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    Boster Bio millipore ifitm1 rabbit
    Figure 4. Interferon-inducible transmembrane protein 1 <t>(IFITM1)</t> is among the most regulated genes on S100A6 knockdown and a key regulator of S100A6 cell signaling. A, Reverse transcription polymerase chain reaction analysis for IFITM1 from RNA samples used in the illumina beadarrays was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when repli- cates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for IFITM1 (n=6 individual; *P<0.05 vs corresponding control siRNA-treated cells, Friedman test with subsequent Dunn correction for multiple comparisons). C, Left, S100A6, S100A6/IFITM1 knockdown, and control HUVECs were stimulated with VEGF-A for 24 h, and EdU incorporation was measured as EdU- positive cells vs the total number of cells (ie, nuclear DAPI-stained cells; n=6 individual experiments; *P<0.05 vs corresponding control siRNA-treated cells and #P=0.062 vs S100A6 siRNA-treated cells with 24 h of VEGF-A stimulation, Friedman test for repeated measures, Wilcoxon signed-rank test with subsequent correction for individual time-point comparisons). Right, Representative immunofluorescence images of serum/growth factor starved and VEGF-A–stimulated control, S100A6, and S100A6/IFITM1 knockdown HUVECs after EdU detection (×20 magnification; scale bar, 40 µm; green=EdU and blue=nuclear DAPI).
    Millipore Ifitm1 Rabbit, supplied by Boster Bio, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pa1112/Anti-IFITM1+Antibody+Picoband/10__1161_slash_atvbaha__115__306415-286-49-55
    Average 92 stars, based on 3 article reviews
    millipore ifitm1 rabbit - by Bioz Stars, 2026-09
    92/100 stars

    Images

    1) Product Images from "S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling"

    Article Title: S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    doi: 10.1161/atvbaha.115.306415

    Figure 4. Interferon-inducible transmembrane protein 1 (IFITM1) is among the most regulated genes on S100A6 knockdown and a key regulator of S100A6 cell signaling. A, Reverse transcription polymerase chain reaction analysis for IFITM1 from RNA samples used in the illumina beadarrays was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when repli- cates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for IFITM1 (n=6 individual; *P<0.05 vs corresponding control siRNA-treated cells, Friedman test with subsequent Dunn correction for multiple comparisons). C, Left, S100A6, S100A6/IFITM1 knockdown, and control HUVECs were stimulated with VEGF-A for 24 h, and EdU incorporation was measured as EdU- positive cells vs the total number of cells (ie, nuclear DAPI-stained cells; n=6 individual experiments; *P<0.05 vs corresponding control siRNA-treated cells and #P=0.062 vs S100A6 siRNA-treated cells with 24 h of VEGF-A stimulation, Friedman test for repeated measures, Wilcoxon signed-rank test with subsequent correction for individual time-point comparisons). Right, Representative immunofluorescence images of serum/growth factor starved and VEGF-A–stimulated control, S100A6, and S100A6/IFITM1 knockdown HUVECs after EdU detection (×20 magnification; scale bar, 40 µm; green=EdU and blue=nuclear DAPI).
    Figure Legend Snippet: Figure 4. Interferon-inducible transmembrane protein 1 (IFITM1) is among the most regulated genes on S100A6 knockdown and a key regulator of S100A6 cell signaling. A, Reverse transcription polymerase chain reaction analysis for IFITM1 from RNA samples used in the illumina beadarrays was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when repli- cates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for IFITM1 (n=6 individual; *P<0.05 vs corresponding control siRNA-treated cells, Friedman test with subsequent Dunn correction for multiple comparisons). C, Left, S100A6, S100A6/IFITM1 knockdown, and control HUVECs were stimulated with VEGF-A for 24 h, and EdU incorporation was measured as EdU- positive cells vs the total number of cells (ie, nuclear DAPI-stained cells; n=6 individual experiments; *P<0.05 vs corresponding control siRNA-treated cells and #P=0.062 vs S100A6 siRNA-treated cells with 24 h of VEGF-A stimulation, Friedman test for repeated measures, Wilcoxon signed-rank test with subsequent correction for individual time-point comparisons). Right, Representative immunofluorescence images of serum/growth factor starved and VEGF-A–stimulated control, S100A6, and S100A6/IFITM1 knockdown HUVECs after EdU detection (×20 magnification; scale bar, 40 µm; green=EdU and blue=nuclear DAPI).

    Techniques Used: Knockdown, Reverse Transcription, Polymerase Chain Reaction, Control, Staining, Immunofluorescence

    Figure 5. Elevated interferon-inducible transmembrane protein 1 (IFITM1) expression and antiproliferative phenotype in S100A6-depleted endothelial cells is caused by signal transducers and activators of transcription 1 (STAT1) activation. A, Reverse transcription polymerase chain reaction analysis for STAT1 from RNA samples used in the gene array was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when replicates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for STAT1 and Tyr-701–phosphorylated STAT1 (n=6 individual experiments; *P<0.05 vs corresponding control siRNA- treated cells, Friedman test and subsequent Dunn correction for multiple comparisons). C, Left, Cell lysates from S100A6, S100A6/STAT1 knockdown, and control HUVECs were collected after over night starvation and after VEGF-A stimulation and were immunoblotted for Ki67 (n=6 individual experiments) and IFITM1 (n=4 individual experiments for 0-h time point and n=6 individual experiments for 24-h time point; #P<0.05 vs corresponding control siRNA-treated cells, *P≤0.05 vs S100A6 siRNA-treated cells at 24 h, Friedman–Dunn test). Right, Representative cell culture images from S100A6, S100A6/STAT1 knockdown, and control HUVECs 24 h after VEGF-A stimulation (×4 magnification) and representative IF images from the corresponding experiment’s EdU incorporation (×20 magnification; scale bar, 50 µm; red=EdU and blue=nuclear DAPI).
    Figure Legend Snippet: Figure 5. Elevated interferon-inducible transmembrane protein 1 (IFITM1) expression and antiproliferative phenotype in S100A6-depleted endothelial cells is caused by signal transducers and activators of transcription 1 (STAT1) activation. A, Reverse transcription polymerase chain reaction analysis for STAT1 from RNA samples used in the gene array was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when replicates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for STAT1 and Tyr-701–phosphorylated STAT1 (n=6 individual experiments; *P<0.05 vs corresponding control siRNA- treated cells, Friedman test and subsequent Dunn correction for multiple comparisons). C, Left, Cell lysates from S100A6, S100A6/STAT1 knockdown, and control HUVECs were collected after over night starvation and after VEGF-A stimulation and were immunoblotted for Ki67 (n=6 individual experiments) and IFITM1 (n=4 individual experiments for 0-h time point and n=6 individual experiments for 24-h time point; #P<0.05 vs corresponding control siRNA-treated cells, *P≤0.05 vs S100A6 siRNA-treated cells at 24 h, Friedman–Dunn test). Right, Representative cell culture images from S100A6, S100A6/STAT1 knockdown, and control HUVECs 24 h after VEGF-A stimulation (×4 magnification) and representative IF images from the corresponding experiment’s EdU incorporation (×20 magnification; scale bar, 50 µm; red=EdU and blue=nuclear DAPI).

    Techniques Used: Expressing, Activation Assay, Reverse Transcription, Polymerase Chain Reaction, Control, Knockdown, Cell Culture

    Figure 7. Novel signaling module linking S100A6 to control of signal transducers and activators of transcription 1 (STAT1) signaling. A, Cell lysates from S100A6 adenovirus (FLAG tagged) and control GFP adenovirus-infected human umbilical vein endothelial cells (HUVECs; 48 h) were collected and immunoblotted for interferon-inducible transmembrane protein 1 (IFITM1), protein inhibitor of acti- vated STAT 1 (PIAS1), STAT1, S100A6, and FLAG (n=3 individual experiments). B, The schematic highlights the molecular control of antiproliferative STAT1 signaling in endothelial cells (ECs) via S100A6. Our findings indicate that S100A6 might constitutively facilitate the activity of the STAT1 suppressor PIAS (1). Loss of S100A6 expression subsequently entails STAT1 activation via loss of PIAS activity inde- pendent of Janus kinases 1 and 2 (JAK1/2), SH2-containing phosphatase (SHP), and suppressors of cytokine signaling (SOCS) signaling (2). STAT1 in turn activates a set of antiproliferative genes, including IFITM1 besides others (3) that oppose cell cycle entry in part most likely via previously described p53/p21 activation. Of note, vascular endothelial growth factor A (VEGF-A)–mediated proliferative signaling pathways, such as extracellular signal–regulated kinase (ERK1/2) and AKT, are not affected by antiproliferative STAT1 signaling, indicating downstream competitive integration of pro- and antiproliferative signals for cell cycle decision entry. Colored elements highlight in silico predicted and experimentally validated components of the novel S100A6/STAT1/IFITM1 signaling module. Grey elements are not affected by S100A6 signaling as predicted by in silico modeling and experimental validation. In summary, S100A6 might be indispensable for timely cell cycle entry by attenuating antiproliferative STAT1 signaling.
    Figure Legend Snippet: Figure 7. Novel signaling module linking S100A6 to control of signal transducers and activators of transcription 1 (STAT1) signaling. A, Cell lysates from S100A6 adenovirus (FLAG tagged) and control GFP adenovirus-infected human umbilical vein endothelial cells (HUVECs; 48 h) were collected and immunoblotted for interferon-inducible transmembrane protein 1 (IFITM1), protein inhibitor of acti- vated STAT 1 (PIAS1), STAT1, S100A6, and FLAG (n=3 individual experiments). B, The schematic highlights the molecular control of antiproliferative STAT1 signaling in endothelial cells (ECs) via S100A6. Our findings indicate that S100A6 might constitutively facilitate the activity of the STAT1 suppressor PIAS (1). Loss of S100A6 expression subsequently entails STAT1 activation via loss of PIAS activity inde- pendent of Janus kinases 1 and 2 (JAK1/2), SH2-containing phosphatase (SHP), and suppressors of cytokine signaling (SOCS) signaling (2). STAT1 in turn activates a set of antiproliferative genes, including IFITM1 besides others (3) that oppose cell cycle entry in part most likely via previously described p53/p21 activation. Of note, vascular endothelial growth factor A (VEGF-A)–mediated proliferative signaling pathways, such as extracellular signal–regulated kinase (ERK1/2) and AKT, are not affected by antiproliferative STAT1 signaling, indicating downstream competitive integration of pro- and antiproliferative signals for cell cycle decision entry. Colored elements highlight in silico predicted and experimentally validated components of the novel S100A6/STAT1/IFITM1 signaling module. Grey elements are not affected by S100A6 signaling as predicted by in silico modeling and experimental validation. In summary, S100A6 might be indispensable for timely cell cycle entry by attenuating antiproliferative STAT1 signaling.

    Techniques Used: Control, Infection, Activity Assay, Expressing, Activation Assay, Protein-Protein interactions, In Silico, Biomarker Discovery

    Related Articles

    Western Blot:

    Article Title: S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling
    Article Snippet: .. Primary antibodies Antibody Donor Dilution WB/IF Manufacturer AKT mouse 1:1000 #2966, Cell Signaling pAKT rabbit 1:1000 #9271, Cell Signaling ERK rabbit 1:2000 #9102 , Cell Signaling Lerchenmüller et al. Materials and Methods pERK mouse 1:2000 #9106, Cell Signaling PECAM-1 goat 1:50 (IF) sc-1506, Santa Cruz GAPDH mouse 1:20000 MAB374, Millipore IFITM1 rabbit 1:1000/ 1:100 PA1112, Boster Bio IFITM1 rabbit 1:50 (IF) PA5-20989,Thermofisher JAK1 mouse 1:1000 ab75744, Abcam pJAK1 rabbit 1:1000 ab138005, Abcam JAK2 rabbit 1:1000 #3230, Cell Signaling pJAK2 rabbit 1:1000 #3771, Cell Signaling Ki67 mouse 1:1000/ 1:100 14-5699, eBioscience NFKB-p65 rabbit 1:1000 #8242, Cell Signaling pNFKB-p65 mouse 1:1000 #3036, Cell Signaling PIAS1 rabbit 1:500 ab32219, Abcam POLE mouse 1:1000 H00005426, Novus STAT1 rabbit 1:500/ 1:50 ab2415, Abcam pSTAT1 mouse 1:1000 ab29045, Abcam STAT2 rabbit 1:1000 #4594, Cell Signaling S100A6 rabbit 1:1000/ 1:100 ab181975, Abcam S100A6 mouse 1.50 (IF) S5049, Sigma S100A4 rabbit 1:1000 A5114, Dako S100B rabbit 1:1000 A5110, Dako Secondary antibodies Antibody Donor Dilution Manufacturer IR Dye 680 vs rabbit goat 1:20000 LI-COR IR Dye 800 vs mouse goat 1:20000 LI-COR Alexa Fluor 488 rabbit goat 1:300 Invitrogen Alexa Fluor 680 mouse goat 1:300 Invitrogen Alexa Fluor 488 mouse donkey 1:200 Invitrogen Alexa Fluor 594 goat donkey 1:200 Invitrogen Alexa Fluor 555 goat donkey 1:200 Invitrogen Alexa Fluor 647 mouse donkey 1:200 Invitrogen HRP antibody vs rabbit goat 1:2000 Santa Cruz HRP antibody vs mouse goat 1:2000 Santa Cruz Microarray data acquisition and analysis Microarray measurements were recorded for siRNA control and S100A6 knockdown HUVECs under VEGF-stimulation in a time course (time points 0h, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h for S100A6 siRNA and 0h, 1h, 4h, 8h, 12h, 24h for siRNA control). ..

    Microarray:

    Article Title: S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling
    Article Snippet: .. Primary antibodies Antibody Donor Dilution WB/IF Manufacturer AKT mouse 1:1000 #2966, Cell Signaling pAKT rabbit 1:1000 #9271, Cell Signaling ERK rabbit 1:2000 #9102 , Cell Signaling Lerchenmüller et al. Materials and Methods pERK mouse 1:2000 #9106, Cell Signaling PECAM-1 goat 1:50 (IF) sc-1506, Santa Cruz GAPDH mouse 1:20000 MAB374, Millipore IFITM1 rabbit 1:1000/ 1:100 PA1112, Boster Bio IFITM1 rabbit 1:50 (IF) PA5-20989,Thermofisher JAK1 mouse 1:1000 ab75744, Abcam pJAK1 rabbit 1:1000 ab138005, Abcam JAK2 rabbit 1:1000 #3230, Cell Signaling pJAK2 rabbit 1:1000 #3771, Cell Signaling Ki67 mouse 1:1000/ 1:100 14-5699, eBioscience NFKB-p65 rabbit 1:1000 #8242, Cell Signaling pNFKB-p65 mouse 1:1000 #3036, Cell Signaling PIAS1 rabbit 1:500 ab32219, Abcam POLE mouse 1:1000 H00005426, Novus STAT1 rabbit 1:500/ 1:50 ab2415, Abcam pSTAT1 mouse 1:1000 ab29045, Abcam STAT2 rabbit 1:1000 #4594, Cell Signaling S100A6 rabbit 1:1000/ 1:100 ab181975, Abcam S100A6 mouse 1.50 (IF) S5049, Sigma S100A4 rabbit 1:1000 A5114, Dako S100B rabbit 1:1000 A5110, Dako Secondary antibodies Antibody Donor Dilution Manufacturer IR Dye 680 vs rabbit goat 1:20000 LI-COR IR Dye 800 vs mouse goat 1:20000 LI-COR Alexa Fluor 488 rabbit goat 1:300 Invitrogen Alexa Fluor 680 mouse goat 1:300 Invitrogen Alexa Fluor 488 mouse donkey 1:200 Invitrogen Alexa Fluor 594 goat donkey 1:200 Invitrogen Alexa Fluor 555 goat donkey 1:200 Invitrogen Alexa Fluor 647 mouse donkey 1:200 Invitrogen HRP antibody vs rabbit goat 1:2000 Santa Cruz HRP antibody vs mouse goat 1:2000 Santa Cruz Microarray data acquisition and analysis Microarray measurements were recorded for siRNA control and S100A6 knockdown HUVECs under VEGF-stimulation in a time course (time points 0h, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h for S100A6 siRNA and 0h, 1h, 4h, 8h, 12h, 24h for siRNA control). ..

    Control:

    Article Title: S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling
    Article Snippet: .. Primary antibodies Antibody Donor Dilution WB/IF Manufacturer AKT mouse 1:1000 #2966, Cell Signaling pAKT rabbit 1:1000 #9271, Cell Signaling ERK rabbit 1:2000 #9102 , Cell Signaling Lerchenmüller et al. Materials and Methods pERK mouse 1:2000 #9106, Cell Signaling PECAM-1 goat 1:50 (IF) sc-1506, Santa Cruz GAPDH mouse 1:20000 MAB374, Millipore IFITM1 rabbit 1:1000/ 1:100 PA1112, Boster Bio IFITM1 rabbit 1:50 (IF) PA5-20989,Thermofisher JAK1 mouse 1:1000 ab75744, Abcam pJAK1 rabbit 1:1000 ab138005, Abcam JAK2 rabbit 1:1000 #3230, Cell Signaling pJAK2 rabbit 1:1000 #3771, Cell Signaling Ki67 mouse 1:1000/ 1:100 14-5699, eBioscience NFKB-p65 rabbit 1:1000 #8242, Cell Signaling pNFKB-p65 mouse 1:1000 #3036, Cell Signaling PIAS1 rabbit 1:500 ab32219, Abcam POLE mouse 1:1000 H00005426, Novus STAT1 rabbit 1:500/ 1:50 ab2415, Abcam pSTAT1 mouse 1:1000 ab29045, Abcam STAT2 rabbit 1:1000 #4594, Cell Signaling S100A6 rabbit 1:1000/ 1:100 ab181975, Abcam S100A6 mouse 1.50 (IF) S5049, Sigma S100A4 rabbit 1:1000 A5114, Dako S100B rabbit 1:1000 A5110, Dako Secondary antibodies Antibody Donor Dilution Manufacturer IR Dye 680 vs rabbit goat 1:20000 LI-COR IR Dye 800 vs mouse goat 1:20000 LI-COR Alexa Fluor 488 rabbit goat 1:300 Invitrogen Alexa Fluor 680 mouse goat 1:300 Invitrogen Alexa Fluor 488 mouse donkey 1:200 Invitrogen Alexa Fluor 594 goat donkey 1:200 Invitrogen Alexa Fluor 555 goat donkey 1:200 Invitrogen Alexa Fluor 647 mouse donkey 1:200 Invitrogen HRP antibody vs rabbit goat 1:2000 Santa Cruz HRP antibody vs mouse goat 1:2000 Santa Cruz Microarray data acquisition and analysis Microarray measurements were recorded for siRNA control and S100A6 knockdown HUVECs under VEGF-stimulation in a time course (time points 0h, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h for S100A6 siRNA and 0h, 1h, 4h, 8h, 12h, 24h for siRNA control). ..

    Knockdown:

    Article Title: S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling
    Article Snippet: .. Primary antibodies Antibody Donor Dilution WB/IF Manufacturer AKT mouse 1:1000 #2966, Cell Signaling pAKT rabbit 1:1000 #9271, Cell Signaling ERK rabbit 1:2000 #9102 , Cell Signaling Lerchenmüller et al. Materials and Methods pERK mouse 1:2000 #9106, Cell Signaling PECAM-1 goat 1:50 (IF) sc-1506, Santa Cruz GAPDH mouse 1:20000 MAB374, Millipore IFITM1 rabbit 1:1000/ 1:100 PA1112, Boster Bio IFITM1 rabbit 1:50 (IF) PA5-20989,Thermofisher JAK1 mouse 1:1000 ab75744, Abcam pJAK1 rabbit 1:1000 ab138005, Abcam JAK2 rabbit 1:1000 #3230, Cell Signaling pJAK2 rabbit 1:1000 #3771, Cell Signaling Ki67 mouse 1:1000/ 1:100 14-5699, eBioscience NFKB-p65 rabbit 1:1000 #8242, Cell Signaling pNFKB-p65 mouse 1:1000 #3036, Cell Signaling PIAS1 rabbit 1:500 ab32219, Abcam POLE mouse 1:1000 H00005426, Novus STAT1 rabbit 1:500/ 1:50 ab2415, Abcam pSTAT1 mouse 1:1000 ab29045, Abcam STAT2 rabbit 1:1000 #4594, Cell Signaling S100A6 rabbit 1:1000/ 1:100 ab181975, Abcam S100A6 mouse 1.50 (IF) S5049, Sigma S100A4 rabbit 1:1000 A5114, Dako S100B rabbit 1:1000 A5110, Dako Secondary antibodies Antibody Donor Dilution Manufacturer IR Dye 680 vs rabbit goat 1:20000 LI-COR IR Dye 800 vs mouse goat 1:20000 LI-COR Alexa Fluor 488 rabbit goat 1:300 Invitrogen Alexa Fluor 680 mouse goat 1:300 Invitrogen Alexa Fluor 488 mouse donkey 1:200 Invitrogen Alexa Fluor 594 goat donkey 1:200 Invitrogen Alexa Fluor 555 goat donkey 1:200 Invitrogen Alexa Fluor 647 mouse donkey 1:200 Invitrogen HRP antibody vs rabbit goat 1:2000 Santa Cruz HRP antibody vs mouse goat 1:2000 Santa Cruz Microarray data acquisition and analysis Microarray measurements were recorded for siRNA control and S100A6 knockdown HUVECs under VEGF-stimulation in a time course (time points 0h, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h for S100A6 siRNA and 0h, 1h, 4h, 8h, 12h, 24h for siRNA control). ..



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    Figure 4. Interferon-inducible transmembrane protein 1 <t>(IFITM1)</t> is among the most regulated genes on S100A6 knockdown and a key regulator of S100A6 cell signaling. A, Reverse transcription polymerase chain reaction analysis for IFITM1 from RNA samples used in the illumina beadarrays was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when repli- cates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for IFITM1 (n=6 individual; *P<0.05 vs corresponding control siRNA-treated cells, Friedman test with subsequent Dunn correction for multiple comparisons). C, Left, S100A6, S100A6/IFITM1 knockdown, and control HUVECs were stimulated with VEGF-A for 24 h, and EdU incorporation was measured as EdU- positive cells vs the total number of cells (ie, nuclear DAPI-stained cells; n=6 individual experiments; *P<0.05 vs corresponding control siRNA-treated cells and #P=0.062 vs S100A6 siRNA-treated cells with 24 h of VEGF-A stimulation, Friedman test for repeated measures, Wilcoxon signed-rank test with subsequent correction for individual time-point comparisons). Right, Representative immunofluorescence images of serum/growth factor starved and VEGF-A–stimulated control, S100A6, and S100A6/IFITM1 knockdown HUVECs after EdU detection (×20 magnification; scale bar, 40 µm; green=EdU and blue=nuclear DAPI).
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    Figure 4. Interferon-inducible transmembrane protein 1 <t>(IFITM1)</t> is among the most regulated genes on S100A6 knockdown and a key regulator of S100A6 cell signaling. A, Reverse transcription polymerase chain reaction analysis for IFITM1 from RNA samples used in the illumina beadarrays was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when repli- cates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for IFITM1 (n=6 individual; *P<0.05 vs corresponding control siRNA-treated cells, Friedman test with subsequent Dunn correction for multiple comparisons). C, Left, S100A6, S100A6/IFITM1 knockdown, and control HUVECs were stimulated with VEGF-A for 24 h, and EdU incorporation was measured as EdU- positive cells vs the total number of cells (ie, nuclear DAPI-stained cells; n=6 individual experiments; *P<0.05 vs corresponding control siRNA-treated cells and #P=0.062 vs S100A6 siRNA-treated cells with 24 h of VEGF-A stimulation, Friedman test for repeated measures, Wilcoxon signed-rank test with subsequent correction for individual time-point comparisons). Right, Representative immunofluorescence images of serum/growth factor starved and VEGF-A–stimulated control, S100A6, and S100A6/IFITM1 knockdown HUVECs after EdU detection (×20 magnification; scale bar, 40 µm; green=EdU and blue=nuclear DAPI).
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    Figure 4. Interferon-inducible transmembrane protein 1 <t>(IFITM1)</t> is among the most regulated genes on S100A6 knockdown and a key regulator of S100A6 cell signaling. A, Reverse transcription polymerase chain reaction analysis for IFITM1 from RNA samples used in the illumina beadarrays was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when repli- cates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for IFITM1 (n=6 individual; *P<0.05 vs corresponding control siRNA-treated cells, Friedman test with subsequent Dunn correction for multiple comparisons). C, Left, S100A6, S100A6/IFITM1 knockdown, and control HUVECs were stimulated with VEGF-A for 24 h, and EdU incorporation was measured as EdU- positive cells vs the total number of cells (ie, nuclear DAPI-stained cells; n=6 individual experiments; *P<0.05 vs corresponding control siRNA-treated cells and #P=0.062 vs S100A6 siRNA-treated cells with 24 h of VEGF-A stimulation, Friedman test for repeated measures, Wilcoxon signed-rank test with subsequent correction for individual time-point comparisons). Right, Representative immunofluorescence images of serum/growth factor starved and VEGF-A–stimulated control, S100A6, and S100A6/IFITM1 knockdown HUVECs after EdU detection (×20 magnification; scale bar, 40 µm; green=EdU and blue=nuclear DAPI).
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    Figure 4. Interferon-inducible transmembrane protein 1 <t>(IFITM1)</t> is among the most regulated genes on S100A6 knockdown and a key regulator of S100A6 cell signaling. A, Reverse transcription polymerase chain reaction analysis for IFITM1 from RNA samples used in the illumina beadarrays was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when repli- cates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for IFITM1 (n=6 individual; *P<0.05 vs corresponding control siRNA-treated cells, Friedman test with subsequent Dunn correction for multiple comparisons). C, Left, S100A6, S100A6/IFITM1 knockdown, and control HUVECs were stimulated with VEGF-A for 24 h, and EdU incorporation was measured as EdU- positive cells vs the total number of cells (ie, nuclear DAPI-stained cells; n=6 individual experiments; *P<0.05 vs corresponding control siRNA-treated cells and #P=0.062 vs S100A6 siRNA-treated cells with 24 h of VEGF-A stimulation, Friedman test for repeated measures, Wilcoxon signed-rank test with subsequent correction for individual time-point comparisons). Right, Representative immunofluorescence images of serum/growth factor starved and VEGF-A–stimulated control, S100A6, and S100A6/IFITM1 knockdown HUVECs after EdU detection (×20 magnification; scale bar, 40 µm; green=EdU and blue=nuclear DAPI).
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    Figure 4. Interferon-inducible transmembrane protein 1 (IFITM1) is among the most regulated genes on S100A6 knockdown and a key regulator of S100A6 cell signaling. A, Reverse transcription polymerase chain reaction analysis for IFITM1 from RNA samples used in the illumina beadarrays was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when repli- cates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for IFITM1 (n=6 individual; *P<0.05 vs corresponding control siRNA-treated cells, Friedman test with subsequent Dunn correction for multiple comparisons). C, Left, S100A6, S100A6/IFITM1 knockdown, and control HUVECs were stimulated with VEGF-A for 24 h, and EdU incorporation was measured as EdU- positive cells vs the total number of cells (ie, nuclear DAPI-stained cells; n=6 individual experiments; *P<0.05 vs corresponding control siRNA-treated cells and #P=0.062 vs S100A6 siRNA-treated cells with 24 h of VEGF-A stimulation, Friedman test for repeated measures, Wilcoxon signed-rank test with subsequent correction for individual time-point comparisons). Right, Representative immunofluorescence images of serum/growth factor starved and VEGF-A–stimulated control, S100A6, and S100A6/IFITM1 knockdown HUVECs after EdU detection (×20 magnification; scale bar, 40 µm; green=EdU and blue=nuclear DAPI).

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling

    doi: 10.1161/atvbaha.115.306415

    Figure Lengend Snippet: Figure 4. Interferon-inducible transmembrane protein 1 (IFITM1) is among the most regulated genes on S100A6 knockdown and a key regulator of S100A6 cell signaling. A, Reverse transcription polymerase chain reaction analysis for IFITM1 from RNA samples used in the illumina beadarrays was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when repli- cates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for IFITM1 (n=6 individual; *P<0.05 vs corresponding control siRNA-treated cells, Friedman test with subsequent Dunn correction for multiple comparisons). C, Left, S100A6, S100A6/IFITM1 knockdown, and control HUVECs were stimulated with VEGF-A for 24 h, and EdU incorporation was measured as EdU- positive cells vs the total number of cells (ie, nuclear DAPI-stained cells; n=6 individual experiments; *P<0.05 vs corresponding control siRNA-treated cells and #P=0.062 vs S100A6 siRNA-treated cells with 24 h of VEGF-A stimulation, Friedman test for repeated measures, Wilcoxon signed-rank test with subsequent correction for individual time-point comparisons). Right, Representative immunofluorescence images of serum/growth factor starved and VEGF-A–stimulated control, S100A6, and S100A6/IFITM1 knockdown HUVECs after EdU detection (×20 magnification; scale bar, 40 µm; green=EdU and blue=nuclear DAPI).

    Article Snippet: Primary antibodies Antibody Donor Dilution WB/IF Manufacturer AKT mouse 1:1000 #2966, Cell Signaling pAKT rabbit 1:1000 #9271, Cell Signaling ERK rabbit 1:2000 #9102 , Cell Signaling Lerchenmüller et al. Materials and Methods pERK mouse 1:2000 #9106, Cell Signaling PECAM-1 goat 1:50 (IF) sc-1506, Santa Cruz GAPDH mouse 1:20000 MAB374, Millipore IFITM1 rabbit 1:1000/ 1:100 PA1112, Boster Bio IFITM1 rabbit 1:50 (IF) PA5-20989,Thermofisher JAK1 mouse 1:1000 ab75744, Abcam pJAK1 rabbit 1:1000 ab138005, Abcam JAK2 rabbit 1:1000 #3230, Cell Signaling pJAK2 rabbit 1:1000 #3771, Cell Signaling Ki67 mouse 1:1000/ 1:100 14-5699, eBioscience NFKB-p65 rabbit 1:1000 #8242, Cell Signaling pNFKB-p65 mouse 1:1000 #3036, Cell Signaling PIAS1 rabbit 1:500 ab32219, Abcam POLE mouse 1:1000 H00005426, Novus STAT1 rabbit 1:500/ 1:50 ab2415, Abcam pSTAT1 mouse 1:1000 ab29045, Abcam STAT2 rabbit 1:1000 #4594, Cell Signaling S100A6 rabbit 1:1000/ 1:100 ab181975, Abcam S100A6 mouse 1.50 (IF) S5049, Sigma S100A4 rabbit 1:1000 A5114, Dako S100B rabbit 1:1000 A5110, Dako Secondary antibodies Antibody Donor Dilution Manufacturer IR Dye 680 vs rabbit goat 1:20000 LI-COR IR Dye 800 vs mouse goat 1:20000 LI-COR Alexa Fluor 488 rabbit goat 1:300 Invitrogen Alexa Fluor 680 mouse goat 1:300 Invitrogen Alexa Fluor 488 mouse donkey 1:200 Invitrogen Alexa Fluor 594 goat donkey 1:200 Invitrogen Alexa Fluor 555 goat donkey 1:200 Invitrogen Alexa Fluor 647 mouse donkey 1:200 Invitrogen HRP antibody vs rabbit goat 1:2000 Santa Cruz HRP antibody vs mouse goat 1:2000 Santa Cruz Microarray data acquisition and analysis Microarray measurements were recorded for siRNA control and S100A6 knockdown HUVECs under VEGF-stimulation in a time course (time points 0h, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h for S100A6 siRNA and 0h, 1h, 4h, 8h, 12h, 24h for siRNA control).

    Techniques: Knockdown, Reverse Transcription, Polymerase Chain Reaction, Control, Staining, Immunofluorescence

    Figure 5. Elevated interferon-inducible transmembrane protein 1 (IFITM1) expression and antiproliferative phenotype in S100A6-depleted endothelial cells is caused by signal transducers and activators of transcription 1 (STAT1) activation. A, Reverse transcription polymerase chain reaction analysis for STAT1 from RNA samples used in the gene array was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when replicates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for STAT1 and Tyr-701–phosphorylated STAT1 (n=6 individual experiments; *P<0.05 vs corresponding control siRNA- treated cells, Friedman test and subsequent Dunn correction for multiple comparisons). C, Left, Cell lysates from S100A6, S100A6/STAT1 knockdown, and control HUVECs were collected after over night starvation and after VEGF-A stimulation and were immunoblotted for Ki67 (n=6 individual experiments) and IFITM1 (n=4 individual experiments for 0-h time point and n=6 individual experiments for 24-h time point; #P<0.05 vs corresponding control siRNA-treated cells, *P≤0.05 vs S100A6 siRNA-treated cells at 24 h, Friedman–Dunn test). Right, Representative cell culture images from S100A6, S100A6/STAT1 knockdown, and control HUVECs 24 h after VEGF-A stimulation (×4 magnification) and representative IF images from the corresponding experiment’s EdU incorporation (×20 magnification; scale bar, 50 µm; red=EdU and blue=nuclear DAPI).

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling

    doi: 10.1161/atvbaha.115.306415

    Figure Lengend Snippet: Figure 5. Elevated interferon-inducible transmembrane protein 1 (IFITM1) expression and antiproliferative phenotype in S100A6-depleted endothelial cells is caused by signal transducers and activators of transcription 1 (STAT1) activation. A, Reverse transcription polymerase chain reaction analysis for STAT1 from RNA samples used in the gene array was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when replicates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for STAT1 and Tyr-701–phosphorylated STAT1 (n=6 individual experiments; *P<0.05 vs corresponding control siRNA- treated cells, Friedman test and subsequent Dunn correction for multiple comparisons). C, Left, Cell lysates from S100A6, S100A6/STAT1 knockdown, and control HUVECs were collected after over night starvation and after VEGF-A stimulation and were immunoblotted for Ki67 (n=6 individual experiments) and IFITM1 (n=4 individual experiments for 0-h time point and n=6 individual experiments for 24-h time point; #P<0.05 vs corresponding control siRNA-treated cells, *P≤0.05 vs S100A6 siRNA-treated cells at 24 h, Friedman–Dunn test). Right, Representative cell culture images from S100A6, S100A6/STAT1 knockdown, and control HUVECs 24 h after VEGF-A stimulation (×4 magnification) and representative IF images from the corresponding experiment’s EdU incorporation (×20 magnification; scale bar, 50 µm; red=EdU and blue=nuclear DAPI).

    Article Snippet: Primary antibodies Antibody Donor Dilution WB/IF Manufacturer AKT mouse 1:1000 #2966, Cell Signaling pAKT rabbit 1:1000 #9271, Cell Signaling ERK rabbit 1:2000 #9102 , Cell Signaling Lerchenmüller et al. Materials and Methods pERK mouse 1:2000 #9106, Cell Signaling PECAM-1 goat 1:50 (IF) sc-1506, Santa Cruz GAPDH mouse 1:20000 MAB374, Millipore IFITM1 rabbit 1:1000/ 1:100 PA1112, Boster Bio IFITM1 rabbit 1:50 (IF) PA5-20989,Thermofisher JAK1 mouse 1:1000 ab75744, Abcam pJAK1 rabbit 1:1000 ab138005, Abcam JAK2 rabbit 1:1000 #3230, Cell Signaling pJAK2 rabbit 1:1000 #3771, Cell Signaling Ki67 mouse 1:1000/ 1:100 14-5699, eBioscience NFKB-p65 rabbit 1:1000 #8242, Cell Signaling pNFKB-p65 mouse 1:1000 #3036, Cell Signaling PIAS1 rabbit 1:500 ab32219, Abcam POLE mouse 1:1000 H00005426, Novus STAT1 rabbit 1:500/ 1:50 ab2415, Abcam pSTAT1 mouse 1:1000 ab29045, Abcam STAT2 rabbit 1:1000 #4594, Cell Signaling S100A6 rabbit 1:1000/ 1:100 ab181975, Abcam S100A6 mouse 1.50 (IF) S5049, Sigma S100A4 rabbit 1:1000 A5114, Dako S100B rabbit 1:1000 A5110, Dako Secondary antibodies Antibody Donor Dilution Manufacturer IR Dye 680 vs rabbit goat 1:20000 LI-COR IR Dye 800 vs mouse goat 1:20000 LI-COR Alexa Fluor 488 rabbit goat 1:300 Invitrogen Alexa Fluor 680 mouse goat 1:300 Invitrogen Alexa Fluor 488 mouse donkey 1:200 Invitrogen Alexa Fluor 594 goat donkey 1:200 Invitrogen Alexa Fluor 555 goat donkey 1:200 Invitrogen Alexa Fluor 647 mouse donkey 1:200 Invitrogen HRP antibody vs rabbit goat 1:2000 Santa Cruz HRP antibody vs mouse goat 1:2000 Santa Cruz Microarray data acquisition and analysis Microarray measurements were recorded for siRNA control and S100A6 knockdown HUVECs under VEGF-stimulation in a time course (time points 0h, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h for S100A6 siRNA and 0h, 1h, 4h, 8h, 12h, 24h for siRNA control).

    Techniques: Expressing, Activation Assay, Reverse Transcription, Polymerase Chain Reaction, Control, Knockdown, Cell Culture

    Figure 7. Novel signaling module linking S100A6 to control of signal transducers and activators of transcription 1 (STAT1) signaling. A, Cell lysates from S100A6 adenovirus (FLAG tagged) and control GFP adenovirus-infected human umbilical vein endothelial cells (HUVECs; 48 h) were collected and immunoblotted for interferon-inducible transmembrane protein 1 (IFITM1), protein inhibitor of acti- vated STAT 1 (PIAS1), STAT1, S100A6, and FLAG (n=3 individual experiments). B, The schematic highlights the molecular control of antiproliferative STAT1 signaling in endothelial cells (ECs) via S100A6. Our findings indicate that S100A6 might constitutively facilitate the activity of the STAT1 suppressor PIAS (1). Loss of S100A6 expression subsequently entails STAT1 activation via loss of PIAS activity inde- pendent of Janus kinases 1 and 2 (JAK1/2), SH2-containing phosphatase (SHP), and suppressors of cytokine signaling (SOCS) signaling (2). STAT1 in turn activates a set of antiproliferative genes, including IFITM1 besides others (3) that oppose cell cycle entry in part most likely via previously described p53/p21 activation. Of note, vascular endothelial growth factor A (VEGF-A)–mediated proliferative signaling pathways, such as extracellular signal–regulated kinase (ERK1/2) and AKT, are not affected by antiproliferative STAT1 signaling, indicating downstream competitive integration of pro- and antiproliferative signals for cell cycle decision entry. Colored elements highlight in silico predicted and experimentally validated components of the novel S100A6/STAT1/IFITM1 signaling module. Grey elements are not affected by S100A6 signaling as predicted by in silico modeling and experimental validation. In summary, S100A6 might be indispensable for timely cell cycle entry by attenuating antiproliferative STAT1 signaling.

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling

    doi: 10.1161/atvbaha.115.306415

    Figure Lengend Snippet: Figure 7. Novel signaling module linking S100A6 to control of signal transducers and activators of transcription 1 (STAT1) signaling. A, Cell lysates from S100A6 adenovirus (FLAG tagged) and control GFP adenovirus-infected human umbilical vein endothelial cells (HUVECs; 48 h) were collected and immunoblotted for interferon-inducible transmembrane protein 1 (IFITM1), protein inhibitor of acti- vated STAT 1 (PIAS1), STAT1, S100A6, and FLAG (n=3 individual experiments). B, The schematic highlights the molecular control of antiproliferative STAT1 signaling in endothelial cells (ECs) via S100A6. Our findings indicate that S100A6 might constitutively facilitate the activity of the STAT1 suppressor PIAS (1). Loss of S100A6 expression subsequently entails STAT1 activation via loss of PIAS activity inde- pendent of Janus kinases 1 and 2 (JAK1/2), SH2-containing phosphatase (SHP), and suppressors of cytokine signaling (SOCS) signaling (2). STAT1 in turn activates a set of antiproliferative genes, including IFITM1 besides others (3) that oppose cell cycle entry in part most likely via previously described p53/p21 activation. Of note, vascular endothelial growth factor A (VEGF-A)–mediated proliferative signaling pathways, such as extracellular signal–regulated kinase (ERK1/2) and AKT, are not affected by antiproliferative STAT1 signaling, indicating downstream competitive integration of pro- and antiproliferative signals for cell cycle decision entry. Colored elements highlight in silico predicted and experimentally validated components of the novel S100A6/STAT1/IFITM1 signaling module. Grey elements are not affected by S100A6 signaling as predicted by in silico modeling and experimental validation. In summary, S100A6 might be indispensable for timely cell cycle entry by attenuating antiproliferative STAT1 signaling.

    Article Snippet: Primary antibodies Antibody Donor Dilution WB/IF Manufacturer AKT mouse 1:1000 #2966, Cell Signaling pAKT rabbit 1:1000 #9271, Cell Signaling ERK rabbit 1:2000 #9102 , Cell Signaling Lerchenmüller et al. Materials and Methods pERK mouse 1:2000 #9106, Cell Signaling PECAM-1 goat 1:50 (IF) sc-1506, Santa Cruz GAPDH mouse 1:20000 MAB374, Millipore IFITM1 rabbit 1:1000/ 1:100 PA1112, Boster Bio IFITM1 rabbit 1:50 (IF) PA5-20989,Thermofisher JAK1 mouse 1:1000 ab75744, Abcam pJAK1 rabbit 1:1000 ab138005, Abcam JAK2 rabbit 1:1000 #3230, Cell Signaling pJAK2 rabbit 1:1000 #3771, Cell Signaling Ki67 mouse 1:1000/ 1:100 14-5699, eBioscience NFKB-p65 rabbit 1:1000 #8242, Cell Signaling pNFKB-p65 mouse 1:1000 #3036, Cell Signaling PIAS1 rabbit 1:500 ab32219, Abcam POLE mouse 1:1000 H00005426, Novus STAT1 rabbit 1:500/ 1:50 ab2415, Abcam pSTAT1 mouse 1:1000 ab29045, Abcam STAT2 rabbit 1:1000 #4594, Cell Signaling S100A6 rabbit 1:1000/ 1:100 ab181975, Abcam S100A6 mouse 1.50 (IF) S5049, Sigma S100A4 rabbit 1:1000 A5114, Dako S100B rabbit 1:1000 A5110, Dako Secondary antibodies Antibody Donor Dilution Manufacturer IR Dye 680 vs rabbit goat 1:20000 LI-COR IR Dye 800 vs mouse goat 1:20000 LI-COR Alexa Fluor 488 rabbit goat 1:300 Invitrogen Alexa Fluor 680 mouse goat 1:300 Invitrogen Alexa Fluor 488 mouse donkey 1:200 Invitrogen Alexa Fluor 594 goat donkey 1:200 Invitrogen Alexa Fluor 555 goat donkey 1:200 Invitrogen Alexa Fluor 647 mouse donkey 1:200 Invitrogen HRP antibody vs rabbit goat 1:2000 Santa Cruz HRP antibody vs mouse goat 1:2000 Santa Cruz Microarray data acquisition and analysis Microarray measurements were recorded for siRNA control and S100A6 knockdown HUVECs under VEGF-stimulation in a time course (time points 0h, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h for S100A6 siRNA and 0h, 1h, 4h, 8h, 12h, 24h for siRNA control).

    Techniques: Control, Infection, Activity Assay, Expressing, Activation Assay, Protein-Protein interactions, In Silico, Biomarker Discovery

    Figure 4. Interferon-inducible transmembrane protein 1 (IFITM1) is among the most regulated genes on S100A6 knockdown and a key regulator of S100A6 cell signaling. A, Reverse transcription polymerase chain reaction analysis for IFITM1 from RNA samples used in the illumina beadarrays was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when repli- cates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for IFITM1 (n=6 individual; *P<0.05 vs corresponding control siRNA-treated cells, Friedman test with subsequent Dunn correction for multiple comparisons). C, Left, S100A6, S100A6/IFITM1 knockdown, and control HUVECs were stimulated with VEGF-A for 24 h, and EdU incorporation was measured as EdU- positive cells vs the total number of cells (ie, nuclear DAPI-stained cells; n=6 individual experiments; *P<0.05 vs corresponding control siRNA-treated cells and #P=0.062 vs S100A6 siRNA-treated cells with 24 h of VEGF-A stimulation, Friedman test for repeated measures, Wilcoxon signed-rank test with subsequent correction for individual time-point comparisons). Right, Representative immunofluorescence images of serum/growth factor starved and VEGF-A–stimulated control, S100A6, and S100A6/IFITM1 knockdown HUVECs after EdU detection (×20 magnification; scale bar, 40 µm; green=EdU and blue=nuclear DAPI).

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling

    doi: 10.1161/atvbaha.115.306415

    Figure Lengend Snippet: Figure 4. Interferon-inducible transmembrane protein 1 (IFITM1) is among the most regulated genes on S100A6 knockdown and a key regulator of S100A6 cell signaling. A, Reverse transcription polymerase chain reaction analysis for IFITM1 from RNA samples used in the illumina beadarrays was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when repli- cates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for IFITM1 (n=6 individual; *P<0.05 vs corresponding control siRNA-treated cells, Friedman test with subsequent Dunn correction for multiple comparisons). C, Left, S100A6, S100A6/IFITM1 knockdown, and control HUVECs were stimulated with VEGF-A for 24 h, and EdU incorporation was measured as EdU- positive cells vs the total number of cells (ie, nuclear DAPI-stained cells; n=6 individual experiments; *P<0.05 vs corresponding control siRNA-treated cells and #P=0.062 vs S100A6 siRNA-treated cells with 24 h of VEGF-A stimulation, Friedman test for repeated measures, Wilcoxon signed-rank test with subsequent correction for individual time-point comparisons). Right, Representative immunofluorescence images of serum/growth factor starved and VEGF-A–stimulated control, S100A6, and S100A6/IFITM1 knockdown HUVECs after EdU detection (×20 magnification; scale bar, 40 µm; green=EdU and blue=nuclear DAPI).

    Article Snippet: Primary antibodies Antibody Donor Dilution WB/IF Manufacturer AKT mouse 1:1000 #2966, Cell Signaling pAKT rabbit 1:1000 #9271, Cell Signaling ERK rabbit 1:2000 #9102 , Cell Signaling Lerchenmüller et al. Materials and Methods pERK mouse 1:2000 #9106, Cell Signaling PECAM-1 goat 1:50 (IF) sc-1506, Santa Cruz GAPDH mouse 1:20000 MAB374, Millipore IFITM1 rabbit 1:1000/ 1:100 PA1112, Boster Bio IFITM1 rabbit 1:50 (IF) PA5-20989,Thermofisher JAK1 mouse 1:1000 ab75744, Abcam pJAK1 rabbit 1:1000 ab138005, Abcam JAK2 rabbit 1:1000 #3230, Cell Signaling pJAK2 rabbit 1:1000 #3771, Cell Signaling Ki67 mouse 1:1000/ 1:100 14-5699, eBioscience NFKB-p65 rabbit 1:1000 #8242, Cell Signaling pNFKB-p65 mouse 1:1000 #3036, Cell Signaling PIAS1 rabbit 1:500 ab32219, Abcam POLE mouse 1:1000 H00005426, Novus STAT1 rabbit 1:500/ 1:50 ab2415, Abcam pSTAT1 mouse 1:1000 ab29045, Abcam STAT2 rabbit 1:1000 #4594, Cell Signaling S100A6 rabbit 1:1000/ 1:100 ab181975, Abcam S100A6 mouse 1.50 (IF) S5049, Sigma S100A4 rabbit 1:1000 A5114, Dako S100B rabbit 1:1000 A5110, Dako Secondary antibodies Antibody Donor Dilution Manufacturer IR Dye 680 vs rabbit goat 1:20000 LI-COR IR Dye 800 vs mouse goat 1:20000 LI-COR Alexa Fluor 488 rabbit goat 1:300 Invitrogen Alexa Fluor 680 mouse goat 1:300 Invitrogen Alexa Fluor 488 mouse donkey 1:200 Invitrogen Alexa Fluor 594 goat donkey 1:200 Invitrogen Alexa Fluor 555 goat donkey 1:200 Invitrogen Alexa Fluor 647 mouse donkey 1:200 Invitrogen HRP antibody vs rabbit goat 1:2000 Santa Cruz HRP antibody vs mouse goat 1:2000 Santa Cruz Microarray data acquisition and analysis Microarray measurements were recorded for siRNA control and S100A6 knockdown HUVECs under VEGF-stimulation in a time course (time points 0h, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h for S100A6 siRNA and 0h, 1h, 4h, 8h, 12h, 24h for siRNA control).

    Techniques: Knockdown, Reverse Transcription, Polymerase Chain Reaction, Control, Staining, Immunofluorescence

    Figure 5. Elevated interferon-inducible transmembrane protein 1 (IFITM1) expression and antiproliferative phenotype in S100A6-depleted endothelial cells is caused by signal transducers and activators of transcription 1 (STAT1) activation. A, Reverse transcription polymerase chain reaction analysis for STAT1 from RNA samples used in the gene array was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when replicates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for STAT1 and Tyr-701–phosphorylated STAT1 (n=6 individual experiments; *P<0.05 vs corresponding control siRNA- treated cells, Friedman test and subsequent Dunn correction for multiple comparisons). C, Left, Cell lysates from S100A6, S100A6/STAT1 knockdown, and control HUVECs were collected after over night starvation and after VEGF-A stimulation and were immunoblotted for Ki67 (n=6 individual experiments) and IFITM1 (n=4 individual experiments for 0-h time point and n=6 individual experiments for 24-h time point; #P<0.05 vs corresponding control siRNA-treated cells, *P≤0.05 vs S100A6 siRNA-treated cells at 24 h, Friedman–Dunn test). Right, Representative cell culture images from S100A6, S100A6/STAT1 knockdown, and control HUVECs 24 h after VEGF-A stimulation (×4 magnification) and representative IF images from the corresponding experiment’s EdU incorporation (×20 magnification; scale bar, 50 µm; red=EdU and blue=nuclear DAPI).

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling

    doi: 10.1161/atvbaha.115.306415

    Figure Lengend Snippet: Figure 5. Elevated interferon-inducible transmembrane protein 1 (IFITM1) expression and antiproliferative phenotype in S100A6-depleted endothelial cells is caused by signal transducers and activators of transcription 1 (STAT1) activation. A, Reverse transcription polymerase chain reaction analysis for STAT1 from RNA samples used in the gene array was carried out to confirm the results of the transcriptome time series analysis (error bars were drawn when replicates were available). B, Cell lysates from control and S100A6 siRNA-treated human umbilical vein endothelial cells (HUVECs) after a time course of vascular endothelial growth factor A (VEGF-A) stimulation were collected and immunoblotted for STAT1 and Tyr-701–phosphorylated STAT1 (n=6 individual experiments; *P<0.05 vs corresponding control siRNA- treated cells, Friedman test and subsequent Dunn correction for multiple comparisons). C, Left, Cell lysates from S100A6, S100A6/STAT1 knockdown, and control HUVECs were collected after over night starvation and after VEGF-A stimulation and were immunoblotted for Ki67 (n=6 individual experiments) and IFITM1 (n=4 individual experiments for 0-h time point and n=6 individual experiments for 24-h time point; #P<0.05 vs corresponding control siRNA-treated cells, *P≤0.05 vs S100A6 siRNA-treated cells at 24 h, Friedman–Dunn test). Right, Representative cell culture images from S100A6, S100A6/STAT1 knockdown, and control HUVECs 24 h after VEGF-A stimulation (×4 magnification) and representative IF images from the corresponding experiment’s EdU incorporation (×20 magnification; scale bar, 50 µm; red=EdU and blue=nuclear DAPI).

    Article Snippet: Primary antibodies Antibody Donor Dilution WB/IF Manufacturer AKT mouse 1:1000 #2966, Cell Signaling pAKT rabbit 1:1000 #9271, Cell Signaling ERK rabbit 1:2000 #9102 , Cell Signaling Lerchenmüller et al. Materials and Methods pERK mouse 1:2000 #9106, Cell Signaling PECAM-1 goat 1:50 (IF) sc-1506, Santa Cruz GAPDH mouse 1:20000 MAB374, Millipore IFITM1 rabbit 1:1000/ 1:100 PA1112, Boster Bio IFITM1 rabbit 1:50 (IF) PA5-20989,Thermofisher JAK1 mouse 1:1000 ab75744, Abcam pJAK1 rabbit 1:1000 ab138005, Abcam JAK2 rabbit 1:1000 #3230, Cell Signaling pJAK2 rabbit 1:1000 #3771, Cell Signaling Ki67 mouse 1:1000/ 1:100 14-5699, eBioscience NFKB-p65 rabbit 1:1000 #8242, Cell Signaling pNFKB-p65 mouse 1:1000 #3036, Cell Signaling PIAS1 rabbit 1:500 ab32219, Abcam POLE mouse 1:1000 H00005426, Novus STAT1 rabbit 1:500/ 1:50 ab2415, Abcam pSTAT1 mouse 1:1000 ab29045, Abcam STAT2 rabbit 1:1000 #4594, Cell Signaling S100A6 rabbit 1:1000/ 1:100 ab181975, Abcam S100A6 mouse 1.50 (IF) S5049, Sigma S100A4 rabbit 1:1000 A5114, Dako S100B rabbit 1:1000 A5110, Dako Secondary antibodies Antibody Donor Dilution Manufacturer IR Dye 680 vs rabbit goat 1:20000 LI-COR IR Dye 800 vs mouse goat 1:20000 LI-COR Alexa Fluor 488 rabbit goat 1:300 Invitrogen Alexa Fluor 680 mouse goat 1:300 Invitrogen Alexa Fluor 488 mouse donkey 1:200 Invitrogen Alexa Fluor 594 goat donkey 1:200 Invitrogen Alexa Fluor 555 goat donkey 1:200 Invitrogen Alexa Fluor 647 mouse donkey 1:200 Invitrogen HRP antibody vs rabbit goat 1:2000 Santa Cruz HRP antibody vs mouse goat 1:2000 Santa Cruz Microarray data acquisition and analysis Microarray measurements were recorded for siRNA control and S100A6 knockdown HUVECs under VEGF-stimulation in a time course (time points 0h, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h for S100A6 siRNA and 0h, 1h, 4h, 8h, 12h, 24h for siRNA control).

    Techniques: Expressing, Activation Assay, Reverse Transcription, Polymerase Chain Reaction, Control, Knockdown, Cell Culture

    Figure 7. Novel signaling module linking S100A6 to control of signal transducers and activators of transcription 1 (STAT1) signaling. A, Cell lysates from S100A6 adenovirus (FLAG tagged) and control GFP adenovirus-infected human umbilical vein endothelial cells (HUVECs; 48 h) were collected and immunoblotted for interferon-inducible transmembrane protein 1 (IFITM1), protein inhibitor of acti- vated STAT 1 (PIAS1), STAT1, S100A6, and FLAG (n=3 individual experiments). B, The schematic highlights the molecular control of antiproliferative STAT1 signaling in endothelial cells (ECs) via S100A6. Our findings indicate that S100A6 might constitutively facilitate the activity of the STAT1 suppressor PIAS (1). Loss of S100A6 expression subsequently entails STAT1 activation via loss of PIAS activity inde- pendent of Janus kinases 1 and 2 (JAK1/2), SH2-containing phosphatase (SHP), and suppressors of cytokine signaling (SOCS) signaling (2). STAT1 in turn activates a set of antiproliferative genes, including IFITM1 besides others (3) that oppose cell cycle entry in part most likely via previously described p53/p21 activation. Of note, vascular endothelial growth factor A (VEGF-A)–mediated proliferative signaling pathways, such as extracellular signal–regulated kinase (ERK1/2) and AKT, are not affected by antiproliferative STAT1 signaling, indicating downstream competitive integration of pro- and antiproliferative signals for cell cycle decision entry. Colored elements highlight in silico predicted and experimentally validated components of the novel S100A6/STAT1/IFITM1 signaling module. Grey elements are not affected by S100A6 signaling as predicted by in silico modeling and experimental validation. In summary, S100A6 might be indispensable for timely cell cycle entry by attenuating antiproliferative STAT1 signaling.

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling

    doi: 10.1161/atvbaha.115.306415

    Figure Lengend Snippet: Figure 7. Novel signaling module linking S100A6 to control of signal transducers and activators of transcription 1 (STAT1) signaling. A, Cell lysates from S100A6 adenovirus (FLAG tagged) and control GFP adenovirus-infected human umbilical vein endothelial cells (HUVECs; 48 h) were collected and immunoblotted for interferon-inducible transmembrane protein 1 (IFITM1), protein inhibitor of acti- vated STAT 1 (PIAS1), STAT1, S100A6, and FLAG (n=3 individual experiments). B, The schematic highlights the molecular control of antiproliferative STAT1 signaling in endothelial cells (ECs) via S100A6. Our findings indicate that S100A6 might constitutively facilitate the activity of the STAT1 suppressor PIAS (1). Loss of S100A6 expression subsequently entails STAT1 activation via loss of PIAS activity inde- pendent of Janus kinases 1 and 2 (JAK1/2), SH2-containing phosphatase (SHP), and suppressors of cytokine signaling (SOCS) signaling (2). STAT1 in turn activates a set of antiproliferative genes, including IFITM1 besides others (3) that oppose cell cycle entry in part most likely via previously described p53/p21 activation. Of note, vascular endothelial growth factor A (VEGF-A)–mediated proliferative signaling pathways, such as extracellular signal–regulated kinase (ERK1/2) and AKT, are not affected by antiproliferative STAT1 signaling, indicating downstream competitive integration of pro- and antiproliferative signals for cell cycle decision entry. Colored elements highlight in silico predicted and experimentally validated components of the novel S100A6/STAT1/IFITM1 signaling module. Grey elements are not affected by S100A6 signaling as predicted by in silico modeling and experimental validation. In summary, S100A6 might be indispensable for timely cell cycle entry by attenuating antiproliferative STAT1 signaling.

    Article Snippet: Primary antibodies Antibody Donor Dilution WB/IF Manufacturer AKT mouse 1:1000 #2966, Cell Signaling pAKT rabbit 1:1000 #9271, Cell Signaling ERK rabbit 1:2000 #9102 , Cell Signaling Lerchenmüller et al. Materials and Methods pERK mouse 1:2000 #9106, Cell Signaling PECAM-1 goat 1:50 (IF) sc-1506, Santa Cruz GAPDH mouse 1:20000 MAB374, Millipore IFITM1 rabbit 1:1000/ 1:100 PA1112, Boster Bio IFITM1 rabbit 1:50 (IF) PA5-20989,Thermofisher JAK1 mouse 1:1000 ab75744, Abcam pJAK1 rabbit 1:1000 ab138005, Abcam JAK2 rabbit 1:1000 #3230, Cell Signaling pJAK2 rabbit 1:1000 #3771, Cell Signaling Ki67 mouse 1:1000/ 1:100 14-5699, eBioscience NFKB-p65 rabbit 1:1000 #8242, Cell Signaling pNFKB-p65 mouse 1:1000 #3036, Cell Signaling PIAS1 rabbit 1:500 ab32219, Abcam POLE mouse 1:1000 H00005426, Novus STAT1 rabbit 1:500/ 1:50 ab2415, Abcam pSTAT1 mouse 1:1000 ab29045, Abcam STAT2 rabbit 1:1000 #4594, Cell Signaling S100A6 rabbit 1:1000/ 1:100 ab181975, Abcam S100A6 mouse 1.50 (IF) S5049, Sigma S100A4 rabbit 1:1000 A5114, Dako S100B rabbit 1:1000 A5110, Dako Secondary antibodies Antibody Donor Dilution Manufacturer IR Dye 680 vs rabbit goat 1:20000 LI-COR IR Dye 800 vs mouse goat 1:20000 LI-COR Alexa Fluor 488 rabbit goat 1:300 Invitrogen Alexa Fluor 680 mouse goat 1:300 Invitrogen Alexa Fluor 488 mouse donkey 1:200 Invitrogen Alexa Fluor 594 goat donkey 1:200 Invitrogen Alexa Fluor 555 goat donkey 1:200 Invitrogen Alexa Fluor 647 mouse donkey 1:200 Invitrogen HRP antibody vs rabbit goat 1:2000 Santa Cruz HRP antibody vs mouse goat 1:2000 Santa Cruz Microarray data acquisition and analysis Microarray measurements were recorded for siRNA control and S100A6 knockdown HUVECs under VEGF-stimulation in a time course (time points 0h, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h for S100A6 siRNA and 0h, 1h, 4h, 8h, 12h, 24h for siRNA control).

    Techniques: Control, Infection, Activity Assay, Expressing, Activation Assay, Protein-Protein interactions, In Silico, Biomarker Discovery