Journal: Cancer research

Article Title: OCTN1 is a high-affinity carrier of nucleoside analogs

doi: 10.1158/0008-5472.CAN-16-2548

Figure Lengend Snippet: Characterization of the transport of various AML-directed therapeutics (concentration, 1 μM; 5 min uptake) was performed in HEK293 cells transfected with an empty vector (VC) or OCTN1 (A). A comparison of OCTN1- and ENT1-mediated transport of cytarabine was done in HEK293 cells (B). Time-dependence of cytarabine (Ara-C) transport by OCTN1 at early time points (range, 10–300 s) (C). Sensitivity of OCTN1-mediated cytarabine transport to temperature, sodium, pH, and inhibitors (D). Concentration-dependent transport of cytarabine (1–50 μM; 5 min uptake) by OCTN1 (E), and these data shown as an Eadie-Hofstee transformation (F). Data are shown as mean values (symbols) and SEM (error bars), using 9–60 observations per group. Solid lines represent a fit of the experimental data to a non-linear maximum-effect model or linear regression. Abbreviations: v, transport velocity; [S], substrate concentration; Km, Michaelis-Menten constant; Vmax, maximal velocity.

Article Snippet: Reconstructed OCTN1 and ENT1 cDNAs (Origene) with a Flag-tag were subcloned into the pMIG II vector, engineered from a MSCV-IRES-GFP vector.

Techniques: Concentration Assay, Transfection, Plasmid Preparation, Comparison, Transformation Assay

Journal: Cancer research

Article Title: OCTN1 is a high-affinity carrier of nucleoside analogs

doi: 10.1158/0008-5472.CAN-16-2548

Figure Lengend Snippet: Gene expression of OCTN1 and ENT1 (two probe sets) in primary AML blast samples from pediatric patients used in the survival analysis shown in Fig. 1A–B, as determined by microarray analysis (A). Each column represents an individual primary sample, and columns are categorized by cytogenetic AML subtypes of prognostic relevance. (B) Protein expression of OCTN1 in a panel of 12 AML cell lines. (C) Cellular uptake of cytarabine (Ara-C; 1 μM; 5 min uptake) with or without NBMPR pre-incubation was measured in OCI-AML3 cells 48 hours after transfection with a non-targeting (NT) control siRNA or a siRNA targeting OCTN1. Results are shown as cytarabine uptake as compared to cells transfected with NT siRNA. Data are representative of two independent experiments done in triplicate. OCTN1 protein expression was determined by western blot from membrane extraction of OCI-AML3 cells 48 hours post-transfection, and the transferrin receptor served as loading control. The relative expression difference of OCTN1 after RNAi is indicated by the numbers above the lanes.

Article Snippet: Reconstructed OCTN1 and ENT1 cDNAs (Origene) with a Flag-tag were subcloned into the pMIG II vector, engineered from a MSCV-IRES-GFP vector.

Techniques: Gene Expression, Microarray, Expressing, Incubation, Transfection, Control, Western Blot, Membrane, Extraction

Journal: bioRxiv

Article Title: Promoting axon regeneration by enhancing the non-coding function of the injury-responsive coding gene Gpr151

doi: 10.1101/2021.02.19.431965

Figure Lengend Snippet: (a) Coomassie staining image of SDS-PAGE from pull-down assay. Control, Biotin-AMP; 5’UTR, Biotin- Gpr151 5’UTR RNA. The labels indicated the identified proteins from mass spectrophotometry analysis. The identified peptides and the scores were in Supplementary Data 2. (b) Alignment of 5’UTR sequences of mouse, rat and human GPR151 gene. Green box, nAAGnA, CSDE1-binding consensus motif , , yellow shadow, the start codon. (c) Western blot analysis of pull-down assay. Total protein lysates prepared from embryonic cultured DRG neurons were incubated with control (Biotin-AMP) or 5’UTR (Biotin-5’UTR-41mer RNA) for pull-down assay and subjected to western blot analysis with anti-CSDE1 and anti- GAPDH antibody. (d) Western blot analysis of pull-down assay with the baits, mouse 5’UTR or human 5’UTR RNA of GPR151 . NonO, a nuclear RNA-binding protein, one of the identified binding proteins was also immunoblotted as an internal control. (e) Western blot analysis of pull- down assay with the baits, mouse 5’UTR or ⊗CSDE1 RNA. ⊗CSDE1 RNA was the CAAGAA- CSDE1-binding motif -deleted RNA. The numbers indicated the fluorescence intensities of the corresponding immunoblot bands from the infrared dye (IR-dye) detection with Odyssey (Leica) and measured by ImageStudio software (Leica). (f) Illustration of CSDE1 immunoprecipitation and RT-qPCR analysis for (g). Mouse sciatic nerves injury was introduced by crushing and L4,5 DRGs were dissected at 24 hours after injury. Total tissues extracts were prepared and incubated with anti-CSDE1 antibody or normal rabbit IgG isotype control. The immunoprecipitants were subjected to RNA extraction and RT-qPCR to analyze the relative enrichments of Gpr151 mRNA- associated with CSDE1 from uninjured or injured DRG tissues. The RNA-seq analysis results were indicated as “Total RNA” along the IP result with injury-dependent fold change . (g) Relative enrichment of Gpr151 mRNA levels analyzed by RT-qPCR (n=3; *** p <0.001 by t-test; nd, not detected; mean±SEM). IP, immunoprecipitation; IgG, normal rabbit IgG isotype control antibody; black, uninjured, gray. injured. Vim and Fabp7 were the internal controls genes known to interact with CSDE1 , showing that they did not respond to sciatic nerve injury. (h) In vitro replating assay of control (scrambled), 5’UTR overexpressed (5’UTR), CSDE1 knocked down (CSDE1 KD), 5’UTR and CSDE1 overexpressed (5’UTR+CSDE1 OE), 5’UTR overexpressed and CSDE1 knocked down (5’UTR+CSDE1 KD) neurons. The images were representative images of raw microscopic fields. Scale bar, 100μm. (i) Average of relative neurite length of (h). (n=4 replicates; total cell numbers analyzed; n=133, 132, 133, 131, 115 for control, 5’UTR, CSDE1 KD, 5’UTR+CSDE1 KD, 5’UTR+CSDE1 OE; * p <0.05, ** p <0.01, ns, not significant by t -test; mean±SEM). (j) Western blot analysis of CSDE1 from cultured embryonic DRG neurons lysates. Lentivirus from pLKO-sh CSDE1 and/or FUGW-CSDE1-myc/DDK was transduced to the cultures at DIV2 to knock down and/or overexpress CSDE1. The numbers indicated the fluorescence intensities of the IR-dye western blot bands. Endogenous and exogenous CSDE1 was labeled and indicated as arrows.

Article Snippet: To overexpress CSDE1, mouse Csde1 ORF clone (Origene, MR210719, NM_144901, Myc-DDK-tagged) was sub-cloned into FUGW vector including its original Myc-DDK-tag using AgeI/EcoRI restriction sites.

Techniques: Staining, SDS Page, Pull Down Assay, Control, Spectrophotometry, Binding Assay, Western Blot, Cell Culture, Incubation, RNA Binding Assay, Fluorescence, Software, Immunoprecipitation, Quantitative RT-PCR, RNA Extraction, RNA Sequencing, In Vitro, Knockdown, Labeling

Journal: bioRxiv

Article Title: Promoting axon regeneration by enhancing the non-coding function of the injury-responsive coding gene Gpr151

doi: 10.1101/2021.02.19.431965

Figure Lengend Snippet: (a) Illustration of the secondary structure of 5’UTR of Gpr151 wild type (5’UTR WT ), mutant 5’UTR with single-stranded CSDE1-binding motif (5’UTRm), no CSDE1-binding motif and no stem-loop (h), single-stranded and no stem-loop (Δ) mutant. (b) Western blot analysis of pull-down assay with the baits of (a). (c and d) Western blot analysis of pull-down assay with the competitor Δ for mouse and human 5’UTR. The competitor RNA Δ was synthetic RNA identical to (a) without biotin-conjugation. NonO was immunoblotted as an internal control of pull-down analysis. (e) Representative images of in vitro replating assay result of control, 5’UTR and 5’UTRm. Scale bar, 100μm. (f) Statistical analysis of relative average axon length from (e) (n=97, 104, 98 cells for control, 5’UTR WT and 5’UTRm; * p <0.05, *** p <0.001 by ANOVA followed by Tukey tests; mean±SEM). (g) Gapdh -normalized level of GFP, 5’UTR WT and 5’UTRm by RT-qPCR analysis. GFP was expressed under CMV promoter from the second ORF in the lentiviral vector and its mRNA level was monitored as an internal control of viral transduction efficiency (n=3 replicates; ns, not significant by ANOVA followed by Tukey tests; mean±SEM). (h) Illustration of experimental time line of spot-culture, immunoprecipitation and Nanopore direct RNA sequencing. Mouse embryonic DRG neurons were cultured by spot-culture method as described. Spot-cultured neurons were infected by control or 5’UTRm lentivirus at DIV2 and lysed for CSDE1-immunoprecipitation at DIV5. RNA was extracted from the immunoprecipitants and subjected to library preparation for Nanopore direct RNA sequencing. (i) Volcano plot with the x-axis of -log 10 P -value and y-axis of CSDE1-dissociation factor. Two replicates of the sequencing results were subjected to edgeR analysis for analyzing differential level of transcripts associated with CSDE1. P -value was calculated from log 2 -fold change with two biological replicates. CSDE1-dissociation factor was defined as -log 2 [CSDE1-associated transcript level from 5’UTRm-expressing neurons / CSDE1-associated transcript level from control neurons]. Red dot indicated transcripts dissociated from CSDE1 when 5’UTRm was overexpressed (248 transcripts). Blue indicated transcripts showing enhanced association with CSDE1, reversely (282 transcripts). See also Supplementary Data 3. (j) The red and the blue transcripts from (i) was re-plotted with the x-axis of log 2 FC of RiboTag -seq result and the y-axis of log 2 FC of bulk-seq result. Green box indicated the transcripts with both values less than 1, 76.1% of total transcripts (248+282).

Article Snippet: To overexpress CSDE1, mouse Csde1 ORF clone (Origene, MR210719, NM_144901, Myc-DDK-tagged) was sub-cloned into FUGW vector including its original Myc-DDK-tag using AgeI/EcoRI restriction sites.

Techniques: Mutagenesis, Binding Assay, Western Blot, Pull Down Assay, Conjugation Assay, Control, In Vitro, Quantitative RT-PCR, Plasmid Preparation, Transduction, Immunoprecipitation, RNA Sequencing, Cell Culture, Infection, Sequencing, Expressing

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of FGFR1 and FGFR4 on lung squamous carcinoma cell lines. See also Supplementary Figure S1. Growth curves in 10% FBS (a) and soft agar assays (b) of FGFR4-overexpressing lung squamous carcinoma cell lines. (c) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR4-overexpressing lung squamous carcinoma cell lines compared to empty-vector-expressing cell lines after stimulation with FBS. Growth curves in 0.5% FBS (d) and soft agar assays (e) of FGFR1- and FGFR4-silenced H520 cells (lung squamous cell carcinoma). (f) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR1- and FGFR4-silenced H520 cells. All experiments were reproduced a minimum of three times in the laboratory, and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. Silencing of either gene was performed using two different shRNAs, referred to as “a” and “b”. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). ADC = Adenocarcinoma, SCC = Squamous cell carcinoma, I = Immortalised, KRAS = KRAS-mutated, EGFR = EGFR-mutated, ALK = ALK translocation bearer, TN = “Triple negative” (referring to the absence of alterations in KRAS, EGFR and ALK), EV = empty vector control, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, scramble = scrambled shRNA control, shFGFR1 = FGFR1 shRNA, shFGFR4 = FGFR4 shRNA, FBS = foetal bovine serum. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Activation Assay, Plasmid Preparation, Expressing, Control, Standard Deviation, MANN-WHITNEY, Translocation Assay, shRNA, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of FGFR1 and FGFR4 on non-squamous lung cell lines. See also Supplementary Figure S2. Growth curves in 10% FBS (a) and soft agar assays (b) of the FGFR1- or FGFR4-overexpressing lung adenocarcinoma cell lines H2009 and H3122. (c) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR1-overexpressing H2009 and H3122 cells compared to empty-vector-expressing cells. Growth curves in 10% FBS (d) and soft agar assays (e) of FGFR1- and FGFR4-silenced A549 cells (adenocarcinoma cell line). (f) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR1- and FGFR4-silenced A549 cells. All experiments were reproduced a minimum of three times in the laboratory, and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. For western blots, cells were serum-starved for five hours prior to protein extraction. For the serum-stimulated conditions, serum-starved cells were incubated in serum-containing complete medium for 15 min before protein extraction. Silencing of either gene was performed using two different shRNAs, referred to as “a” and “b”. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). EV = empty vector control, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, scramble = scrambled shRNA control, shFGFR1 = FGFR1 shRNA, shFGFR4 = FGFR4 shRNA, FBS = foetal bovine serum. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Activation Assay, Plasmid Preparation, Expressing, Control, Standard Deviation, Protein Extraction, Incubation, MANN-WHITNEY, shRNA, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of N-cadherin on the pro-oncogenic role of FGFR1 and FGFR4. See also Supplementary Figures S3 and S4. (a) Western blots of N-cadherin and E-cadherin protein expression in our lung cell line panel. To assess the expression of these proteins in the 18 cell lines, different blots were performed in parallel with an internal reference sample and the assembled images are shown. 10% FBS growth curves (b) and soft agar assays (c) of H2009 and H3122 cells overexpressing N-cadherin and either FGFR1 or FGFR4. (d) Western blot analysis of the activation of FGFR-related signalling pathways in these cell lines. All experiments were reproduced a minimum of three times in the laboratory and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001) non-SCC = non-Squamous, SCC = Squamous cell carcinoma, I = Immortalised, KRAS = KRAS-mutated, EGFR = EGFR-mutated, ALK = ALK translocation bearer, TN = “Triple negative” (referring to the absence of alterations in KRAS, EGFR and ALK), EV1 = empty vector 1, EV2 = empty vector 2, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, CDH2 = N -cadherin-overexpressing. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Expressing, Activation Assay, Plasmid Preparation, Control, Standard Deviation, MANN-WHITNEY, Translocation Assay, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of N-cadherin on the pro-oncogenic role of FGFR1 and FGFR4 and the interaction of N-cadherin with FGFR1 and FGFR4. See also Supplementary Figure S4. (a) 0.5% FBS growth curves for FGFR1-overexpressing and N-cadherin-silenced (left) or FGFR4-overexpressing and N-cadherin-silenced (right) NL20 cells. (b) Soft agar assays of FGFR-overexpressing and N-cadherin-silenced NL20 cells. (c) Western blot analysis of the activation of FGFR-related signalling pathways in these cell lines. (d) Xenograft tumour volumes of the FGFR1, FGFR4 and N-cadherin interaction models in the immortalised NL20 cell line. (e) Proximity ligation assays (PLA) to assess the physical interaction of N-cadherin with FGFR1 (upper panel) or FGFR4 (lower panel). The interactions detected were quantified and normalised by cell number for each condition. As controls to distinguish signal from noise, interactions were quantified in the single antibody conditions (labelled as “N-cadherin”, “FGFR1” and “FGFR4”). To assess the interaction between the two proteins, both antibodies were used, either N-cadherin + FGFR1 (upper panel) or N-cadherin + FGFR4 (lower panel), in the condition labelled as “combo”. Representative images and quantifications are shown. (f) Co-immunoprecipitation of N-cadherin with FGFR1 and with FGFR4 in the H520 cell line. (g) Kaplan-Meier curves of overall survival (OS) for the entire NSCLC patient cohort ( N = 109). Patients were grouped based on FGFR1 and N-cadherin expression levels or on FGFR4 and N-cadherin expression levels. (h) OS curve of patients in the cohort with high expression of FGFR1 and/or FGFR4 stratified by N-cadherin expression levels. In each analysis, for the FGFR1 and N-cadherin genes, the cut-off point was the median mRNA expression value for that variable. For FGFR4, the cut-off point was the first-quartile mRNA expression value in the TCGA adenocarcinoma cohort. The Kaplan-Meier method was used for survival analyses of the clinical data and cell line xenograft experiments, with a Cox proportional hazards model used to adjust for explanatory variables. A log Rank analysis was used to analyse differences in survival between groups. To obtain the hazard ratio values, the Cox proportional hazards model was used. All in vitro experiments were reproduced a minimum of three times in the laboratory, and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. N-cadherin silencing was performed using two different shRNAs. Results generated with the alternative shRNA are shown in Supplementary Figure S3 c-e. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). EV1 = empty vector 1, EV2 = empty vector 2, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, CDH2 = N -cadherin-overexpressing, scramble = scrambled shRNA control, shCDH2 = silenced with N-cadherin shRNA. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Activation Assay, Ligation, Immunoprecipitation, Expressing, In Vitro, Plasmid Preparation, Control, Standard Deviation, Generated, shRNA, MANN-WHITNEY, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: RNAseq analysis of TCGA lung adenocarcinoma and squamous cell carcinoma datasets. (a) Differential gene expression analysis in FGFR1/4high-CDH2high ( n = 145) versus FGFR1/4high-CDH2low ( n = 94) patients. The gene dataset was filtered by discarding genes whose expression was dependant on CDH2 high/low status irrespective of FGFR1 and/or FGFR4 expression (FGFR1/4low-CDH2high patients, n = 21). Parameters were set up as logFC>1, B >0. (b) Query of defined gene expression signature against Gene Ontology. The results shown here are based in whole or in part upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/ .

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Gene Expression, Expressing, Generated

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Predictive potential of N-cadherin expression for anti-FGFR therapy in vitro . See also Supplementary Figure S5. Treatment for 72 h with AZD4547 or BGJ398 at a concentration of 0.5 or 1 µM was applied to cells with high endogenous expression of FGFR1 and/or FGFR4, with high or low endogenous expression of N-cadherin (a) and to cells either exogenously expressing FGFR1 or FGFR4, alone or in combination with N-cadherin, or to cells with high endogenous expression of the three genes with N-cadherin downregulation (b). All experiments were reproduced a minimum of three times in the laboratory. For growth curves, a representative figure is shown and the mean and standard deviation for the technical replicates are indicated. N-cadherin silencing was performed using two different shRNAs to avoid off-target effects. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). EV1 = empty vector 1, EV2 = empty vector 2, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, CDH2 = N -cadherin-overexpressing, scramble = scrambled shRNA control, shCDH2 = silenced with N-cadherin shRNA.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Expressing, In Vitro, Concentration Assay, Standard Deviation, MANN-WHITNEY, Plasmid Preparation, shRNA, Control

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: FGFR efficacy in N-cadherin, FGFR1 and/or FGFR4 co-expressing patient-derived xenografts (PDXs). See also Supplementary Figure S6. (a) Western blot showing FGFR1, FGFR4 and N-cadherin protein expression in five different lung PDXs. AZD4547 treatment of low (b) and high (c) N-cadherin-expressing PDXs. (d) Results of the PDX treatments in terms of tumour versus control volume (T/C), and complete regressions. T/C values are expressed as percentages. (e) Graph showing the median variation in tumour volume from the initial volume, for every model, calculated as the increase or decrease in volume and expressed as a percentage. (f) Western blot showing the effects of AZD4547 treatment on FGFR-related signalling pathways in one low-N-cadherin-expressing (TP13) and one high-N-cadherin-expressing (TP114) adenocarcinoma PDX. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Expressing, Derivative Assay, Western Blot, Control, MANN-WHITNEY, Molecular Weight

Journal: Cardiovascular research

Article Title: ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

doi: 10.1093/cvr/cvae026

Figure Lengend Snippet: Figure 3 Glycolytic control enzymes are targets of ISGylation. (A) Schematic representation of glycolysis depicting identified ISGylated enzymes and modi- fication sites within CVB3-infected mouse hearts. (B + C) Validation of ISG15–modification of hexokinase-2 (HK2) and phosphofructokinase (PFK). HeLa cells were transfected with a four-plasmid combination (HA-ISG15, Ube1L, Ube2L6, Herc5) and FLAG-tagged HK2 (B) or PFK (C). FLAG-immunoprecipitation was performed prior to Western blot analysis. Arrows point toward enriched target and modification sites, as indicated. (D + E) R mutants of HK2 ISGylation site K419 (D) and PFK ISGylation sites K372/K727 (E) were generated. Transfection and immunoprecipitation were performed as described in (B + C). ISGylation patterns of HK2 K419R (D) and PFK K372R/K727R (E) were compared by Western blotting. Targets and modification bands are indicated by arrows and brackets.

Article Snippet: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 1 List of plasmid sources for ISGylation targets Gene Protein name NCBI ref seq Vector Tag Source Aldoa Aldolase A (ALDO) NM_007438.4 pCMV3 C-terminal FLAG Sinobiological, Beijing, China Gapdh Glyceraldehyde–3–phosphate dehydrogenase (GAPDH) NM_008084.1 pCMV6 C-terminal FLAG OriGene, Rockville, MD, USA Hk2 Hexokinase 2 (HK2) NM_013820.3 pCMV6 C-terminal FLAG OriGene, Rockville, MD, USA Ldha Lactate dehydrogenase A (LDH) NM_010699.2 pCMV3 C-terminal FLAG Sinobiological, Beijing, China Pfkm Phosphofructokinase muscle form (PFK1) NM_001163487.1 pCMV3 C-terminal FLAG Sinobiological, Beijing, China .

Techniques: Control, Infection, Biomarker Discovery, Modification, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Generated

Journal: Cardiovascular research

Article Title: ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

doi: 10.1093/cvr/cvae026

Figure Lengend Snippet: Figure 4 Impact of ISG15/ISGylation on HK2 and PFK1 activity. (A–C) ISG15-deficient HeLa cells were transfected with a four-plasmid combination (HA–ISG15 or GFP, together with Ube1L, Ube2L6, and Herc5) and FLAG-tagged HK2 (B) or PFK1 (C) or their respective K to R site mutants. HK2 and PFK1 were enriched by FLAG-immunoprecipitation prior to enzyme activity measurement of HK2 (n = 4) and PFK1 (n = 3). Measurements [mU/µg] were normalized to baseline activity. Statistical comparisons were achieved by one-tailed and two-tailed t-tests. (D–E) Lysine 419 is located close to the substrate (Glc) binding site in HK2 (surface representation), as revealed by an already determined enzyme structure (PDB ID 5hg129. Consequently, ISG15 (cartoon representation, PDB 1z2m30 bound to K419 with its C-terminal domain would cover this substrate binding site. The provided visualization is not a complex model between HK2 and ISG15, rather a putative orientation is implied. Computational docking of ISG15 to K419 is not feasible using known template structures, since HK2 is always in a substrate bound conformation, while ISG15 would likely bind to an unbound (apo-) HK2 conformation, which can be drastically different. Such structural template is not available; therefore, this scheme is an approximation. However, specific ISG15-bound protease structures (PDB: 5w8u31. (F) shows that ISG15 can be bound into a cleft-like structure arrangement of the target protein, as would be the case supposed here for the HK2-ISG15 assembling at K419. (G) A similar observation can be made at a structural PFK1 model (surface representation, two subunits (orange, beige)) with bound ligands (e.g. Frc6P) where several lysine residues are located close to the ligand-binding sites (red). ISG15 fused with the C-terminal domain to one of these lysine residues would hamper substrate binding, whereby two ISG15 molecules should be bound into cleft-like structural arrangements in a spatial fit-in manner. The visualization again does not show a computational fully fused and modelled ISG15-PFK1 complex, but is a by-hand oriented approximation due to a missing enzyme template structure in a non-substrated state. Therefore, the structural PFK1 conformation accessible for ISG15 is unknown (as for HK2) and cannot be simulated without further information. (H) The lysine residues K372 and K727 identified in this study as ISGylation sites were experimentally excluded to have a functional impact on PFK1 activity and are indeed more distantly located to the substrate binding regions. Note: The visualized PFK1 protein model is derived from a phosphofructokinase structure of Staphylococcus aureus (PDB 5xz832. The sequence of the protein was substituted by mouse PFK1 amino acid sequence for homology (sequence similarity ∼83%, BLOSUM 62 matrix). Model representa- tions were created using the PyMol Molecular Graphics System Version 1.3 (Schrödinger, LLC, New York, NY).

Article Snippet: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 1 List of plasmid sources for ISGylation targets Gene Protein name NCBI ref seq Vector Tag Source Aldoa Aldolase A (ALDO) NM_007438.4 pCMV3 C-terminal FLAG Sinobiological, Beijing, China Gapdh Glyceraldehyde–3–phosphate dehydrogenase (GAPDH) NM_008084.1 pCMV6 C-terminal FLAG OriGene, Rockville, MD, USA Hk2 Hexokinase 2 (HK2) NM_013820.3 pCMV6 C-terminal FLAG OriGene, Rockville, MD, USA Ldha Lactate dehydrogenase A (LDH) NM_010699.2 pCMV3 C-terminal FLAG Sinobiological, Beijing, China Pfkm Phosphofructokinase muscle form (PFK1) NM_001163487.1 pCMV3 C-terminal FLAG Sinobiological, Beijing, China .

Techniques: Activity Assay, Transfection, Plasmid Preparation, Immunoprecipitation, One-tailed Test, Two Tailed Test, Binding Assay, Ligand Binding Assay, Functional Assay, Derivative Assay, Sequencing

Journal: Journal of Cell Science

Article Title: The Rho family GEF FARP2 is activated by aPKCι to control tight junction formation and polarity

doi: 10.1242/jcs.223743

Figure Lengend Snippet: FARP2 is a RIPR-dependent substrate of aPKCɩ that is responsible for maintaining tight junctions and polarity. (A,B) FARP1 and FARP2 co-precipitate with aPKC. HCT116 cells were co-transfected with plasmids expressing FLAG-tagged FARP1 (A) or FARP2 (B) and GFP, GFP-tagged aPKCι or GFP-tagged aPKCι containing a RIPR to AIPA mutation (R480A/R483A). Immunoprecipitates were analysed with the indicated antibodies. Images are of representative blots of n =3. (C) GFP–PKCι phosphorylates FARP1 and FARP2 in cells. HCT116 cells were co-transfected with plasmids expressing FLAG-tagged FARP1 or FARP2, and GFP or GFP-tagged aPKCι. Immunoprecipitates (IP) were analysed via ProQ diamond staining or with the indicated antibodies. (D) FARP2 and not FARP1 is involved in junctional establishment after Ca 2+ switch. Caco-2 cells were subjected to siRNA treatment (p represents the use of ON-TARGETplus SMARTpool siRNA, Dharmacon), processed for Ca 2+ switch immunofluorescence and stained for the junctional marker ZO-1. A representative example of n =3 experiments with six coverslips per immunofluorescence experiment is shown. (E) FARP1 depletion has no effect on junctional permeability as indicated by a Ca 2+ switch TER assay. A representative example of n =3 experiments is shown. (F) FARP2 depletion has a substantial effect on junctional permeability as indicated by a Ca 2+ switch transepithelial assay. A representative example of n =3 experiments with six samples per experiment is shown. (G) 3D lumen formation in a CaCo2 model is disturbed upon knockdown of either FARP2, Cdc42 or PKCι. CaCo2 cells were grown on a Matrigel-coated surface as described in the Materials and Methods. Cysts were stained for ZO-1 (green), F-actin (red) as indicated and Hoechst 33342 (stained according to manufacturer's instructions; Sigma-Aldrich) (blue). (H) Quantification of the proportion of single lumen cysts for experiments as in G. n ≥100 cysts were counted per experiment. Results are mean±s.d. ns, not significant ( P >0.05); *** P ≤0.001; **** P ≤0.0001 (unpaired t -test). siCtrl, control siRNA. Scale bars: 20 μm.

Article Snippet: C-terminal FLAG–Myc-tagged FARP1 (RC208329) and FARP2 (RC216784) human cDNA constructs were purchased from OriGene.

Techniques: Transfection, Expressing, Mutagenesis, Staining, Immunofluorescence, Marker, Permeability, Knockdown, Control

Journal: Journal of Cell Science

Article Title: The Rho family GEF FARP2 is activated by aPKCι to control tight junction formation and polarity

doi: 10.1242/jcs.223743

Figure Lengend Snippet: FARP2 is required for efficient initiation of junction formation. (A) Individual siRNA oligonucleotides directed at FARP2 cause severe disruption of ZO-1 during junction establishment (see also Fig. S2). A representative example or n =3 with six samples per experiment is shown. (B) FARP2, aPKC and Cdc42 siRNA deconvolution in a Ca 2+ switch assay. The TER is severely disrupted, indicative of loss of junctional integrity. A representative example of n =3 experiments with five samples per experiment is shown. (C) De novo junction formation in EGF-stimulated A431 cells. Pooled siRNA (denoted by p, siGenome Pools) directed at FARP2, aPKC or Cdc42 results in junctional impairment, indicated by the loss of integrity of ZO-1. A representative example or n =3 with six samples per experiment is shown. Results in B are mean±s.d. **** P ≤0.0001 (unpaired t -test). siCtrl, control siRNA. Scale bars: 20 μm.

Article Snippet: C-terminal FLAG–Myc-tagged FARP1 (RC208329) and FARP2 (RC216784) human cDNA constructs were purchased from OriGene.

Techniques: Disruption, Control

Journal: Journal of Cell Science

Article Title: The Rho family GEF FARP2 is activated by aPKCι to control tight junction formation and polarity

doi: 10.1242/jcs.223743

Figure Lengend Snippet: Molecular function of FARP2 and the effect of aPKCι-mediated phosphorylation. (A) G-LISA assay assessing the levels of active Cdc42 in CaCo2 cells transfected with either control siRNA (siCtrl) or siRNA targeting FARP2; n =3. (B) FARP2 depletion impairs localisation of Cdc42-GTP and ZO1 at cell–cell junctions. A representative example of n =2 experiments with five samples per experiment. (C) FARP2 is phosphorylated by aPKCι. FARP2 WT or mutants were expressed in HCT116 cells with or without aPKCι and immunoprecipitated (IP). Phosphorylation at S340 and S370 was assessed using antibodies that recognise the sequence context of each site. The use of aPKCι-specific inhibitor CRT0066854 (10 µM, 60 min) confirmed aPKCι-mediated phosphorylation. Representative blots of n =2 experiments are shown. (D) Active aPKCι phosphorylates FARP2 without requiring its regulatory region. HCT116 cells were co-transfected with WT FARP2 or mutants as indicated with or without aPKCι or its kinase domain (K.Dom.). FARP2 was immunoprecipitated, and phosphorylation at S340 and S370 was assessed as in C. Representative blots of n =2 experiments are shown. (E,F) Mutation of the S340 and S370 phosphorylation sites in FARP2 prevents siRNA-resistant FARP2 from rescuing the altered ZO-1 localisation phenotype observed upon FARP2 depletion. The location of ZO-1 is indicated (left panels; red in merge) alongside the GFP expression (right panels; green in merge). A representative example of n =3 experiments with six samples per experiment is shown. A quantitative analysis is shown in the histogram (F) as indicated for the different rescue constructs. (G) Levels of active Cdc42 during a Ca 2+ switch. The effects of FARP2 or aPKCι knockdown at 8 h post Ca 2+ re-addition result in severe depletion of Cdc42-GTP. A representative example of n =3 experiments with six samples per experiment is shown. (H) Levels of active Cdc42 are rescued by both the WT and mutant constructs. A representative example of n =2 experiments with eight samples per experiment is shown. Results are mean±s.d. * P ≤0.05; ** P ≤0.01; *** P ≤0.001; **** P ≤0.0001 (unpaired t -test). Scale bars: 20 μm.

Article Snippet: C-terminal FLAG–Myc-tagged FARP1 (RC208329) and FARP2 (RC216784) human cDNA constructs were purchased from OriGene.

Techniques: Phospho-proteomics, Transfection, Control, Immunoprecipitation, Sequencing, Mutagenesis, Expressing, Construct, Knockdown

Journal: Journal of Cell Science

Article Title: The Rho family GEF FARP2 is activated by aPKCι to control tight junction formation and polarity

doi: 10.1242/jcs.223743

Figure Lengend Snippet: Hypothetical model of a feedback activation mechanism for aPKC during junction establishment and maintenance. FARP2 associates with aPKC via a kinase domain RIPR-motif–FARP2FERM-FA interaction. Phosphorylation of FARP2 at S340 and S370 (red circles; dashed arrow) in the FA domain results in dissociation of the complex and promotes localised function of FARP2 at the junctions (indicated by the curly bracket), where it activates Cdc42. Activated Cdc42 (ovals with red to green transition; Ccd42 as a possible partner in membrane-associated aPKC–Par6 complexes is depicted in a faded shade) can activate downstream effectors such as aPKC containing complexes. Maintained aPKC activity results in continuous FARP2 phosphorylation, resulting in a positive-feedback cycle necessary to initiate and maintain junctions. FARP2 is also active independently of phosphorylation as depicted in the model. PPtase, phosphatase mediating dephosphorylation of FARP2.

Article Snippet: C-terminal FLAG–Myc-tagged FARP1 (RC208329) and FARP2 (RC216784) human cDNA constructs were purchased from OriGene.

Techniques: Activation Assay, Phospho-proteomics, Membrane, Activity Assay, De-Phosphorylation Assay