labeling, in the number of S100β + cells, and the level of S100β mRNA in TSA-treated relative to untreated cultures in C57BL/6 wild-type and p53 or p19 mutant mice. (F) Senescent phenotype of P8 cultures is rescued by treatment with 50 μ m anacardic acid (AA). Data are shown as mean ± SEM of 3 independent cultures from each strain or treatment (* P

    Journal: Aging Cell

    Article Title: Regulation of the p19Arf/p53 pathway by histone acetylation underlies neural stem cell behavior in senescence-prone SAMP8 mice

    doi: 10.1111/acel.12328

    Figure Lengend Snippet: Inhibition of HDACs in neurospheres induces senescence. (A) Left: Representative immunoblot for Ac-p53 and p19 in R1 neurospheres treated with TSA or VPA. Right : Densitometric quantification reveals a TSA and VPA dose-dependent increase in Ac-p53 and p19 protein levels, relative to Gapdh (in arbitrary units, a.u.). (B) Percentage of BrdU-positive cells in P8, R1, and R1 TSA- or VPA-treated neurospheres. (C) Secondary spheres from R1 mice treated with vehicle (DMSO), 50 n m TSA, or 4 m m VPA. Insets : TSA- and VPA-treated R1 neurospheres exhibit SA β-gal staining. (D) SA β-gal labeling of P8 cells infected with control or p53 shRNA-carrying retroviruses and treated with DMSO or 50 n m TSA (R1 n = 5, P8 n = 5). (E) Fold changes in the number of neurospheres, of SA β-gal + labeling, in the number of S100β + cells, and the level of S100β mRNA in TSA-treated relative to untreated cultures in C57BL/6 wild-type and p53 or p19 mutant mice. (F) Senescent phenotype of P8 cultures is rescued by treatment with 50 μ m anacardic acid (AA). Data are shown as mean ± SEM of 3 independent cultures from each strain or treatment (* P

    Article Snippet: The membranes were blocked 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline supplied with 0.1% Tween 20 (TBS-T) and incubated with rabbit anti-p53 (1:500, Novocastra), ac-p53 (1:1000, Cell Signaling), ac-H3 (1:10 000, Sigma), p16 (1:200, Santa Cruz) or pp53 (S18) (1:500, RyD), rat anti-p19 (1:1000, Abcam), or mouse anti-Gapdh (1:1500, Millipore) antibodies in blocking buffer, o/n at 4 °C.

    Techniques: Inhibition, Mouse Assay, Staining, Labeling, Infection, shRNA, Mutagenesis

    Bcr−/− and abr−/− PASMC show increased proliferation when exposed to hypoxia in vitro . A, Western blot analysis on PASMC lysates from wt , bcr−/− or abr−/− mice with anti-Bcr N20 antibodies or Abr antiserum. GAPDH, loading control. B, Third passage primary PASMC isolated from the intrapulmonary arteries of 5 different mice per genotype (1×10 4 cells/well) were synchronized by serum free medium for 24 hrs, then cultured in medium with 10% FBS for 5 days, after which cells were counted. The Rac inhibitor Z62954982 was added to the indicated samples. * p

    Journal: PLoS ONE

    Article Title: Lack of Bcr and Abr Promotes Hypoxia-Induced Pulmonary Hypertension in Mice

    doi: 10.1371/journal.pone.0049756

    Figure Lengend Snippet: Bcr−/− and abr−/− PASMC show increased proliferation when exposed to hypoxia in vitro . A, Western blot analysis on PASMC lysates from wt , bcr−/− or abr−/− mice with anti-Bcr N20 antibodies or Abr antiserum. GAPDH, loading control. B, Third passage primary PASMC isolated from the intrapulmonary arteries of 5 different mice per genotype (1×10 4 cells/well) were synchronized by serum free medium for 24 hrs, then cultured in medium with 10% FBS for 5 days, after which cells were counted. The Rac inhibitor Z62954982 was added to the indicated samples. * p

    Article Snippet: Western blot analysis was done with Abr antiserum (1∶200) and Bcr (N-20)(1∶500, Santa Cruz) or GAPDH antibodies (1∶5000, Millipore).

    Techniques: In Vitro, Western Blot, Mouse Assay, Isolation, Cell Culture

    OXPHOS protein expression in breast cancer cell lines. A) Cells were maintained in normal growth medium and 40 μg of whole cell extract proteins were separated on NuPAGE 4-12% Bis-Tris gels. A representative western blot is shown. For A and C, the membranes were stripped and re-probed for GAPDH for normalization. B) Values are the ratio of target protein/ GAPDH normalized to each value for MCF-7, set to one. Values are the average of 5 separate experiments ± SEM. C) Western blot for UQCRC2. D) Complex III activity was measured in triplicate in three biological replicate samples for each cell line. Thus values are the avg. of 9 separate determinations ± SEM. For B, C, and D: * p

    Journal: Experimental cell research

    Article Title: Nuclear respiratory factor-1 and bioenergetics in tamoxifen-resistant breast cancer cells

    doi: 10.1016/j.yexcr.2016.08.006

    Figure Lengend Snippet: OXPHOS protein expression in breast cancer cell lines. A) Cells were maintained in normal growth medium and 40 μg of whole cell extract proteins were separated on NuPAGE 4-12% Bis-Tris gels. A representative western blot is shown. For A and C, the membranes were stripped and re-probed for GAPDH for normalization. B) Values are the ratio of target protein/ GAPDH normalized to each value for MCF-7, set to one. Values are the average of 5 separate experiments ± SEM. C) Western blot for UQCRC2. D) Complex III activity was measured in triplicate in three biological replicate samples for each cell line. Thus values are the avg. of 9 separate determinations ± SEM. For B, C, and D: * p

    Article Snippet: Antibodies were purchased as follows: NRF-1, Rockland Immunochemicals, Inc (Pottstown, PA, USA); TFAM (DO1P), Abnova; total OXPHOS WB antibody cocktail (Abcam, Cambridge, MA, USA);α-tubulin, Neomarkers; β-actin, Sigma-Aldrich; GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA); UQCRC2 (PA530204, Thermo Fisher, Waltham, MA, USA).

    Techniques: Expressing, Western Blot, Activity Assay

    Unfolded Protein Response activation in TA UPR activation was analyzed by immunoblotting with indicated antibodies in TA muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, and p‐Eif2a protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Journal: The EMBO Journal

    Article Title: Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone‐responsive myopathy

    doi: 10.15252/embj.201899576

    Figure Lengend Snippet: Unfolded Protein Response activation in TA UPR activation was analyzed by immunoblotting with indicated antibodies in TA muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, and p‐Eif2a protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Article Snippet: Reagents The following primary antibodies were used: anti‐p62 (SQSTM) (Abnova), anti‐LAMP2 (Abcam), anti‐FGF21 (abcam), anti‐Bip (BD Biosciences), anti‐Gapdh (Santa Cruz), anti‐SREBP1c (Santa Cruz), anti‐SREBP2 (abcam), anti‐ATF6 (abcam), anti‐LC3 (Nanotools), anti‐Tom20 (SantaCruz), anti‐lipin1 (SantaCruz, sc‐376874).

    Techniques: Activation Assay, Mouse Assay

    Lipin1 deficiency triggers an ER stress in muscles and leads to Unfolded Protein Response activation UPR activation was analyzed by immunoblotting with indicated antibodies in GC muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p‐Eif2a, and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Journal: The EMBO Journal

    Article Title: Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone‐responsive myopathy

    doi: 10.15252/embj.201899576

    Figure Lengend Snippet: Lipin1 deficiency triggers an ER stress in muscles and leads to Unfolded Protein Response activation UPR activation was analyzed by immunoblotting with indicated antibodies in GC muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p‐Eif2a, and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Article Snippet: Reagents The following primary antibodies were used: anti‐p62 (SQSTM) (Abnova), anti‐LAMP2 (Abcam), anti‐FGF21 (abcam), anti‐Bip (BD Biosciences), anti‐Gapdh (Santa Cruz), anti‐SREBP1c (Santa Cruz), anti‐SREBP2 (abcam), anti‐ATF6 (abcam), anti‐LC3 (Nanotools), anti‐Tom20 (SantaCruz), anti‐lipin1 (SantaCruz, sc‐376874).

    Techniques: Activation Assay, Mouse Assay

    TRF2 binding on hTERT promoter is independent of telomere looping A . Schematic for insertion of Gaussia luciferase gene, driven by (+33 to-1276) bp hTERT promoter inserted at CCR5 safe harbour locus (46Mb away from nearest telomere using CRISPR/Cas9) in HEK293T cells. B . Effect of TRF2 silencing or over-expression of DNA binding mutants of TRF2, TRF2-DelB, TRF2-DelM and TRF2-DelB DelM on Gaussia luciferase activity, normalized over total protein C . qRT-PCR following TRF2 ChIP on the exogenously inserted hTERT promoter at CCR5 locus ; normalized over mock (IgG) ( GAPDH promoter-negative control for TRF2 occupancy) D . qRT-PCR following ChIP for shelterin complex proteins TRF1, POT1 and RAP1, on hTERT promoter insert in HEK293T cells E . qRT-PCR following ChIP for TRF1, POT1 and RAP1 on endogenous hTERT promoter in HT1080 cells (in D-E: Chromosome 5p (interstitial telomeric sequence) ITS site-positive control and GAPDH promoter-negative control). All error bars represent ± standard deviations from mean values and p values have been calculated by paired /un paired T-test. (*: p

    Journal: bioRxiv

    Article Title: Human Telomerase Expression is under Direct Transcriptional Control of the Telomere-binding-factor TRF2

    doi: 10.1101/2020.01.15.907626

    Figure Lengend Snippet: TRF2 binding on hTERT promoter is independent of telomere looping A . Schematic for insertion of Gaussia luciferase gene, driven by (+33 to-1276) bp hTERT promoter inserted at CCR5 safe harbour locus (46Mb away from nearest telomere using CRISPR/Cas9) in HEK293T cells. B . Effect of TRF2 silencing or over-expression of DNA binding mutants of TRF2, TRF2-DelB, TRF2-DelM and TRF2-DelB DelM on Gaussia luciferase activity, normalized over total protein C . qRT-PCR following TRF2 ChIP on the exogenously inserted hTERT promoter at CCR5 locus ; normalized over mock (IgG) ( GAPDH promoter-negative control for TRF2 occupancy) D . qRT-PCR following ChIP for shelterin complex proteins TRF1, POT1 and RAP1, on hTERT promoter insert in HEK293T cells E . qRT-PCR following ChIP for TRF1, POT1 and RAP1 on endogenous hTERT promoter in HT1080 cells (in D-E: Chromosome 5p (interstitial telomeric sequence) ITS site-positive control and GAPDH promoter-negative control). All error bars represent ± standard deviations from mean values and p values have been calculated by paired /un paired T-test. (*: p

    Article Snippet: After blocking the membrane was incubated with primary antibodies-anti-TRF2 antibody (Novus Biological), anti-TERT antibody (Abcam), anti-REST(Millipore), anti-EZH2(CST) and anti-GAPDH antibody (Santa-cruz).

    Techniques: Binding Assay, Luciferase, CRISPR, Over Expression, Activity Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Negative Control, Sequencing, Positive Control

    Effects of Meth on dopamine receptors and sigma-1 receptor in CD4 + T-cells. ( A ) CD4 + T-cells were untreated or treated with 100 µM Meth for different time points (5 mins-24 hours), lysed and the protein extracts were analyzed for the expression of various dopamine receptors and sigma-1 receptor. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( B ) Fold change in the pixel density of sigma-1 receptor expression in ( A ). All values normalized to untreated sample. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). ( C ) CD4 + T-cells were untreated or treated with 10 µM sigma-1 receptor inhibitor (σ1 R inh.) for 1 hour, then treated with or without 100 µM Meth for 1 hour, followed by lysis and analysis of protein extracts for the indicated activated signaling molecules by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( D ) HIV-1 p24 titer on day 3 after HIV-1 infection in unstimulated and stimulated CD4 + T-cells pretreated with or without sigma-1 receptor inhibitor (σ1 R inh.) and treated in the presence or absence of Meth. Data represent the mean ± SD of 3 independent experiments (**p ≤ 0.01, ***p ≤ 0.001).

    Journal: Scientific Reports

    Article Title: Methamphetamine functions as a novel CD4+ T-cell activator via the sigma-1 receptor to enhance HIV-1 infection

    doi: 10.1038/s41598-018-35757-x

    Figure Lengend Snippet: Effects of Meth on dopamine receptors and sigma-1 receptor in CD4 + T-cells. ( A ) CD4 + T-cells were untreated or treated with 100 µM Meth for different time points (5 mins-24 hours), lysed and the protein extracts were analyzed for the expression of various dopamine receptors and sigma-1 receptor. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( B ) Fold change in the pixel density of sigma-1 receptor expression in ( A ). All values normalized to untreated sample. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). ( C ) CD4 + T-cells were untreated or treated with 10 µM sigma-1 receptor inhibitor (σ1 R inh.) for 1 hour, then treated with or without 100 µM Meth for 1 hour, followed by lysis and analysis of protein extracts for the indicated activated signaling molecules by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( D ) HIV-1 p24 titer on day 3 after HIV-1 infection in unstimulated and stimulated CD4 + T-cells pretreated with or without sigma-1 receptor inhibitor (σ1 R inh.) and treated in the presence or absence of Meth. Data represent the mean ± SD of 3 independent experiments (**p ≤ 0.01, ***p ≤ 0.001).

    Article Snippet: GW182, D1DR, D2DR, D3DR, D4DR, Sigma-1 Receptor, and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Expressing, Lysis, Western Blot, Infection

    Meth induced degradation of Ago1 and altered structural integrity of P-bodies: ( A ) CD4 + T-cells were untreated or treated with Meth (100 µM) for 0, 4 and 24 hours, lysed and Ago1 expression was analyzed by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S4 ( B ) CD4 + T-cell lysates in ( A ) were immunoprecipitated with Ago1 antibody and subjected to Western blot analysis using Ubiquitin antibody. Ago1 served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. ( C ) CD4 + T-cell lysates in (A) were immunoprecipitated with GW182 antibody (upper panel) or Ago1 antibody (lower panel) and subjected to Western blot analysis using Ago1 (upper panel) or GW182 (lower panel) antibodies. B-Actin served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. Full-length blots are presented in Supplementary Fig. S4 ( D ) Confocal images of GW182 and Ago1 interaction in CD4 + T-cells, untreated or treated with Meth (100 µM) for 24 hours. Scale bar = 10 µm. Results are representative of 3 independent experiments.

    Journal: Scientific Reports

    Article Title: Methamphetamine functions as a novel CD4+ T-cell activator via the sigma-1 receptor to enhance HIV-1 infection

    doi: 10.1038/s41598-018-35757-x

    Figure Lengend Snippet: Meth induced degradation of Ago1 and altered structural integrity of P-bodies: ( A ) CD4 + T-cells were untreated or treated with Meth (100 µM) for 0, 4 and 24 hours, lysed and Ago1 expression was analyzed by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S4 ( B ) CD4 + T-cell lysates in ( A ) were immunoprecipitated with Ago1 antibody and subjected to Western blot analysis using Ubiquitin antibody. Ago1 served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. ( C ) CD4 + T-cell lysates in (A) were immunoprecipitated with GW182 antibody (upper panel) or Ago1 antibody (lower panel) and subjected to Western blot analysis using Ago1 (upper panel) or GW182 (lower panel) antibodies. B-Actin served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. Full-length blots are presented in Supplementary Fig. S4 ( D ) Confocal images of GW182 and Ago1 interaction in CD4 + T-cells, untreated or treated with Meth (100 µM) for 24 hours. Scale bar = 10 µm. Results are representative of 3 independent experiments.

    Article Snippet: GW182, D1DR, D2DR, D3DR, D4DR, Sigma-1 Receptor, and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Expressing, Western Blot, Immunoprecipitation

    Knockdown of CPSF4 inhibits PI3K/AKT, MAPK signaling and activates caspase-dependent apoptotic pathway in H1299 and A549 cells. ( A ) At 72 hours after siRNA treatment, the expression of CPSF4 protein and the total and phosphorylated Akt, PI3K, ERK1/2, JNK and p38 proteins in H1299 and A549 was detected by Western blot. GAPDH served as the loading control. ( B ) Apoptosis in H1299 and A549 was determined by flow cytometry 72 h after siRNA transfection using an Annexin V-FITC/PI-staining kit. The representative data from three independent experiments are shown. ( C ) Apoptosis was calculated in terms of the FITC-positive in cells. Results are shown as the mean ± SD of three independent experiments (*, P

    Journal: PLoS ONE

    Article Title: Upregulation of Cleavage and Polyadenylation Specific Factor 4 in Lung Adenocarcinoma and Its Critical Role for Cancer Cell Survival and Proliferation

    doi: 10.1371/journal.pone.0082728

    Figure Lengend Snippet: Knockdown of CPSF4 inhibits PI3K/AKT, MAPK signaling and activates caspase-dependent apoptotic pathway in H1299 and A549 cells. ( A ) At 72 hours after siRNA treatment, the expression of CPSF4 protein and the total and phosphorylated Akt, PI3K, ERK1/2, JNK and p38 proteins in H1299 and A549 was detected by Western blot. GAPDH served as the loading control. ( B ) Apoptosis in H1299 and A549 was determined by flow cytometry 72 h after siRNA transfection using an Annexin V-FITC/PI-staining kit. The representative data from three independent experiments are shown. ( C ) Apoptosis was calculated in terms of the FITC-positive in cells. Results are shown as the mean ± SD of three independent experiments (*, P

    Article Snippet: Western blots were probed with antibodies against CPSF4 (Proteintech Group, Inc., Chicago, USA), phospho-PI3K p85 (Tyr458)/p55 (Tyr199), PI3K, phosphor-Akt (Ser473), Akt, pTyr202/Y204-ERK1/2, ERK1/2, pThr183/Tyr185-SAPK/JNK, SAPK/JNK, cleaved caspase-3, cleaved caspase-9 and GAPDH (Cell Signaling Technology, Beverly, MA).The protein bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).

    Techniques: Expressing, Western Blot, Flow Cytometry, Cytometry, Transfection, Staining

    HGF stimulation is abrogated by IQSEC1 depletion. (A) Western blot of PC3 cells expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 30 minutes using anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, ARF5, ARF6 and GAPDH (shown for Akt blot) antibodies. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. Values, mean ± s.d. n=3 independent experiments. (C) Phase contrast images of PC3 acini expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 96 hours. Scale bars, 100μm. Cartoon, depicts acini phenotype representative of each condition. (D) Quantitation of PC3 acini shown in C. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 4 replicates/condition, 1,400-4,025 acini/condition. (E) Western blots of PC3 cells expressing Scr or IQSEC1 KD4 shRNA treated with cycloheximide (CHX) for various times using anti-IQSEC1, Met and GAPDH (shown for Met blot) antibodies. Quantitation of Met expression levels normalised to time 0 is shown. Values, mean ± s.e. n=3. p-values (one-way ANOVA): *p≤0.05, ***p≤0.001 and ****p≤0.0001.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: HGF stimulation is abrogated by IQSEC1 depletion. (A) Western blot of PC3 cells expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 30 minutes using anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, ARF5, ARF6 and GAPDH (shown for Akt blot) antibodies. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. Values, mean ± s.d. n=3 independent experiments. (C) Phase contrast images of PC3 acini expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 96 hours. Scale bars, 100μm. Cartoon, depicts acini phenotype representative of each condition. (D) Quantitation of PC3 acini shown in C. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 4 replicates/condition, 1,400-4,025 acini/condition. (E) Western blots of PC3 cells expressing Scr or IQSEC1 KD4 shRNA treated with cycloheximide (CHX) for various times using anti-IQSEC1, Met and GAPDH (shown for Met blot) antibodies. Quantitation of Met expression levels normalised to time 0 is shown. Values, mean ± s.e. n=3. p-values (one-way ANOVA): *p≤0.05, ***p≤0.001 and ****p≤0.0001.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Western Blot, Expressing, shRNA, Quantitation Assay

    IQSEC1 isoforms differentially regulate collective invasion. (A) Schema, domain structure of IQSEC1 variants (v) 1-4. All variants possess an IQ motif, SEC7 and PH domains. v1 and v2 contain N-terminal extensions. A nuclear localization signal (NLS) is present in v1-3, lost in v4. v2/v3 share a common C-terminus. Common domains shown in grey, unique domains indicated by colour; blue; v1, pink; v2 and v3, green; v4. (B) Western blot in 2D and 3D using anti-IQSEC1 antibody. GAPDH is shown for IQSEC1 blot. Relative expression of all IQSEC1 bands normalised to 2D RWPE1 is shown. n=6 independent experiments. Values, mean ± s.d. p-values (Student’s t-test), *p ≤0.05; n.s. not significant. (C) Cartoon, isoform specific IQSEC1 functions were identified by comparing the growth and invasion of acini expressing different GFP-IQSEC1 variants upon depletion of endogenous IQSEC1 (shRNA KD4). (D) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4, and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, GFP and GAPDH antibodies. All antibodies used on same membrane. GAPDH is shown for IQSEC1 blot. Different exposures of IQSEC1 (long, short) are presented to demonstrate expression of all variants. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (E) Phase images of acini from cells described in D. GFP positive acini are outlined in yellow. Scale bars, 100μm. (F) Quantitation of images shown in E. Heatmap shows area and compactness measurements as Z-score-normalised to control (+-+-) values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower panels). n=3, 3 replicates/condition, 300-650 acini per condition. (G) PC3 acini described in D were stained for F-actin and nuclei (black and magenta respectively in upper panels) after 4 days. Green arrowheads, protrusions. Scale bars, 20μm. Localisation of GFP-IQSEC1 can be appreciated from FIRE pseudo coloured Look Up Table (FIRE LUT) (middle panels). Magnified images of boxed regions are inset. Cartoon, localization of GFP-IQSEC1 variants in PC3 acini (lower panel). (H) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4 using anti-IQSEC1 (specific for v2) and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. Different exposures of GFP (long, short) are presented showing GFP-IQSEC1 proteins. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (I) Schema, summary of the effect of IQSEC1 KD and expression of GFP-IQSEC1 v2 on growth and protrusive ability of PC3 acini. (J) Endogenous IQSEC1 v2 co-stained with F-actin (green and red respectively in merge). Magnified images of boxed region are shown. Red arrowhead indicates localisation at protrusion tip.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1 isoforms differentially regulate collective invasion. (A) Schema, domain structure of IQSEC1 variants (v) 1-4. All variants possess an IQ motif, SEC7 and PH domains. v1 and v2 contain N-terminal extensions. A nuclear localization signal (NLS) is present in v1-3, lost in v4. v2/v3 share a common C-terminus. Common domains shown in grey, unique domains indicated by colour; blue; v1, pink; v2 and v3, green; v4. (B) Western blot in 2D and 3D using anti-IQSEC1 antibody. GAPDH is shown for IQSEC1 blot. Relative expression of all IQSEC1 bands normalised to 2D RWPE1 is shown. n=6 independent experiments. Values, mean ± s.d. p-values (Student’s t-test), *p ≤0.05; n.s. not significant. (C) Cartoon, isoform specific IQSEC1 functions were identified by comparing the growth and invasion of acini expressing different GFP-IQSEC1 variants upon depletion of endogenous IQSEC1 (shRNA KD4). (D) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4, and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, GFP and GAPDH antibodies. All antibodies used on same membrane. GAPDH is shown for IQSEC1 blot. Different exposures of IQSEC1 (long, short) are presented to demonstrate expression of all variants. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (E) Phase images of acini from cells described in D. GFP positive acini are outlined in yellow. Scale bars, 100μm. (F) Quantitation of images shown in E. Heatmap shows area and compactness measurements as Z-score-normalised to control (+-+-) values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower panels). n=3, 3 replicates/condition, 300-650 acini per condition. (G) PC3 acini described in D were stained for F-actin and nuclei (black and magenta respectively in upper panels) after 4 days. Green arrowheads, protrusions. Scale bars, 20μm. Localisation of GFP-IQSEC1 can be appreciated from FIRE pseudo coloured Look Up Table (FIRE LUT) (middle panels). Magnified images of boxed regions are inset. Cartoon, localization of GFP-IQSEC1 variants in PC3 acini (lower panel). (H) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4 using anti-IQSEC1 (specific for v2) and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. Different exposures of GFP (long, short) are presented showing GFP-IQSEC1 proteins. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (I) Schema, summary of the effect of IQSEC1 KD and expression of GFP-IQSEC1 v2 on growth and protrusive ability of PC3 acini. (J) Endogenous IQSEC1 v2 co-stained with F-actin (green and red respectively in merge). Magnified images of boxed region are shown. Red arrowhead indicates localisation at protrusion tip.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

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

    IQSEC1 regulates growth and invasion in multiple prostate cancer cell lines. (A) Heatmap shows mRNA expression levels, mined from CCLE, of the Met-PI3K-Akt pathway across different prostate cancer cell lines. (B) Western blot of prostate cancer cell lines using anti-IQSEC1, phospho-Y1234/1234 Met, total Met, LRP1, ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. (C) Schema, effect of NAV-2729 on ARF GTPase cycle. (D) Phase images of acini (+ and – HGF) at different time points are shown. Acini were also treated with NAV-2729 for 96 hours. Scale bars, 100μm. (E) Schema, summarizes the effect of each treatment described in D on acini growth and invasion. (F-I) Quantitation of 22Rv1 (F) , LNCaP (G) , DU145 (H) and DU145 + HGF (I) acini formation in the absence or presence of NAV-2729. 22Rv1 and DU145 acini were also expressing LRP1-GFP and IQSEC1-GFP v2 respectively. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=2, replicates/condition, 400-1600 acini/condition.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1 regulates growth and invasion in multiple prostate cancer cell lines. (A) Heatmap shows mRNA expression levels, mined from CCLE, of the Met-PI3K-Akt pathway across different prostate cancer cell lines. (B) Western blot of prostate cancer cell lines using anti-IQSEC1, phospho-Y1234/1234 Met, total Met, LRP1, ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. (C) Schema, effect of NAV-2729 on ARF GTPase cycle. (D) Phase images of acini (+ and – HGF) at different time points are shown. Acini were also treated with NAV-2729 for 96 hours. Scale bars, 100μm. (E) Schema, summarizes the effect of each treatment described in D on acini growth and invasion. (F-I) Quantitation of 22Rv1 (F) , LNCaP (G) , DU145 (H) and DU145 + HGF (I) acini formation in the absence or presence of NAV-2729. 22Rv1 and DU145 acini were also expressing LRP1-GFP and IQSEC1-GFP v2 respectively. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=2, replicates/condition, 400-1600 acini/condition.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Expressing, Western Blot, Quantitation Assay

    IQSEC1 interacts with multiple transmembrane proteins. (A) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-IQSEC1, LRP1, Met, SORL1, RICTOR, Sin1, ARFGAP1, 14-3-3ζ/Δ, PPC6, GFP and GAPDH (shown for GFP blot) antibodies. (B) Western blot of PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (C) Western blot of Epi and EMT14 PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (D) Met immunoprecipitation was carried out on PC3 cells expressing Scr or IQSEC1 KD4 shRNA. Western blot was then performed on lysates (upper panels) and IPs (lower panels) using anti-LRP1, Met, IQSEC1 and GAPDH (shown for IQSEC1 blot) antibodies. Quantitation of LRP1/Met interaction normalised to Scr is shown. Values, mean ± s.d. n=3 independent experiments. p values (one-way ANOVA): n.s. not significant. (E-F) Western blot analysis of PC3 cells expressing Scr or (E) LRP1 and (F) Met shRNA. GAPDH is shown for Akt blots. (G) Quantitation of shape classification of PC3 cells expressing shRNAs for IQSEC1 binding partners as log2 fold change of each phenotype over control (left heatmap). p-values (one-way ANOVA): greyscale values as indicated (right heatmap). n=2, 4 replicates per condition, minimum 14,000 cells imaged/condition. (H) Quantitation of PC3 acini expressing GFP or LRP1-GFP. Heatmaps for area and compactness measurements shown as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 3 replicates, minimum 150 acini/condition. (I) Western blot of PC3 cells expressing SORL1 shRNA. GAPDH is shown for Akt blots. (J) Representative phase contrast images of PC3 acini expressing Scr (also shown in Figure 4H ), SORL1_ 2 and RICTOR_2 shRNA taken after 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum 700 acini/condition. (L-N) Western blots of PC3 cells expressing (L) RICTOR or (M) SIN1 or (N) ARFGAP1 shRNA. GAPDH is shown for Akt blots. (O) Representative phase contrast images of PC3 acini expressing Scr, ARFGAP1_1 and ARFGAP1_2 shRNA taken after 96 hours. Scale bars, 100μm. (P) Quantitation of PC3 acini shown in O. n=2, 4 replicates, minimum 1,100 acini/condition. (Q) Cartoon, depicts representative acini phenotype upon depletion of ARFGAP1 and ARF GTPase cycle.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1 interacts with multiple transmembrane proteins. (A) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-IQSEC1, LRP1, Met, SORL1, RICTOR, Sin1, ARFGAP1, 14-3-3ζ/Δ, PPC6, GFP and GAPDH (shown for GFP blot) antibodies. (B) Western blot of PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (C) Western blot of Epi and EMT14 PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (D) Met immunoprecipitation was carried out on PC3 cells expressing Scr or IQSEC1 KD4 shRNA. Western blot was then performed on lysates (upper panels) and IPs (lower panels) using anti-LRP1, Met, IQSEC1 and GAPDH (shown for IQSEC1 blot) antibodies. Quantitation of LRP1/Met interaction normalised to Scr is shown. Values, mean ± s.d. n=3 independent experiments. p values (one-way ANOVA): n.s. not significant. (E-F) Western blot analysis of PC3 cells expressing Scr or (E) LRP1 and (F) Met shRNA. GAPDH is shown for Akt blots. (G) Quantitation of shape classification of PC3 cells expressing shRNAs for IQSEC1 binding partners as log2 fold change of each phenotype over control (left heatmap). p-values (one-way ANOVA): greyscale values as indicated (right heatmap). n=2, 4 replicates per condition, minimum 14,000 cells imaged/condition. (H) Quantitation of PC3 acini expressing GFP or LRP1-GFP. Heatmaps for area and compactness measurements shown as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 3 replicates, minimum 150 acini/condition. (I) Western blot of PC3 cells expressing SORL1 shRNA. GAPDH is shown for Akt blots. (J) Representative phase contrast images of PC3 acini expressing Scr (also shown in Figure 4H ), SORL1_ 2 and RICTOR_2 shRNA taken after 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum 700 acini/condition. (L-N) Western blots of PC3 cells expressing (L) RICTOR or (M) SIN1 or (N) ARFGAP1 shRNA. GAPDH is shown for Akt blots. (O) Representative phase contrast images of PC3 acini expressing Scr, ARFGAP1_1 and ARFGAP1_2 shRNA taken after 96 hours. Scale bars, 100μm. (P) Quantitation of PC3 acini shown in O. n=2, 4 replicates, minimum 1,100 acini/condition. (Q) Cartoon, depicts representative acini phenotype upon depletion of ARFGAP1 and ARF GTPase cycle.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Immunoprecipitation, Expressing, Western Blot, shRNA, Quantitation Assay, Binding Assay

    IQSEC1 activates ARF5/6 in distinct locations within protrusions. (A) Western blot of PC3 cells expressing Scr, ARF5 or ARF6 shRNA, alone or together, using anti ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. ARF intensity normalised to Scr is quantified. n=3 independent experiments. Values, mean ± s.d. p-values (one-way ANOVA). (B) Phase images of PC3 acini described in A. Yellow outlines indicate shRNA (mVenus) positive acini. Scale bars, 100μm. (C) Quantitation of images shown in B. Quantitation is shown for area and compactness measurements as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 5 replicates/condition, 2880 - 3188 acini quantified/condition. Cartoon, acini phenotype representative of each condition. (D) Western blot of PC3 cells stably co-overexpressing (OX) mNeonGreen (mNG) and TagRFP-T (RFP) (Control) or ARF5-mNG and ARF6-RFP (ARF5/6), and either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, ARF5, ARF6 and GAPDH antibodies were used. Both endogenous and exogenous (OX) ARFs were detected. GAPDH is shown for ARF5 blot. (E) Phase images of PC3 acini described in D. Scale bars, 100μm. (F) Quantitation of images shown in E. n=2, 4 replicates/condition, 1,254 – 1,567 acini/condition. (G) PC3 cells co-overexpressing ARF5-mNG and ARF6-RFP (ARF5/6 OX) were stained for IQSEC1 v2 and merged image of spindle shaped cell shown (left panel). Magnified images of boxed region are shown. White arrowheads indicate areas of colocalization. Scale bars, 20μm. (H) PC3 acini expressing either ARF5-mNG or ARF6-mNG were stained for IQSEC1. Localisation of these proteins is shown using FIRE LUT. Yellow and white arrowheads, colocalization in juxtanuclear region and protrusive tips, respectively. White arrows, lack of colocalization. Scale bars, 5μm. (I) Schema, GTPase cycle of ARF5 and ARF6, site of action of the IQSEC1-inhibiting molecules NAV-2729, the pan ARFGAP-inhibitor QS11, and GTP-loaded ARF detection by a GGA1-NGAT probe. (J) PC3 acini expressing either ARF5-mNG or ARF6-mNG and GGA1-NGAT-RFP were fixed and FIRE LUT of maximum projections (upper panels) and a single Z-slice of boxed regions (lower panels) shown. White and green arrowheads, colocalization in protrusions, white arrows, colocalization in cell body. Scale bars, 10μm. (K-L) PC3 cells expressing (K) ARF5-NG and GGA1-NGAT or (L) ARF6-NG and GGA1-NGAT were stably transfected with either Scr or IQSEC1 KD4 shRNA. Cells were treated with NAV-2729 or QS11 for 24 hours prior to fixation. Quantitation of % overlap of ARF and ARF-GTP probe per cell is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 3 replicates/condition with a minimum of 500 cells/condition. p-values (one-way ANOVA): *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001. (M) Schema, different localization of IQSEC1 and active ARFs in protrusions is shown.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1 activates ARF5/6 in distinct locations within protrusions. (A) Western blot of PC3 cells expressing Scr, ARF5 or ARF6 shRNA, alone or together, using anti ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. ARF intensity normalised to Scr is quantified. n=3 independent experiments. Values, mean ± s.d. p-values (one-way ANOVA). (B) Phase images of PC3 acini described in A. Yellow outlines indicate shRNA (mVenus) positive acini. Scale bars, 100μm. (C) Quantitation of images shown in B. Quantitation is shown for area and compactness measurements as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 5 replicates/condition, 2880 - 3188 acini quantified/condition. Cartoon, acini phenotype representative of each condition. (D) Western blot of PC3 cells stably co-overexpressing (OX) mNeonGreen (mNG) and TagRFP-T (RFP) (Control) or ARF5-mNG and ARF6-RFP (ARF5/6), and either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, ARF5, ARF6 and GAPDH antibodies were used. Both endogenous and exogenous (OX) ARFs were detected. GAPDH is shown for ARF5 blot. (E) Phase images of PC3 acini described in D. Scale bars, 100μm. (F) Quantitation of images shown in E. n=2, 4 replicates/condition, 1,254 – 1,567 acini/condition. (G) PC3 cells co-overexpressing ARF5-mNG and ARF6-RFP (ARF5/6 OX) were stained for IQSEC1 v2 and merged image of spindle shaped cell shown (left panel). Magnified images of boxed region are shown. White arrowheads indicate areas of colocalization. Scale bars, 20μm. (H) PC3 acini expressing either ARF5-mNG or ARF6-mNG were stained for IQSEC1. Localisation of these proteins is shown using FIRE LUT. Yellow and white arrowheads, colocalization in juxtanuclear region and protrusive tips, respectively. White arrows, lack of colocalization. Scale bars, 5μm. (I) Schema, GTPase cycle of ARF5 and ARF6, site of action of the IQSEC1-inhibiting molecules NAV-2729, the pan ARFGAP-inhibitor QS11, and GTP-loaded ARF detection by a GGA1-NGAT probe. (J) PC3 acini expressing either ARF5-mNG or ARF6-mNG and GGA1-NGAT-RFP were fixed and FIRE LUT of maximum projections (upper panels) and a single Z-slice of boxed regions (lower panels) shown. White and green arrowheads, colocalization in protrusions, white arrows, colocalization in cell body. Scale bars, 10μm. (K-L) PC3 cells expressing (K) ARF5-NG and GGA1-NGAT or (L) ARF6-NG and GGA1-NGAT were stably transfected with either Scr or IQSEC1 KD4 shRNA. Cells were treated with NAV-2729 or QS11 for 24 hours prior to fixation. Quantitation of % overlap of ARF and ARF-GTP probe per cell is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 3 replicates/condition with a minimum of 500 cells/condition. p-values (one-way ANOVA): *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001. (M) Schema, different localization of IQSEC1 and active ARFs in protrusions is shown.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Western Blot, Expressing, shRNA, Quantitation Assay, Stable Transfection, Staining, Transfection

    IQSEC1-LRP1 complex regulates Met endocytic trafficking. (A) Western blot of PC3 cells co-expressing mNG and RFP (Control) or ARF5-mNG and ARF6-RFP (ARF5/6) with either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, and GAPDH (sample control) antibodies were used. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. n=2 independent experiments. Values, mean ± s.d. (C) Schema, effect of IQSEC1 on Met trafficking. (D) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were incubated with a Met-647 fluorescent antibody at 4°C (Surface) prior to stimulation with HGF for either 10 or 30 minutes (Internalisation). PC3 cells were also treated with chloroquine to allow accumulation of surface-derived Met (black) for 1 hour at 17°C prior to stimulation with HGF for either 10 or 30 minutes (Recycling). Cells were stained with F-actin (green outlines) and Hoechst (nuclei in magenta). Magnified images of boxed regions are inset. Scale bars 20µm. (E) Intensity of Met antibody in each cell was quantified (Z-score normalised); shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (F) Intensity of Met antibody in membrane, cytoplasmic and juxtanuclear regions was quantified. Line graphs show relative region intensities compared to the intensity of the whole cell (Z-score normalised). n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (G) Schema, sub-cellular re-localisation of active Met upon HGF treatment. (H) PC3 cells expressing Scr, IQSEC1 KD4, LRP1 or SORL1 shRNA were stimulated with HGF for 30 minutes. Cells were stained for phospho-Met (black), F-actin (green outlines) and Hoechst/nuclei (magenta). Magnified images of boxed regions are shown (lower panels). Cartoon, sub-cellular localization of active Met under different conditions. (I-J) Quantitation of spot intensity (Z-score normalised) is shown for images in H in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p-values (one-way ANOVA). (K) Schema, regulation of Met internalisation, but not recycling, by IQSEC1 and LRP1. All p values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1-LRP1 complex regulates Met endocytic trafficking. (A) Western blot of PC3 cells co-expressing mNG and RFP (Control) or ARF5-mNG and ARF6-RFP (ARF5/6) with either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, and GAPDH (sample control) antibodies were used. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. n=2 independent experiments. Values, mean ± s.d. (C) Schema, effect of IQSEC1 on Met trafficking. (D) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were incubated with a Met-647 fluorescent antibody at 4°C (Surface) prior to stimulation with HGF for either 10 or 30 minutes (Internalisation). PC3 cells were also treated with chloroquine to allow accumulation of surface-derived Met (black) for 1 hour at 17°C prior to stimulation with HGF for either 10 or 30 minutes (Recycling). Cells were stained with F-actin (green outlines) and Hoechst (nuclei in magenta). Magnified images of boxed regions are inset. Scale bars 20µm. (E) Intensity of Met antibody in each cell was quantified (Z-score normalised); shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (F) Intensity of Met antibody in membrane, cytoplasmic and juxtanuclear regions was quantified. Line graphs show relative region intensities compared to the intensity of the whole cell (Z-score normalised). n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (G) Schema, sub-cellular re-localisation of active Met upon HGF treatment. (H) PC3 cells expressing Scr, IQSEC1 KD4, LRP1 or SORL1 shRNA were stimulated with HGF for 30 minutes. Cells were stained for phospho-Met (black), F-actin (green outlines) and Hoechst/nuclei (magenta). Magnified images of boxed regions are shown (lower panels). Cartoon, sub-cellular localization of active Met under different conditions. (I-J) Quantitation of spot intensity (Z-score normalised) is shown for images in H in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p-values (one-way ANOVA). (K) Schema, regulation of Met internalisation, but not recycling, by IQSEC1 and LRP1. All p values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Western Blot, Expressing, shRNA, Quantitation Assay, Incubation, Derivative Assay, Staining

    IQSEC1 is a scaffold for Met signalling. (A) Schematic, IQSEC1 chimeras and variants display distinct phenotypes in 3D culture. GFP-trap immunoprecipitation was performed on PC3 cells expressing these proteins followed by tryptic digestion “on-beads” and LC/MS/MS analysis. IQSEC1 domain-specific interactions were identified and sorted by mRNA expression compared in paired PC3 subclones. (B) STRING network analysis of IQSEC1 binding partners identified by MS visualized using Cytoscape. Most IQSEC1 binding partners could be clustered into 8 protein complexes (colour coded). (C) Schema, indicates the protein complex each binding partner is associated with. Heatmap shows the fold change of IQSEC1 interactors from panel B binding to different IQSEC1 domains over GFP control. Values are −log 2 (FC), very high = −7 to −5, high = −5 to −2.5, medium −2.5 to −1, low = −1 to −0.05, no binding > 0.05. p-values are indicated in grey heatmap. The specific IQSEC1 domain to which each interactor binds is also depicted. Interactions were sorted according to the fold change in mRNA of non-invasive PC3 subclones (GS689.Li, EMT) compared to invasive subclones (PC3E, Epi) (RNAseq). (D) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-Met, LRP1, GFP, IQSEC1 and GAPDH antibodies. GAPDH is shown for LRP1 blot. Quantitation of LRP1/IQSEC1 and Met/IQSEC1 interactions for each GFP-trap are shown. n=3 and n=4, respectively. Values, mean ± s.d. p values (one-way ANOVA): **p≤0.01 and n.s. not significant. (E) Cartoon, depicts domain specific binding of IQSEC1 interactors. (F) PC3 acini expressing ARF5-mNG or ARF6-mNG were stained for Met and LRP1. FIRE LUT images are displayed with magnified images of boxed regions shown in lower panels. White arrowheads indicate localisation in protrusion tips. Scale bars, 5µm (upper) and 10µm (lower). (G) Schema, colocalization of IQSEC1-ARF interactors in protrusive tips. (H) Phase images of PC3 acini expressing Scr, LRP1_1, Met_2, SORL1_1, RICTOR_1 or SIN1_2 shRNA after 96 hours. Scale bars, 100μm. (I) Quantitation of images shown in H. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates/condition, 700-2,400 acini/condition. Cartoon, depicts acini phenotype representative of each condition.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1 is a scaffold for Met signalling. (A) Schematic, IQSEC1 chimeras and variants display distinct phenotypes in 3D culture. GFP-trap immunoprecipitation was performed on PC3 cells expressing these proteins followed by tryptic digestion “on-beads” and LC/MS/MS analysis. IQSEC1 domain-specific interactions were identified and sorted by mRNA expression compared in paired PC3 subclones. (B) STRING network analysis of IQSEC1 binding partners identified by MS visualized using Cytoscape. Most IQSEC1 binding partners could be clustered into 8 protein complexes (colour coded). (C) Schema, indicates the protein complex each binding partner is associated with. Heatmap shows the fold change of IQSEC1 interactors from panel B binding to different IQSEC1 domains over GFP control. Values are −log 2 (FC), very high = −7 to −5, high = −5 to −2.5, medium −2.5 to −1, low = −1 to −0.05, no binding > 0.05. p-values are indicated in grey heatmap. The specific IQSEC1 domain to which each interactor binds is also depicted. Interactions were sorted according to the fold change in mRNA of non-invasive PC3 subclones (GS689.Li, EMT) compared to invasive subclones (PC3E, Epi) (RNAseq). (D) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-Met, LRP1, GFP, IQSEC1 and GAPDH antibodies. GAPDH is shown for LRP1 blot. Quantitation of LRP1/IQSEC1 and Met/IQSEC1 interactions for each GFP-trap are shown. n=3 and n=4, respectively. Values, mean ± s.d. p values (one-way ANOVA): **p≤0.01 and n.s. not significant. (E) Cartoon, depicts domain specific binding of IQSEC1 interactors. (F) PC3 acini expressing ARF5-mNG or ARF6-mNG were stained for Met and LRP1. FIRE LUT images are displayed with magnified images of boxed regions shown in lower panels. White arrowheads indicate localisation in protrusion tips. Scale bars, 5µm (upper) and 10µm (lower). (G) Schema, colocalization of IQSEC1-ARF interactors in protrusive tips. (H) Phase images of PC3 acini expressing Scr, LRP1_1, Met_2, SORL1_1, RICTOR_1 or SIN1_2 shRNA after 96 hours. Scale bars, 100μm. (I) Quantitation of images shown in H. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates/condition, 700-2,400 acini/condition. Cartoon, depicts acini phenotype representative of each condition.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Immunoprecipitation, Expressing, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Western Blot, Quantitation Assay, Staining, shRNA

    Upregulation of IQSEC1 is associated with tumorigenesis. (A) Schema, prostate cell lines forming non-invasive or invasive 3D acini in extracellular matrix (ECM). (B) Cartoon, phenotype of typical RWPE-1 and RWPE-2 acini. Confocal (F-actin (red) and nuclei (blue)) and brightfield images show RWPE-1 and RWPE-2 acini at 120 hours. Arrowheads, protrusions. Scale bar, 20μm. (C) Schema, PC3 acini form (grow) and invade (protrusions) through ECM over time. Phase contrast images of PC3 acini where higher magnification of boxed region at different time points is shown. Arrowheads, protrusions. Scale bar, 100μm. (D) Cartoon, ARF GTPase cycle. (E) Heatmap representation of mRNA expression. Data are normalized to RWPE-1 and presented as the log2-transformed fold change compared to the average of all values. Bar graphs summarise fold changes of ARF and IQSEC mRNA levels. n=3 technical replicates. Values, mean ± s.d. p-values (Student’s t-test). (F) Schema, elevated activation of ARF GTPases in PC3 cells by GEFs such as IQSEC1. (G) Graph generated using RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) comparing IQSEC1 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (H) Western blot analysis of androgen receptor (AR) proficient or deficient prostate cell lines using anti-ARF1, ARF6, pan-IQSEC1 isoform and GAPDH (as sample control) antibodies. (I) Quantitation of IQSEC1 expression levels in either 52 normal or 487 tumour samples mined from The Cancer Genome Atlas (TCGA). Box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. p-values (one-way ANOVA). (J) IQSEC1 gene expression from 487 primary prostate tumours (TCGA) was evaluated for outcome prediction from (recurrence-free) survival data. Patient samples were clustered into quartiles based on normalized gene expression. Highest expression, red, lowest expression, blue. Heatmap shows clustering of expression values. p values (Logrank). All p-values: *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: Upregulation of IQSEC1 is associated with tumorigenesis. (A) Schema, prostate cell lines forming non-invasive or invasive 3D acini in extracellular matrix (ECM). (B) Cartoon, phenotype of typical RWPE-1 and RWPE-2 acini. Confocal (F-actin (red) and nuclei (blue)) and brightfield images show RWPE-1 and RWPE-2 acini at 120 hours. Arrowheads, protrusions. Scale bar, 20μm. (C) Schema, PC3 acini form (grow) and invade (protrusions) through ECM over time. Phase contrast images of PC3 acini where higher magnification of boxed region at different time points is shown. Arrowheads, protrusions. Scale bar, 100μm. (D) Cartoon, ARF GTPase cycle. (E) Heatmap representation of mRNA expression. Data are normalized to RWPE-1 and presented as the log2-transformed fold change compared to the average of all values. Bar graphs summarise fold changes of ARF and IQSEC mRNA levels. n=3 technical replicates. Values, mean ± s.d. p-values (Student’s t-test). (F) Schema, elevated activation of ARF GTPases in PC3 cells by GEFs such as IQSEC1. (G) Graph generated using RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) comparing IQSEC1 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (H) Western blot analysis of androgen receptor (AR) proficient or deficient prostate cell lines using anti-ARF1, ARF6, pan-IQSEC1 isoform and GAPDH (as sample control) antibodies. (I) Quantitation of IQSEC1 expression levels in either 52 normal or 487 tumour samples mined from The Cancer Genome Atlas (TCGA). Box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. p-values (one-way ANOVA). (J) IQSEC1 gene expression from 487 primary prostate tumours (TCGA) was evaluated for outcome prediction from (recurrence-free) survival data. Patient samples were clustered into quartiles based on normalized gene expression. Highest expression, red, lowest expression, blue. Heatmap shows clustering of expression values. p values (Logrank). All p-values: *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Expressing, Transformation Assay, Activation Assay, Generated, Western Blot, Quantitation Assay

    IQSEC1-ARF signalling controls phosphoinositide generation in invasive protrusion tips. (A) Schema, signalling pathways in protrusive tips of PC3 acini. (B) PC3 acini expressing mNeonGreen tagged PH-PLCδ or PH-Grp1 were fixed after 3 days. PC3 acini were also stained with phospho-S473 Akt antibody. FIRE LUT used to show localisation and intensity of mNeonGreen or pAkt. Magnified images of boxed regions are also shown. White arrowheads, localisation at protrusive tips. Scale bars, 10 µm. Cartoon, spatial PIP production in protrusive tips. (C) Quantitation of cortical enrichment of PI(4,5)P 2 (upper panel) or PIP 3 per cell in the presence or absence of IQSEC1 is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, > 500 cells/condition. p-values (one-way ANOVA): ****p≤0.0001. (D) Cartoon, PIPK targeting in presence or absence of IQSEC1. (E) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control), Myr-FLAG-PIP5Kα, Myr-FLAG-PIP5Kβ or Myr-FLAG-PI3Kβ and either Scr or IQSEC1 shRNA (KD4). Anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies were used. GAPDH is shown for IQSEC1 blot. (F) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control) or Myr-Akt1 and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. (G) Phase images of PC3 acini described in E are shown. Scr acini were also treated with NAV-2729 (IQSEC1 inhibitor). Scale bars, 100μm. (H) Quantitation of images shown in G. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=3 with 4 replicates per condition. Between 1,693 and 2,435 acini quantified per condition. Cartoon, acini phenotype representative of each condition. (I) Phase images of PC3 acini described in F after 96 hours. Scr acini were also treated with IQSEC1-inhibiting compound NAV-2729. Scale bars, 100μm. (J) Quantitation of images shown in I. n=2 with 4 replicates/condition, 1,287-2,363 acini/condition. Cartoon, acini phenotype representative of each condition. (K) Schema, summarizes the relationship between location and level of cortical PIP 3 and 2D and 3D PC3 phenotype.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1-ARF signalling controls phosphoinositide generation in invasive protrusion tips. (A) Schema, signalling pathways in protrusive tips of PC3 acini. (B) PC3 acini expressing mNeonGreen tagged PH-PLCδ or PH-Grp1 were fixed after 3 days. PC3 acini were also stained with phospho-S473 Akt antibody. FIRE LUT used to show localisation and intensity of mNeonGreen or pAkt. Magnified images of boxed regions are also shown. White arrowheads, localisation at protrusive tips. Scale bars, 10 µm. Cartoon, spatial PIP production in protrusive tips. (C) Quantitation of cortical enrichment of PI(4,5)P 2 (upper panel) or PIP 3 per cell in the presence or absence of IQSEC1 is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, > 500 cells/condition. p-values (one-way ANOVA): ****p≤0.0001. (D) Cartoon, PIPK targeting in presence or absence of IQSEC1. (E) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control), Myr-FLAG-PIP5Kα, Myr-FLAG-PIP5Kβ or Myr-FLAG-PI3Kβ and either Scr or IQSEC1 shRNA (KD4). Anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies were used. GAPDH is shown for IQSEC1 blot. (F) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control) or Myr-Akt1 and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. (G) Phase images of PC3 acini described in E are shown. Scr acini were also treated with NAV-2729 (IQSEC1 inhibitor). Scale bars, 100μm. (H) Quantitation of images shown in G. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=3 with 4 replicates per condition. Between 1,693 and 2,435 acini quantified per condition. Cartoon, acini phenotype representative of each condition. (I) Phase images of PC3 acini described in F after 96 hours. Scr acini were also treated with IQSEC1-inhibiting compound NAV-2729. Scale bars, 100μm. (J) Quantitation of images shown in I. n=2 with 4 replicates/condition, 1,287-2,363 acini/condition. Cartoon, acini phenotype representative of each condition. (K) Schema, summarizes the relationship between location and level of cortical PIP 3 and 2D and 3D PC3 phenotype.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

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

    IQSEC1-ARF controls cortical PI(4,5)P 2 generation, which is required for production of PIP 3. (A) PC3 cells expressing mNeonGreen tagged PH-PLCδ (PIP 2 ) or PH-Grp1 (PIP 3 ) and either Scr or IQSEC1 shRNA KD4 were fixed after 2 days. FIRE LUT is used to show localisation and intensity of GFP in magnified images of boxed regions. White arrowhead indicates localisation at protrusive tip. Scale bars, 20µm. (B-C) Quantitation of (B) PI(4,5)P 2 or (C) PIP 3 cortical enrichment in spindle, spread and round cells expressing either Scr or IQSEC1 KD4 shRNA is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=3, 4 replicates/condition. 1330/1049 and 2222/1182 cells analysed for Src/ IQSEC1 KD4 shRNA respectively in B and C. p-values (one-way ANOVA). (D) Cartoon, summary of cortical PI(4,5)P 2 and PIP 3 localization in different cell shapes. (E) Western blot of PC3 cells treated with LY294002 (pan PI3K), AZD8835 (PI3Kα), AZD8186 (PI3Kβ), AS605240 (PI3Kγ), Cal-101 (PI3Kδ) and AktII (Akt) inhibitors for 24 hours. Anti-phospho-S473 Akt, Akt and GAPDH antibodies were used. (F) Phase contrast images of PC3 acini described in E. Scale bars, 100μm. (G) Cartoon, depicts acini phenotype representative of each condition. (H) Quantitation of PC3 acini shown in F. Heatmaps show area and compactness measurements as Z-score-normalised values (upper heatmaps). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates, minimum of 350 acini/condition. (I) Western blot of PC3 cells expressing Scr, PIP5K1α or PIP5K1β shRNA using anti-PIP5K, IQSEC1, phospho-S473 Akt, Akt and GAPDH (shown for Akt blots) antibodies. (J) Phase contrast images of PC3 acini expressing Scr, PIP5K1α or PIP5K1β shRNA at 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum of 1,348 acini/condition. Cartoon, depicts acini phenotype representative of each condition. (L) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were stained for pS473 Akt (black) and Hoechst /nuclei (magenta). Magnified images of boxed regions are shown in lower panels. Blue arrowheads indicate localisation of active Akt. Scale bars, 10μm. (M-N) Quantitation of pAkt (M) intensity or (N) area in spots in the juxtanuclear, cytoplasm and periphery of PC3 cells expressing either Scr or IQSEC1 KD4 shRNA. Box-and-whiskers plots: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition. p values (Mann Whitney). (O) Cartoon, depicting phospho-Akt intensity, in the presence or absence of IQSEC1, at different subcellular locations. All p-values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1-ARF controls cortical PI(4,5)P 2 generation, which is required for production of PIP 3. (A) PC3 cells expressing mNeonGreen tagged PH-PLCδ (PIP 2 ) or PH-Grp1 (PIP 3 ) and either Scr or IQSEC1 shRNA KD4 were fixed after 2 days. FIRE LUT is used to show localisation and intensity of GFP in magnified images of boxed regions. White arrowhead indicates localisation at protrusive tip. Scale bars, 20µm. (B-C) Quantitation of (B) PI(4,5)P 2 or (C) PIP 3 cortical enrichment in spindle, spread and round cells expressing either Scr or IQSEC1 KD4 shRNA is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=3, 4 replicates/condition. 1330/1049 and 2222/1182 cells analysed for Src/ IQSEC1 KD4 shRNA respectively in B and C. p-values (one-way ANOVA). (D) Cartoon, summary of cortical PI(4,5)P 2 and PIP 3 localization in different cell shapes. (E) Western blot of PC3 cells treated with LY294002 (pan PI3K), AZD8835 (PI3Kα), AZD8186 (PI3Kβ), AS605240 (PI3Kγ), Cal-101 (PI3Kδ) and AktII (Akt) inhibitors for 24 hours. Anti-phospho-S473 Akt, Akt and GAPDH antibodies were used. (F) Phase contrast images of PC3 acini described in E. Scale bars, 100μm. (G) Cartoon, depicts acini phenotype representative of each condition. (H) Quantitation of PC3 acini shown in F. Heatmaps show area and compactness measurements as Z-score-normalised values (upper heatmaps). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates, minimum of 350 acini/condition. (I) Western blot of PC3 cells expressing Scr, PIP5K1α or PIP5K1β shRNA using anti-PIP5K, IQSEC1, phospho-S473 Akt, Akt and GAPDH (shown for Akt blots) antibodies. (J) Phase contrast images of PC3 acini expressing Scr, PIP5K1α or PIP5K1β shRNA at 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum of 1,348 acini/condition. Cartoon, depicts acini phenotype representative of each condition. (L) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were stained for pS473 Akt (black) and Hoechst /nuclei (magenta). Magnified images of boxed regions are shown in lower panels. Blue arrowheads indicate localisation of active Akt. Scale bars, 10μm. (M-N) Quantitation of pAkt (M) intensity or (N) area in spots in the juxtanuclear, cytoplasm and periphery of PC3 cells expressing either Scr or IQSEC1 KD4 shRNA. Box-and-whiskers plots: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition. p values (Mann Whitney). (O) Cartoon, depicting phospho-Akt intensity, in the presence or absence of IQSEC1, at different subcellular locations. All p-values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Expressing, shRNA, Quantitation Assay, Western Blot, Staining, MANN-WHITNEY

    Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on JNK phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; GAPDH antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p

    Journal: Neurochemistry International

    Article Title: Effect of Glutamate and Riluzole on Manganese-Induced Apoptotic Cell Signaling in Neuronally Differentiated Mouse P19 Cells

    doi: 10.1016/j.neuint.2012.04.015

    Figure Lengend Snippet: Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on JNK phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; GAPDH antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p

    Article Snippet: Phospho-JNK antibody, anti-GAPDH antibody and secondary antibody for Western blots were obtained from Cell Signaling (Danvers, MA) and Western lightning plus substrate to develop immunoblots was from Perkin Elmer (Waltham, MA).

    Techniques: Western Blot

    The miR-320d and FoxM1 expression level in GCA tissues and the adjacent normal tissues. RT-qPCR analysis of miR-320d expression level ( a ) and FoxM1 mRNA level ( b ) in 60 GCA tissues and the adjacent non-cancerous tissues. c Correlation between miR-320d expression and FoxM1 mRNA expression in 60 GCA tissues ( r = − 2.94, P = 0.023). Representative IHC images ( d ) and western-blot analysis ( e ) of FoxM1 protein in GCA and normal tissues. f IHC scores of FoxM1 protein in GCA and normal tissues evaluated by the independent pathologists (n = 4). g Quantification of FoxM1 level by western-blot (n = 6). GAPDH was normalized as 100%. The * represents significant difference from GCA tissues to the normal tissues (**: P

    Journal: Cell & Bioscience

    Article Title: MicroRNA-320d regulates tumor growth and invasion by promoting FoxM1 and predicts poor outcome in gastric cardiac adenocarcinoma

    doi: 10.1186/s13578-020-00439-7

    Figure Lengend Snippet: The miR-320d and FoxM1 expression level in GCA tissues and the adjacent normal tissues. RT-qPCR analysis of miR-320d expression level ( a ) and FoxM1 mRNA level ( b ) in 60 GCA tissues and the adjacent non-cancerous tissues. c Correlation between miR-320d expression and FoxM1 mRNA expression in 60 GCA tissues ( r = − 2.94, P = 0.023). Representative IHC images ( d ) and western-blot analysis ( e ) of FoxM1 protein in GCA and normal tissues. f IHC scores of FoxM1 protein in GCA and normal tissues evaluated by the independent pathologists (n = 4). g Quantification of FoxM1 level by western-blot (n = 6). GAPDH was normalized as 100%. The * represents significant difference from GCA tissues to the normal tissues (**: P

    Article Snippet: The total proteins were incubated with FoxM1 primary antibody (1:1000, Abcam, USA) and GAPDH antibody (Cell signaling, USA) at 4 °C for overnight.

    Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Western Blot

    The regulatory relationship of miR-320d and FoxM1 in GCA cell lines. a RT-qPCR analysis of miR-320d and FoxM1 expression levels in GCA cell SK-GT2 and OE-19. b The expression of miR-320d in OE-19 cell that transfected with miR-320d mimics or in SK-GT2 cell with miR-320d inhibitor transfection. Western blot images ( c ) or quantification ( d ) of FoxM1 expression in GCA cells with or without transfection. The empty vector (EV) was set as control, and GAPDH was normalized as 100%. The * represents significant difference from miR-320d vector-transfected cells to EV-transfected cells (*: P

    Journal: Cell & Bioscience

    Article Title: MicroRNA-320d regulates tumor growth and invasion by promoting FoxM1 and predicts poor outcome in gastric cardiac adenocarcinoma

    doi: 10.1186/s13578-020-00439-7

    Figure Lengend Snippet: The regulatory relationship of miR-320d and FoxM1 in GCA cell lines. a RT-qPCR analysis of miR-320d and FoxM1 expression levels in GCA cell SK-GT2 and OE-19. b The expression of miR-320d in OE-19 cell that transfected with miR-320d mimics or in SK-GT2 cell with miR-320d inhibitor transfection. Western blot images ( c ) or quantification ( d ) of FoxM1 expression in GCA cells with or without transfection. The empty vector (EV) was set as control, and GAPDH was normalized as 100%. The * represents significant difference from miR-320d vector-transfected cells to EV-transfected cells (*: P

    Article Snippet: The total proteins were incubated with FoxM1 primary antibody (1:1000, Abcam, USA) and GAPDH antibody (Cell signaling, USA) at 4 °C for overnight.

    Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Plasmid Preparation

    AA6 prevents metastasization targeting the TET–miR200–Zeb1/CtBP1–MMP3 axis. a Relative enrichment of 5mC in selected CCpGG sites of miR-200 family promoter regions for cluster 1 (left panel) and cluster 2 (right panel) in 4T1-injected mice DNA treated with AA6 (50 mg/kg; grey bars) versus control mice DNA (black bars); n = 5. b Pri-miR-200 cluster 1 (left panel) and cluster 2 (right panel) level of AA6 (50 mg/kg; grey bars) treated 4T1-injected mice expressed as fold-induction compared to untreated mice (black bars); n = 3. c Cluster 1 (miR-200b, miR-200a and miR-429; left panel) and cluster 2 (miR-200c and miR-141; right panel) expression in 4T1-injected mice treated with AA6 (50 mg/kg; grey bars), the graph represents average fold changes versus controls (black bars); n = 4. d Cluster 1 (miR-200b, miR-200a and miR-429; left panel) and cluster 2 (miR-200c and miR-141; right panel) expression in 4T1 cells treated with AA6 (50 µM; grey bars) for 6, 16, and 24 h, bar graphs represent average fold changes versus vehicle-treated cells (black bars); n = 4. e Representative WB (left panel) and relative densitometry (right panel; n = 5) of ZEB1 protein level in AA6 (50 mg/kg; grey bar) treated mice compared to controls (black bar). GAPDH and Red Ponceau were used as loading controls. f , g Zeb1 mRNA expression levels ( f ) and representative western blotting analysis of ZEB1 protein expression ( g ) in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); the right panel shows the relative densitometry as fold-change versus vehicle. α-tubulin was used as loading control; n = 4. h Representative WB (left panel) and relative densitometry (right panel) of ZEB1 protein expression level in AA6 treated 4T1 cells compared to vehicle-treated cells after transfection either with scramble-LNA (vehicle: black bar; AA6 50 µM: light grey bar) or anti-miR-200c-LNA (vehicle: dark grey bar; AA6 50 µM: medium grey bar). α-tubulin was used as loading control; n = 4. Data are presented as mean ± SE; * p

    Journal: Cell Death & Disease

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis

    doi: 10.1038/s41419-018-0802-8

    Figure Lengend Snippet: AA6 prevents metastasization targeting the TET–miR200–Zeb1/CtBP1–MMP3 axis. a Relative enrichment of 5mC in selected CCpGG sites of miR-200 family promoter regions for cluster 1 (left panel) and cluster 2 (right panel) in 4T1-injected mice DNA treated with AA6 (50 mg/kg; grey bars) versus control mice DNA (black bars); n = 5. b Pri-miR-200 cluster 1 (left panel) and cluster 2 (right panel) level of AA6 (50 mg/kg; grey bars) treated 4T1-injected mice expressed as fold-induction compared to untreated mice (black bars); n = 3. c Cluster 1 (miR-200b, miR-200a and miR-429; left panel) and cluster 2 (miR-200c and miR-141; right panel) expression in 4T1-injected mice treated with AA6 (50 mg/kg; grey bars), the graph represents average fold changes versus controls (black bars); n = 4. d Cluster 1 (miR-200b, miR-200a and miR-429; left panel) and cluster 2 (miR-200c and miR-141; right panel) expression in 4T1 cells treated with AA6 (50 µM; grey bars) for 6, 16, and 24 h, bar graphs represent average fold changes versus vehicle-treated cells (black bars); n = 4. e Representative WB (left panel) and relative densitometry (right panel; n = 5) of ZEB1 protein level in AA6 (50 mg/kg; grey bar) treated mice compared to controls (black bar). GAPDH and Red Ponceau were used as loading controls. f , g Zeb1 mRNA expression levels ( f ) and representative western blotting analysis of ZEB1 protein expression ( g ) in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); the right panel shows the relative densitometry as fold-change versus vehicle. α-tubulin was used as loading control; n = 4. h Representative WB (left panel) and relative densitometry (right panel) of ZEB1 protein expression level in AA6 treated 4T1 cells compared to vehicle-treated cells after transfection either with scramble-LNA (vehicle: black bar; AA6 50 µM: light grey bar) or anti-miR-200c-LNA (vehicle: dark grey bar; AA6 50 µM: medium grey bar). α-tubulin was used as loading control; n = 4. Data are presented as mean ± SE; * p

    Article Snippet: Nitrocellulose blotting membranes were probed with the following antibodies: ZEB-1 (Santa Cruz), CtBP-1 (Cell Signaling), GPNMB (Thermo Fisher Sc.), MMP-3 (BIOSS), SRC (Cell Signaling), KGDH (alias OGDH, Genetex), TET-1 (Genetex), TET-2 (Santa Cruz) and TET-3 (Novus Biologicals) flag (SIGMA), α-tubulin (Cell Signaling), GAPDH (abcam), Grb2 (Santa Cruz).

    Techniques: Injection, Mouse Assay, Expressing, Western Blot, Transfection

    AA6 administration decreases metastasis-associated transcripts and interferes with 4T1 cells migration. a Heatmap showing the 53 most differentially regulated genes in tumour mass derived from AA6 injected mice (50 mg/kg), or untreated mice; n = 3 each group. Yellow and blue represent over- and under-expressed genes, respectively. b mRNA expression analysis of Matrix metallopeptidase 3 (Mmp3), Glycoprotein transmembrane non-metastatic B (Gpnmb), C-terminal binding protein 1 (Ctbp1), Plasminogen activator, urokinase receptor (Plaur) and Rous sarcoma oncogene (Src) genes in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. c Representative western blot (upper panels) and relative densitometry (lower panel) of MMP3, GPNMB, CtBP1 and SRC protein levels in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). GRB2 and GAPDH were used as loading controls; n = 5 each group. d Representative phase contrast microscopy images (upper panel) depicting 4T1 cells motility after 24 h treatment with AA6 (50 µM) or vehicle alone; the graph (lower panel) shows the percentage of closure in 4T1 cells after 24 h treatment with AA6 (50 µM; grey bar) or vehicle (black bar). Scale bar 100 μm; n = 5 each group. e Representative pictures (upper panel) showing 4T1 cell invasiveness after AA6 (50 µM) treatment versus vehicle; the graphs (lower panel) represent migrated cells counted after 24 h treatment with AA6 (50 µM; grey bar) or vehicle alone (black bar). Scale bar 50 μm; n = 3. Data are presented as mean ± SE; * p

    Journal: Cell Death & Disease

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis

    doi: 10.1038/s41419-018-0802-8

    Figure Lengend Snippet: AA6 administration decreases metastasis-associated transcripts and interferes with 4T1 cells migration. a Heatmap showing the 53 most differentially regulated genes in tumour mass derived from AA6 injected mice (50 mg/kg), or untreated mice; n = 3 each group. Yellow and blue represent over- and under-expressed genes, respectively. b mRNA expression analysis of Matrix metallopeptidase 3 (Mmp3), Glycoprotein transmembrane non-metastatic B (Gpnmb), C-terminal binding protein 1 (Ctbp1), Plasminogen activator, urokinase receptor (Plaur) and Rous sarcoma oncogene (Src) genes in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. c Representative western blot (upper panels) and relative densitometry (lower panel) of MMP3, GPNMB, CtBP1 and SRC protein levels in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). GRB2 and GAPDH were used as loading controls; n = 5 each group. d Representative phase contrast microscopy images (upper panel) depicting 4T1 cells motility after 24 h treatment with AA6 (50 µM) or vehicle alone; the graph (lower panel) shows the percentage of closure in 4T1 cells after 24 h treatment with AA6 (50 µM; grey bar) or vehicle (black bar). Scale bar 100 μm; n = 5 each group. e Representative pictures (upper panel) showing 4T1 cell invasiveness after AA6 (50 µM) treatment versus vehicle; the graphs (lower panel) represent migrated cells counted after 24 h treatment with AA6 (50 µM; grey bar) or vehicle alone (black bar). Scale bar 50 μm; n = 3. Data are presented as mean ± SE; * p

    Article Snippet: Nitrocellulose blotting membranes were probed with the following antibodies: ZEB-1 (Santa Cruz), CtBP-1 (Cell Signaling), GPNMB (Thermo Fisher Sc.), MMP-3 (BIOSS), SRC (Cell Signaling), KGDH (alias OGDH, Genetex), TET-1 (Genetex), TET-2 (Santa Cruz) and TET-3 (Novus Biologicals) flag (SIGMA), α-tubulin (Cell Signaling), GAPDH (abcam), Grb2 (Santa Cruz).

    Techniques: Migration, Derivative Assay, Injection, Mouse Assay, Expressing, Binding Assay, Western Blot, Microscopy

    KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p

    Journal: Cell Death & Disease

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis

    doi: 10.1038/s41419-018-0802-8

    Figure Lengend Snippet: KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p

    Article Snippet: Nitrocellulose blotting membranes were probed with the following antibodies: ZEB-1 (Santa Cruz), CtBP-1 (Cell Signaling), GPNMB (Thermo Fisher Sc.), MMP-3 (BIOSS), SRC (Cell Signaling), KGDH (alias OGDH, Genetex), TET-1 (Genetex), TET-2 (Santa Cruz) and TET-3 (Novus Biologicals) flag (SIGMA), α-tubulin (Cell Signaling), GAPDH (abcam), Grb2 (Santa Cruz).

    Techniques: Inhibition, Expressing, In Vivo, In Vitro, Translocation Assay, Injection, Mouse Assay, Western Blot, Activity Assay

    ZEB1 was up-regulated in hESC-derived neural cells. A, a scheme of neural differentiation of hESCs. Scale bar : 50 μm. B, real-time PCR analysis showed the alteration of ZEB1 mRNA levels during neural differentiation of hESCs. The amount of mRNA was normalized to GAPDH levels. Bars represent mean ± S.D. of three experimental replicates. C, Western blotting showed expression of the ZEB1 protein at different stages of neural differentiation from hESCs. β-ACTIN was a loading control; n = 3. D, representative images of immunostaining results using indicated antibodies in hESC-derived neural cells. DAPI was included to show the nuclei (here and after); n = 3. Scale bar : 50 μm. hESC , human embryonic stem cell; EB , embryoid bodies; NSC , neural stem cell; E8 , essential 8 medium; NIM , neural induction medium; NDM , neural differentiation medium.

    Journal: The Journal of Biological Chemistry

    Article Title: Zinc finger E-box–binding homeobox 1 (ZEB1) is required for neural differentiation of human embryonic stem cells

    doi: 10.1074/jbc.RA118.005498

    Figure Lengend Snippet: ZEB1 was up-regulated in hESC-derived neural cells. A, a scheme of neural differentiation of hESCs. Scale bar : 50 μm. B, real-time PCR analysis showed the alteration of ZEB1 mRNA levels during neural differentiation of hESCs. The amount of mRNA was normalized to GAPDH levels. Bars represent mean ± S.D. of three experimental replicates. C, Western blotting showed expression of the ZEB1 protein at different stages of neural differentiation from hESCs. β-ACTIN was a loading control; n = 3. D, representative images of immunostaining results using indicated antibodies in hESC-derived neural cells. DAPI was included to show the nuclei (here and after); n = 3. Scale bar : 50 μm. hESC , human embryonic stem cell; EB , embryoid bodies; NSC , neural stem cell; E8 , essential 8 medium; NIM , neural induction medium; NDM , neural differentiation medium.

    Article Snippet: The following primary antibodies were used: ZEB1 (Millipore, ABN285), ZEB2 (Santa Cruz, sc48789), N-CADHERIN (Abcam, ab18203), SNAIL (Cell Signaling Technology, number 3879), SLUG (Cell Signaling Technology, number 9585), TWIST2 (Abcam, ab50887), FLAG (Sigma, F3165), OCT4 (Abcam, ab19857), NANOG (R & D Systems, AF1997), SOX2 (Millipore, AB5603), PAX6 (Developmental Studies Hybridoma Bank), SOX1 (Millipore, MAB3369), NESTIN (Millipore, MAB5326), βIII-tubulin (TUJ1, Covance, PRB-435P), GAPDH (Abcam, ab9485), β-actin (Proteintech, 60008-1-Ig),and β-tubulin (Cell Signaling Technology, number 2148).

    Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunostaining

    Sustained overexpression of ZEB1 in hESC-induced neural differentiation. A, Western blotting using the indicated antibodies in selected clone #2 of hESCs. GAPDH was the loading control. B, representative phase-contrast images showing different morphologies of H9ESC colonies with ( ZEB1 ) or without (Control) doxycycline in the medium; n = 3. Scale bar : 100 μm. C, images showing the phenotypic alterations of cells at various stages of neural differentiation after overexpression of ZEB1 . Typical rosettes in control and extended neurite-like structures from colonies with ZEB1 overexpression were observed. Alterations of PAX6 and SOX1 on day 15, TUJ1 and MAP2 on day 25 were shown as well. Scale bar , 100 μm. D and E, quantification of TUJ1+ cells and MAP2-positive cells. ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Zinc finger E-box–binding homeobox 1 (ZEB1) is required for neural differentiation of human embryonic stem cells

    doi: 10.1074/jbc.RA118.005498

    Figure Lengend Snippet: Sustained overexpression of ZEB1 in hESC-induced neural differentiation. A, Western blotting using the indicated antibodies in selected clone #2 of hESCs. GAPDH was the loading control. B, representative phase-contrast images showing different morphologies of H9ESC colonies with ( ZEB1 ) or without (Control) doxycycline in the medium; n = 3. Scale bar : 100 μm. C, images showing the phenotypic alterations of cells at various stages of neural differentiation after overexpression of ZEB1 . Typical rosettes in control and extended neurite-like structures from colonies with ZEB1 overexpression were observed. Alterations of PAX6 and SOX1 on day 15, TUJ1 and MAP2 on day 25 were shown as well. Scale bar , 100 μm. D and E, quantification of TUJ1+ cells and MAP2-positive cells. ***, p

    Article Snippet: The following primary antibodies were used: ZEB1 (Millipore, ABN285), ZEB2 (Santa Cruz, sc48789), N-CADHERIN (Abcam, ab18203), SNAIL (Cell Signaling Technology, number 3879), SLUG (Cell Signaling Technology, number 9585), TWIST2 (Abcam, ab50887), FLAG (Sigma, F3165), OCT4 (Abcam, ab19857), NANOG (R & D Systems, AF1997), SOX2 (Millipore, AB5603), PAX6 (Developmental Studies Hybridoma Bank), SOX1 (Millipore, MAB3369), NESTIN (Millipore, MAB5326), βIII-tubulin (TUJ1, Covance, PRB-435P), GAPDH (Abcam, ab9485), β-actin (Proteintech, 60008-1-Ig),and β-tubulin (Cell Signaling Technology, number 2148).

    Techniques: Over Expression, Western Blot

    Inhibition of Ep3 suppresses the growth of A549 cells via inhibition of TGF-β/Smad signaling. The protein expression of (A) TGF-β, (B) p-Smad2 and Smad2, and (C) p-Smad3 and Smad3 were detected by western blotting and normalized to GAPDH levels. (D) Cell viability was assessed by MTT assay. (E) Cell apoptosis was assessed by flow cytometry assay. The data are presented as the mean ± the standard error of the mean of three independent experiments *P

    Journal: Oncology Letters

    Article Title: Inhibition of Ep3 attenuates migration and promotes apoptosis of non-small cell lung cancer cells via suppression of TGF-β/Smad signaling

    doi: 10.3892/ol.2018.9391

    Figure Lengend Snippet: Inhibition of Ep3 suppresses the growth of A549 cells via inhibition of TGF-β/Smad signaling. The protein expression of (A) TGF-β, (B) p-Smad2 and Smad2, and (C) p-Smad3 and Smad3 were detected by western blotting and normalized to GAPDH levels. (D) Cell viability was assessed by MTT assay. (E) Cell apoptosis was assessed by flow cytometry assay. The data are presented as the mean ± the standard error of the mean of three independent experiments *P

    Article Snippet: Membranes were blocked in 5% bovine serum albumin (Sigma Aldrich; Merck KGaA) at room temperature for 1 h, and subsequently immunoblotted with the following primary antibodies according to the recommended dilution concentration: Anti-Ep3 (1:500; cat. no. P8372; Sigma-Aldrich; Merck KGaA), anti-caspase-3 (1:1,000; cat. no. 9662), anti-B cell lymphoma (Bcl)-2 (1:1,000; cat. no. 2872), anti-Bcl-associated × protein (Bax; 1:1,000; cat. no. 2772; all Cell Signaling Technology, Inc., Danvers, MA, USA), anti-MMP-9 (1:500; cat. no. ab58803; Abcam, Cambridge, UK), anti-VEGF (1:1,000; cat. no. V6627; Sigma-Aldrich; Meck KGaA), anti-TGF-β1 (1:1,000; cat. no. SAB4502954; Sigma-Aldrich; Merck KGaA), anti-Smad-2 (1:1,000; cat. no. ab63576), anti-Smad-3 (1:2,000; cat. no. ab40854), anti-phosphorylated (p)-Smad2 (1:800; cat. no. ab53100), anti-p-Smad3 (1:2,000; cat. no. ab52903) and anti-GAPDH (1:2,000; cat. no. ab8245; all Abcam) at 4°C overnight.

    Techniques: Inhibition, Expressing, Western Blot, MTT Assay, Flow Cytometry, Cytometry

    GM1-deficient C6 cells are sensitized to CT by inhibition of sialylation or GSL biosynthesis. (A-E) C6 cells were cultured with the indicated inhibitors for 72 h followed by: (A) Staining was then performed with biotin-CTB, followed by DTAF-streptavidin. Fluorescence was measured by flow cytometry, represented here by MFI. (B) 1 h exposure to CT after which accumulated cAMP was measured by the cAMP-Glo™ luminescence assay. Luminescence signal is inversely proportional to cAMP levels. (C) As in panel A , but stained with biotin-PNA, followed by DTAF-streptavidin (D) Cell lysates were separated by PAGE and probed with biotin-PNA, biotin-CTB, or no biotinylated reagent, followed by streptavidin-peroxidase conjugate and development with chemiluminescent substrate. Equivalent amounts of protein were loaded in each lane and blots were probed with an anti-α-tubulin or anti-GAPDH antibody to confirm equivalent loading. (E) As in panel B , but brefeldin A (BFA) was added 1 h prior to CT addition and was also present during CT induction.

    Journal: PLoS Pathogens

    Article Title: GM1 ganglioside-independent intoxication by Cholera toxin

    doi: 10.1371/journal.ppat.1006862

    Figure Lengend Snippet: GM1-deficient C6 cells are sensitized to CT by inhibition of sialylation or GSL biosynthesis. (A-E) C6 cells were cultured with the indicated inhibitors for 72 h followed by: (A) Staining was then performed with biotin-CTB, followed by DTAF-streptavidin. Fluorescence was measured by flow cytometry, represented here by MFI. (B) 1 h exposure to CT after which accumulated cAMP was measured by the cAMP-Glo™ luminescence assay. Luminescence signal is inversely proportional to cAMP levels. (C) As in panel A , but stained with biotin-PNA, followed by DTAF-streptavidin (D) Cell lysates were separated by PAGE and probed with biotin-PNA, biotin-CTB, or no biotinylated reagent, followed by streptavidin-peroxidase conjugate and development with chemiluminescent substrate. Equivalent amounts of protein were loaded in each lane and blots were probed with an anti-α-tubulin or anti-GAPDH antibody to confirm equivalent loading. (E) As in panel B , but brefeldin A (BFA) was added 1 h prior to CT addition and was also present during CT induction.

    Article Snippet: Blots were incubated in mild stripping buffer (200 mM glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) at 37°C for 45 min and incubated with anti-histone H3 antibody (Abcam, Cat. No. ab1791, 1:2000 dilution, for AAL blot), anti-α-tubulin antibody (Sigma-Aldrich, Cat. No. T6199, 1:10000 dilution, for PNA and CTB blot), or anti-GAPDH antibody (Abcam, Cat. No. ab8245, 1:10000 dilution, for streptavidin-POD blot) at room temperature for 1 h and washed with TBST.

    Techniques: Inhibition, Cell Culture, Staining, CtB Assay, Fluorescence, Flow Cytometry, Cytometry, Luminescence Assay, Polyacrylamide Gel Electrophoresis

    Investigation of mechanisms underlying NMR-detected changes in the levels of choline metabolites and lactate following treatment of SF188 pediatric glioblastoma cells with PI-103 (5×GI 50 ). (A) Representative Western blots showing changes in protein expression levels of enzymes involved in choline metabolism (CHKA) and glucose metabolism including: GLUT1, HK2 and LDHA, at selected time points post treatment with PI-103. GAPDH was used as a loading control. (B) Quantitative measurement of 1 H-NMR detected percentage changes in the levels of lactate (Lac, internal external) and glucose (external) at selected time points post treatment with PI-103 relative to controls, 8 hours n = 2. Results are expressed as percentage of treated to control and presented as the mean ± SD (error bars). Statistically significant different from the control *p≤0.05, **p

    Journal: PLoS ONE

    Article Title: Lactate and Choline Metabolites Detected In Vitro by Nuclear Magnetic Resonance Spectroscopy Are Potential Metabolic Biomarkers for PI3K Inhibition in Pediatric Glioblastoma

    doi: 10.1371/journal.pone.0103835

    Figure Lengend Snippet: Investigation of mechanisms underlying NMR-detected changes in the levels of choline metabolites and lactate following treatment of SF188 pediatric glioblastoma cells with PI-103 (5×GI 50 ). (A) Representative Western blots showing changes in protein expression levels of enzymes involved in choline metabolism (CHKA) and glucose metabolism including: GLUT1, HK2 and LDHA, at selected time points post treatment with PI-103. GAPDH was used as a loading control. (B) Quantitative measurement of 1 H-NMR detected percentage changes in the levels of lactate (Lac, internal external) and glucose (external) at selected time points post treatment with PI-103 relative to controls, 8 hours n = 2. Results are expressed as percentage of treated to control and presented as the mean ± SD (error bars). Statistically significant different from the control *p≤0.05, **p

    Article Snippet: Immunodetection was performed using antibodies against pAKT (Ser473), total AKT, pRPS6 (Ser240/244), total RPS6, HK2 (Cell Signaling), CHKA (Sigma), GLUT1, LDHA (Santa Cruz Biotechnology) and GAPDH (Chemicon).

    Techniques: Nuclear Magnetic Resonance, Western Blot, Expressing

    Chromatin architecture of the Reg locus in adult cortex and pancreas. ( A ) Levels of cohesin were assayed by immunoblot with SA1 and Rad21 antibodies in pancreas from wild-type and SA1 heterozygous mice (two individuals per genotype). GAPDH serves as loading control. ( B ) Table showing the four genes differentially expressed (FDR

    Journal: Nucleic Acids Research

    Article Title: The contribution of cohesin-SA1 to gene expression and chromatin architecture in two murine tissues

    doi: 10.1093/nar/gkv144

    Figure Lengend Snippet: Chromatin architecture of the Reg locus in adult cortex and pancreas. ( A ) Levels of cohesin were assayed by immunoblot with SA1 and Rad21 antibodies in pancreas from wild-type and SA1 heterozygous mice (two individuals per genotype). GAPDH serves as loading control. ( B ) Table showing the four genes differentially expressed (FDR

    Article Snippet: Equal amounts of protein were run in 7.5 Bis/Tris gels followed by western blotting with antibodies against SA1 and Rad21 ( ) and GAPDH (Sigma, G8795).

    Techniques: Mouse Assay

    K nockdown of FTO does not significantly change mRNA levels of genes involved in miRNA biogenesis. The steady-state mRNA levels of DICER , DROSHA , DGCR8 and ADAR were analyzed by qRT-PCR in cells treated with scrambled (scr) and FTO- specific siRNAs, respectively. GAPDH was used as a reference gene. The observed changes were not significant. Merged values of mean ± SD from triplicates per assay for the three independent cell lines FTO1C1, FTO2D4 and FTO3C3 are depicted. FTO kd, FTO -specific siRNA treated cells, scr siRNA, scrambled siRNA treated cells.

    Journal: PLoS ONE

    Article Title: N6-Adenosine Methylation in MiRNAs

    doi: 10.1371/journal.pone.0118438

    Figure Lengend Snippet: K nockdown of FTO does not significantly change mRNA levels of genes involved in miRNA biogenesis. The steady-state mRNA levels of DICER , DROSHA , DGCR8 and ADAR were analyzed by qRT-PCR in cells treated with scrambled (scr) and FTO- specific siRNAs, respectively. GAPDH was used as a reference gene. The observed changes were not significant. Merged values of mean ± SD from triplicates per assay for the three independent cell lines FTO1C1, FTO2D4 and FTO3C3 are depicted. FTO kd, FTO -specific siRNA treated cells, scr siRNA, scrambled siRNA treated cells.

    Article Snippet: The primary antibodies used for western blotting were mouse and rabbit anti-FTO (Abcam, Cambridge, UK; Epitomics, Burlingame CA, USA) and rabbit anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Quantitative RT-PCR

    FTO knockdown in HEK293 cell clones FTO1C1, FTO2D4 and FTO3C3. A) The levels of FTO transcripts were investigated by qRT-PCR. GAPDH was used as a reference gene. Mean ± SD for three pairs of scrambled (scr) and FTO specific siRNAs treated cells are shown. B) Reduced protein levels were revealed by Western blot in all three cell lines transfected with siRNAs targeting FTO mRNA. Exemplary photo is depicted.

    Journal: PLoS ONE

    Article Title: N6-Adenosine Methylation in MiRNAs

    doi: 10.1371/journal.pone.0118438

    Figure Lengend Snippet: FTO knockdown in HEK293 cell clones FTO1C1, FTO2D4 and FTO3C3. A) The levels of FTO transcripts were investigated by qRT-PCR. GAPDH was used as a reference gene. Mean ± SD for three pairs of scrambled (scr) and FTO specific siRNAs treated cells are shown. B) Reduced protein levels were revealed by Western blot in all three cell lines transfected with siRNAs targeting FTO mRNA. Exemplary photo is depicted.

    Article Snippet: The primary antibodies used for western blotting were mouse and rabbit anti-FTO (Abcam, Cambridge, UK; Epitomics, Burlingame CA, USA) and rabbit anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Clone Assay, Quantitative RT-PCR, Western Blot, Transfection

    Deregulation of miRNAs in FTO knockdown cells. Mature miRNAs showing increased (A) and decreased (B) steady state levels in FTO knockdown cells. Normalized RNA-seq read numbers of individual miRNAs in FTO knockdown and scrambled siRNA treated cells were compared. Mean ± SD of three independent experiments are depicted. For verification and further studies, qRT-PCR analyses of selected mature miRNAs (C) and primary miRNA transcripts (D) were performed. We did not use other small RNAs as a reference gene for measuring mature miRNAs levels (as suggested by Life Technologies), since depletion of FTO might have an impact on their levels. Therefore, luciferase RNA was used to generate a standard curve and added to the qRT-PCR assays. GAPDH was used as a reference gene for measuring primary miRNAs transcript levels. Mean ± SD from quadruplicates per assay for three independent cell lines (FTO1C1, FTO2D4 and FTO3C3) are depicted. kd, FTO specific siRNA treated cells, scr, scrambled siRNA treated cells.

    Journal: PLoS ONE

    Article Title: N6-Adenosine Methylation in MiRNAs

    doi: 10.1371/journal.pone.0118438

    Figure Lengend Snippet: Deregulation of miRNAs in FTO knockdown cells. Mature miRNAs showing increased (A) and decreased (B) steady state levels in FTO knockdown cells. Normalized RNA-seq read numbers of individual miRNAs in FTO knockdown and scrambled siRNA treated cells were compared. Mean ± SD of three independent experiments are depicted. For verification and further studies, qRT-PCR analyses of selected mature miRNAs (C) and primary miRNA transcripts (D) were performed. We did not use other small RNAs as a reference gene for measuring mature miRNAs levels (as suggested by Life Technologies), since depletion of FTO might have an impact on their levels. Therefore, luciferase RNA was used to generate a standard curve and added to the qRT-PCR assays. GAPDH was used as a reference gene for measuring primary miRNAs transcript levels. Mean ± SD from quadruplicates per assay for three independent cell lines (FTO1C1, FTO2D4 and FTO3C3) are depicted. kd, FTO specific siRNA treated cells, scr, scrambled siRNA treated cells.

    Article Snippet: The primary antibodies used for western blotting were mouse and rabbit anti-FTO (Abcam, Cambridge, UK; Epitomics, Burlingame CA, USA) and rabbit anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Luciferase

    YY1 and rs398206 affect MX2 expression in melanoma cells. a YY1 was knocked down using four different siRNAs in UACC647 cells, and MX2 levels were measured. GAPDH -normalized MX2 mRNA levels are shown as fold change over those from non-targeting siRNA. Four biological replicates of n = 6 were combined (total n = 24). b CRISPRi using dCAS9-KRAB-MeCP2 and four different gRNAs targeting rs398206 in UACC903 cells. MX2 mRNA levels ( GAPDH -normalized) are shown as fold change over those from non-targeting gRNA. Three biological replicates of n = 6 were combined (total n = 18, except gRNA-3, n = 17). gRNA 1, 3, and 4 directly overlap rs398206. gRNA 2 targets ~25 bp upstream of rs398206. AAVS1 (gRNA targeting adeno-associated virus integration site on Chr19). Box: Median and 25th to 75th percentile. Whisker: 10th to 90th percentile. P -values are shown from one-sample Wilcoxon test (two-sided) for difference from non-targeting siRNA/gRNA. Dotted line denotes the MX2 levels in non-targeting siRNA/gRNA control. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma

    doi: 10.1038/s41467-020-16590-1

    Figure Lengend Snippet: YY1 and rs398206 affect MX2 expression in melanoma cells. a YY1 was knocked down using four different siRNAs in UACC647 cells, and MX2 levels were measured. GAPDH -normalized MX2 mRNA levels are shown as fold change over those from non-targeting siRNA. Four biological replicates of n = 6 were combined (total n = 24). b CRISPRi using dCAS9-KRAB-MeCP2 and four different gRNAs targeting rs398206 in UACC903 cells. MX2 mRNA levels ( GAPDH -normalized) are shown as fold change over those from non-targeting gRNA. Three biological replicates of n = 6 were combined (total n = 18, except gRNA-3, n = 17). gRNA 1, 3, and 4 directly overlap rs398206. gRNA 2 targets ~25 bp upstream of rs398206. AAVS1 (gRNA targeting adeno-associated virus integration site on Chr19). Box: Median and 25th to 75th percentile. Whisker: 10th to 90th percentile. P -values are shown from one-sample Wilcoxon test (two-sided) for difference from non-targeting siRNA/gRNA. Dotted line denotes the MX2 levels in non-targeting siRNA/gRNA control. Source data are provided as a Source Data file.

    Article Snippet: The primary antibodies used were rabbit anti-MX2 (NBP1-81018, Novus Biologicals), and mouse anti-GAPDH (sc-51907, Santa Cruz).

    Techniques: Expressing, Whisker Assay

    The αN4 is an integral part of OASL. ( A ) The crystal structure of OLD with a zoom-in on the αN4 helix. The anchoring residues Val67, Phe70 and Asn72 are indicated and their side chains are shown in sticks. ( B ) An alignment of porcine OAS1 (pOAS1), human OAS1 (hOAS1), human cGAS (hcGAS), human OASL (hOASL), Archaeoglobus fulgidus CCA (AfCCA) and Saccharomyces cerevisiae PAP (yPAP). The residues spanning the αN4 or the αN4-equivalent helices in cGAS, CCA and PAP are indicated. The asterisks indicate the three carboxyl acid residues in the catalytic triad of OAS1, cGAS, CCA and PAP. The residues are colored according to conservation. ( C ) HEK293T OASL neg cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL V67G or OASL N72K in combination with an IFN-β promoter firefly luciferase reporter construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. Error bars indicate standard deviation from three independent experiments. The inset shows an immunoblot against V5 and GAPDH to confirm the expression of the transfected constructs and to ensure that similar amounts were loaded on the gel, respectively. ( D ) HeLa cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL V67G or OASL N72K in combination with an IFN-β promoter firefly luciferase reporter construct and a β-actin promoter Renilla luciferase construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. The measurements are shown as a ratio of firefly to Renilla luciferase activities. Error bars indicate standard deviation from three independent experiments. **: P -value ≤ 0.01. ***: P -value ≤ 0.001.

    Journal: Nucleic Acids Research

    Article Title: Structural and functional analysis reveals that human OASL binds dsRNA to enhance RIG-I signaling

    doi: 10.1093/nar/gkv389

    Figure Lengend Snippet: The αN4 is an integral part of OASL. ( A ) The crystal structure of OLD with a zoom-in on the αN4 helix. The anchoring residues Val67, Phe70 and Asn72 are indicated and their side chains are shown in sticks. ( B ) An alignment of porcine OAS1 (pOAS1), human OAS1 (hOAS1), human cGAS (hcGAS), human OASL (hOASL), Archaeoglobus fulgidus CCA (AfCCA) and Saccharomyces cerevisiae PAP (yPAP). The residues spanning the αN4 or the αN4-equivalent helices in cGAS, CCA and PAP are indicated. The asterisks indicate the three carboxyl acid residues in the catalytic triad of OAS1, cGAS, CCA and PAP. The residues are colored according to conservation. ( C ) HEK293T OASL neg cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL V67G or OASL N72K in combination with an IFN-β promoter firefly luciferase reporter construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. Error bars indicate standard deviation from three independent experiments. The inset shows an immunoblot against V5 and GAPDH to confirm the expression of the transfected constructs and to ensure that similar amounts were loaded on the gel, respectively. ( D ) HeLa cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL V67G or OASL N72K in combination with an IFN-β promoter firefly luciferase reporter construct and a β-actin promoter Renilla luciferase construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. The measurements are shown as a ratio of firefly to Renilla luciferase activities. Error bars indicate standard deviation from three independent experiments. **: P -value ≤ 0.01. ***: P -value ≤ 0.001.

    Article Snippet: The proteins were then transferred to a polyvinylidene flouride (PVDF) membrane (Applichem) and blocked with 5% milk in Tris-buffered saline with 0.05% Tween-20 (TTBS) at room temperature for 1 h. The membrane was washed 3×5 min in TTBS and blotted with mouse anti-V5 (Invitrogen, catalog# R960-25), rabbit anti-GAPDH (Santa Cruz Biotechnology, catalog# sc-25778), mouse anti-His4 (Qiagen, catalog# 34670) or mouse monoclonal anti-FLAG (Sigma-Aldrich, catalog# F3165) at 4°C ON.

    Techniques: Transfection, Plasmid Preparation, Luciferase, Construct, Infection, Standard Deviation, Expressing

    OASL binds to dsRNA and dsRNA binding is essential to enhance RIG-I signaling. ( A ) A constant concentration of OLD was incubated with increasing amounts of 18 bp dsRNA and subsequently analyzed by silver stained native PAGE. OLD was kept constant at 2.4 μM (1 μg) and incubated with 0, 0.13, 0.27, 0.54, 1.1, 2.2, 4.3 or 8.6 μM (1 μg) 18 bp dsRNA. The negative control was performed with 8.6 μM 18 bp dsRNA and no OLD. ( B ) 2.4 μM wild-type OLD or K66E OLD was incubated with 8.6 μM 18 bp dsRNA and analyzed by native PAGE. The proteins were visualized by silver staining or immunoblotting against the 6xHis-tag. Wells: the position of the bottom of the wells in the corresponding native gel. ( C ) A constant concentration of 18 bp dsRNA was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of 18 bp dsRNA was kept constant at 10 μM and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. ( D ) A constant concentration of poly(I:C)-LMW was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of poly(I:C)-LMW was kept constant at 0.2 mg/ml and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. ( E ) HEK293T OASL neg cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL R45E/K66E/R196E/K200E (RKRK), OASL K63E, OASL K66E or OASL S327A in combination with an IFN-β promoter luciferase reporter construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. Error bars indicate standard deviation from three independent experiments. The inset shows an immunoblot against V5 and GAPDH to confirm the expression of the transfected constructs and to ensure that similar amounts were loaded on the gel, respectively. Ns: P -value > 0.05. ***: P -value ≤ 0.001.

    Journal: Nucleic Acids Research

    Article Title: Structural and functional analysis reveals that human OASL binds dsRNA to enhance RIG-I signaling

    doi: 10.1093/nar/gkv389

    Figure Lengend Snippet: OASL binds to dsRNA and dsRNA binding is essential to enhance RIG-I signaling. ( A ) A constant concentration of OLD was incubated with increasing amounts of 18 bp dsRNA and subsequently analyzed by silver stained native PAGE. OLD was kept constant at 2.4 μM (1 μg) and incubated with 0, 0.13, 0.27, 0.54, 1.1, 2.2, 4.3 or 8.6 μM (1 μg) 18 bp dsRNA. The negative control was performed with 8.6 μM 18 bp dsRNA and no OLD. ( B ) 2.4 μM wild-type OLD or K66E OLD was incubated with 8.6 μM 18 bp dsRNA and analyzed by native PAGE. The proteins were visualized by silver staining or immunoblotting against the 6xHis-tag. Wells: the position of the bottom of the wells in the corresponding native gel. ( C ) A constant concentration of 18 bp dsRNA was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of 18 bp dsRNA was kept constant at 10 μM and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. ( D ) A constant concentration of poly(I:C)-LMW was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of poly(I:C)-LMW was kept constant at 0.2 mg/ml and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. ( E ) HEK293T OASL neg cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL R45E/K66E/R196E/K200E (RKRK), OASL K63E, OASL K66E or OASL S327A in combination with an IFN-β promoter luciferase reporter construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. Error bars indicate standard deviation from three independent experiments. The inset shows an immunoblot against V5 and GAPDH to confirm the expression of the transfected constructs and to ensure that similar amounts were loaded on the gel, respectively. Ns: P -value > 0.05. ***: P -value ≤ 0.001.

    Article Snippet: The proteins were then transferred to a polyvinylidene flouride (PVDF) membrane (Applichem) and blocked with 5% milk in Tris-buffered saline with 0.05% Tween-20 (TTBS) at room temperature for 1 h. The membrane was washed 3×5 min in TTBS and blotted with mouse anti-V5 (Invitrogen, catalog# R960-25), rabbit anti-GAPDH (Santa Cruz Biotechnology, catalog# sc-25778), mouse anti-His4 (Qiagen, catalog# 34670) or mouse monoclonal anti-FLAG (Sigma-Aldrich, catalog# F3165) at 4°C ON.

    Techniques: Binding Assay, Concentration Assay, Incubation, Staining, Clear Native PAGE, Negative Control, Silver Staining, Mutagenesis, Transfection, Plasmid Preparation, Luciferase, Construct, Infection, Standard Deviation, Expressing