Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A to C ) Interactions of CDK4 (A) or CDK6 (B) with SMYD2, and SMYD2 with CDK4 or CDK6 (C) in RCTE cells were detected by immunoprecipitation (IP) and immunoblotting (IB). Immunoglobulin G (IgG) was used as a negative control. ( D ) Schematic of GST-SMYD2 fusion protein constructs (top), which was detected using Coomassie blue staining (middle). GST pull-down assays indicated the interaction of GST-SMYD2 fusion proteins with CDK4 and CDK6 (bottom). ( E ) GFP-tagged SET domain–deleted SMYD2 cannot pull down CDK4 and CDK6 in HEK293T cells. ( F to I ) The phosphorylation of SMYD2 was decreased in RCTE cells transfected with siRNAs to CDK4 (F), CDK6 (G), or both (H), and in RCTE cells treated with Abe (10 μM) (I). Knockdown of CDK4 decreased the expression of CDK6, and vice versa for CDK6 in these cells. The quantification analysis ( n = 3) of band intensities was shown in the graphs (bottom), in that the density of each protein band was normalized to actin, and then the value was divided by the value of corresponding siRNA and vehicle band density to actin. The value of the control band to actin was set to 1.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Immunoprecipitation, Western Blot, Negative Control, Construct, Staining, Phospho-proteomics, Transfection, Knockdown, Expressing, Control

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A to D ) Western blot analysis indicated that knockdown of CDK4 and CDK6 with siRNAs (A) or knockdown of SMYD2 with siRNA (B) as well as inhibition of CDK4/6 with Abe (C) or inhibition of SMYD2 with AZ505 (D) decreased the mono-, di-, and trimethylation of histone H3 at lysine 4 (H3K4) and lysine 36 (H3K36) in RCTE cells. The quantification and statistical analysis ( n = 3) were shown in the graph (bottom). * P < 0.01 as compared to each control. ( E and F ) Knockdown (E) or inhibition (F) of SMYD2 decreased the mRNA and protein levels of CDK4 and CDK6 in RCTE cells, examined with qRT-PCR and Western blotting. * P < 0.01 as compared to each control ( n = 3). ns, not significant. ( G ) SMYD2 bound to the promoter of CDK4 and CDK6. ChIP-qPCR analysis was performed with an SMYD2 antibody, or normal rabbit IgG in RCTE cells. ( H ) ChIP assay was performed with mono-, di-, and trimethylated H3K4 and H3K36 antibodies and normal rabbit IgG in RCTE cells.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Western Blot, Knockdown, Inhibition, Control, Quantitative RT-PCR, ChIP-qPCR

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A ) Immunofluorescence staining of primary cilia with α-acetyl-tubulin antibody (α-ac-tubulin) in primary renal epithelial cells isolated from kidneys of wild-type (WT) and Smyd2 flox/flox : Ksp-Cre mice, in which Smyd2 was specifically knocked out (KO) by the kidney-specific promoter (Ksp)–driven Cre recombinase in renal epithelial cells and cultured in serum-free medium for 72 hours before subjected to staining. The percentage of ciliated cells and cilia length was measured and statistically analyzed. Scale bar, 5 μm. Error bars represent the SD. N values represent the numbers of cilia (left graph) and cells (right graph), respectively, for each group. ( B to E ) Knockdown of Smyd2 with siRNA (B) or inhibition of Smyd2 with AZ505 (C) as well as knockdown of CDK4/CDK6 with siRNAs (D) or inhibition of CDK4/CDK6 with Abe (E) in mIMCD3 cells resulted in longer cilia compared to control cells as examined by immunofluorescence with α-acetyl-tubulin and SMYD2 antibodies. Scale bar, 5 μm. Error bars represent the SD. N values represent cilia numbers for each group.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Immunofluorescence, Staining, Isolation, Cell Culture, Knockdown, Inhibition, Control

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A ) Coimmunoprecipitation of endogenous α- and γ-tubulin with SMYD2 in RCTE cells. IgG was used as a negative control. ( B to D ) Coimmunoprecipitation of endogenous α-tubulin (B), γ-tubulin (C), and β-tubulin (D) with overexpressed SMYD2 in HEK293T cells. GFP vector–transfected cells were used as a negative control. ( E ) GST pull-down assays were performed by incubation of GST-SMYD2 fusion protein with 1 μg of recombinant α- or γ-tubulin protein and immunoblotting with an α-tubulin (top) and γ-tubulin antibody (middle). The expression of GST-SMYD2 was detected with Coomassie blue staining (bottom). ( F ) The expression of GST-SMYD2 constructs was detected using Coomassie blue staining (top). GST pull-down assays of GST-SMYD2 fusion proteins incubated with 1 mg of cell lysate from RCTE cells and immunoblotting using α- and γ-tubulin antibodies (bottom). ( G and H ) In vitro methylation assay of α-tubulin (G), γ-tubulin (H), and recombinant SMYD2. Histone H3 was used as a positive control in the in vitro methylation assays. ( I ) Relative intensity of methylated α- and γ-tubulin bands (black box) compared to the intensity of methylated histone H3, which was set to 1 (open box), in the in vitro methylation assays.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Negative Control, Plasmid Preparation, Transfection, Incubation, Recombinant, Western Blot, Expressing, Staining, Construct, In Vitro, Methylation, Positive Control

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A and B ) Knockdown of SMYD2 with siRNA (A) or inhibition of SMYD2 with AZ505 (B) decreased methylation of α-tubulin at K394 but not at K40 as examined by Western blotting with our generated TubK40me3 and TubK394me3 antibodies in RCTE cells. The quantification and statistical analysis ( n = 3) were shown in the graphs (bottom), in which the band density of control siRNA (A) and DMSO (B) was set to 1. ( C ) Western blot of the methylation of α-tubulin at K40 and at K394 with the TubK40me3 and TubK394me3 antibodies in RCTE cells transfected with Flag-tagged SMYD2 and GFP-tagged α-tubulin. The quantification and statistical analysis ( n = 3) were shown in the graph (bottom). ( D ) Representative images of RCTE cells stained with TubK394me3 (left) or SMYD2 (right) and costained with α-acetyl-tubulin/γ-tubulin (red) antibodies and DAPI (blue). Scale bar, 5 μm. ( E ) Representative images of RCTE cells stained with TubK394me3 (left, green) or SMYD2 (right, green) and costained with α-acetyl-tubulin/γ-tubulin (red) and DAPI (blue). Scale bar, 5 μm. Error bars represent the SD. N values represent cilia numbers in (D) and (E).

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Knockdown, Inhibition, Methylation, Western Blot, Generated, Control, Transfection, Staining

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A and B ) Knockdown (A) and inhibition of SMYD2 (B) increased the levels of IFT20 protein (top) and mRNA (bottom) as examined by Western blotting and qRT-PCR in RCTE cells. The quantification and statistical analysis ( n = 3) were shown in the graph (bottom), as are also shown in (B), (C), (E), and (F). * P < 0.01 as compared to controls. ( C ) Overexpression of SMYD2 decreased the levels of IFT20 protein (left) and mRNA (right) as examined by Western blotting and qRT-PCR in HEK293T cells. ( D ) SMYD2 and H3K36me3 antibodies bound to the promoter of IFT20 in RCTE cells as examined with ChIP assay. ( E and F ) Knockdown (E) and inhibition of CDK4/6 (F) increased the levels of IFT20 protein (top) and mRNA (bottom) as examined by Western blotting and qRT-PCR in RCTE cells. n = 3. ( G ) Representative images of RCTE cells stained with IFT20 and α-acetyl-tubulin/γ-tubulin antibodies and DAPI in the presence of AZ505 (middle), Abe (right), and vehicle (left). Scale bar, 5 μm. The statistical analysis was shown in the graph (right). Error bars represent the SD. N values represent cilia numbers.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Knockdown, Inhibition, Western Blot, Quantitative RT-PCR, Over Expression, Staining

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A ) Western blot of SMYD2, CDK4, CDK6, and IFT20 in normal mammary cells MCF10A and breast cancer cells, including MCF7, T47D, MDA-MB468, and MDA-MB231 cells. ( B ) Western blot of SMYD2, IFT20, CDK4, and CDK6 in PH2 and PN24 cells. ( C ) The quantification and statistical analysis ( n = 3) were shown in the graphs corresponding to (top) and (bottom). ( D to F ) Representative images of T47D (top) and MDA-MB468 cells (bottom) (D) as well as PN24 cells (E and F) stained with SMYD2 (green) and α-acetyl-tubulin/γ-tubulin (red) and costained with DAPI (blue) in the presence of the SMYD2 inhibitor AZ505 (middle) and the CDK4/6 inhibitor Abe as well as vehicle. Scale bar, 5 μm. Statistical analysis of the percentage of ciliated breast cancer cells ( n = 200) ( P < 0.01) as well as the percentage of ciliated PN24 cells ( n = 900) and their cilia lengths are shown in the graph (right). Error bars represent the SD.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Western Blot, Staining

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A and B ) Representative images of RCTE (A) and PN24 (B) cells stained with GLI2 (green) and GLI3 (red), and costained with α-acetyl-tubulin/γ-tubulin (purple) antibodies and DAPI (blue). Scale bar, 5 μm. The statistical analysis of the cilia tip localization of GLI2 (left) and GLI3 (right) is shown in the graphs. * P < 0.01. Error bars represent the SD. n = 100 cilia for each group. ( C and D ) Western blot analysis of Ptch1 and GLI1 in RCTE cells, which were treated with or without SAG and cotreated with or without the SMYD2 inhibitor AZ505 (C) and the CDK4/CDK6 inhibitor Abe (D) in serum-free medium for 48 hours. Statistical analysis of the expression ( n = 3) of Ptch1 and GLI1 proteins is shown in the graphs (right). ( E and F ) Western blot analysis of Ptch1 and Gli1 in PN24 cells, which were treated with or without SAG and cotreated with or without the SMYD2 inhibitor AZ505 (E) and the CDK4/CDK6 inhibitor Abe (F) in serum-free medium for 48 hours. Statistical analysis of the expression ( n = 3) of Ptch1 and Gli1 proteins is shown in the graphs (bottom).

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Staining, Western Blot, Expressing

Journal: Science signaling

Article Title: The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

doi: 10.1126/scisignal.aaj1784

Figure Lengend Snippet: Fig. 1. TSP1 isincreased in aging human lung vasculature and induces endothelialcell cycle arrest in vitro. Data for human tissue are shown in green; data for cell experiments are shown in red; data in gray are the corresponding controls. (A) Correlation of TSP1 expression as quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with age in human lung homogenates (n = 7 subjects; r 2=0.69, P = 0.02). Data points are means of three replicates of the same sample. (B) Representative images of TSP1 protein abundance in the intimal and medial layer of human pulmonary arteries of a 73-year-old subject (right; arrows indicate intima) compared to an artery from a 36-year-old subject (left) as determined by immunofluorescence (IF). Scale bar, 100 mm. L, vascular lumen. (C) Quantitation of immunofluorescence images from six subjects (n = 4 samples each, 10 to20 images per sample) is plotted as a linear regression; equation, r 2, and P values are as indicated. MFI, mean fluorescence intensity. (D) Cell cycle profile analysis of vehicle (left) and TSP1- challenged (right) HPAECs measured by propidium iodide (PI) labeling and fluorescence-activated cell sorting (FACS). FL-2, fluorescence channel 2. (E) Quantitative analysis of cell cycle phase distribution of HPAECs. Data are the means ± SEM of 20,000 events (n = 4 biological replicates per treatment group) expressed as percentage. *P < 0.05 for TSP1 chal- lenge compared to vehicle control by Mann-Whitney test of each phase. (F and G) HPAEC proliferation in the presence or absence of TSP1, as measured by trypan blue exclusion assay (F) and MTT assay (G). Data are means ± SEM (n = 3 biological replicates per treatment). P < 0.001 for TSP1 compared to vehicle control by repeated-measures analyses. (H) BrdU incor- poration in HPAECs in the presence or absence of TSP1 at 24 hours. Data are means ± SEM (n = 3 biological replicates per treatment). *P < 0.05 for TSP1 challenge compared to vehicle control by Student’s t test.

Article Snippet: In these experiments, cells were pretreated with peptides for 1 hour before TSP1 (2.2 nM) challenge. siRNA gene silencing and Nox1 plasmid transfection HPAECs were seeded at a density to achieve a 75 to 80% confluence overnight and were subjected to either p53 or Nox1 siRNA transfection (Santa Cruz Biotechnology) using the Lipofectamine 2000 transfection reagent (Invitrogen) according to themanufacturer’s protocol, as documented previously (51).

Techniques: In Vitro, Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Quantitative Proteomics, Immunofluorescence, Quantitation Assay, Fluorescence, Labeling, FACS, Control, MANN-WHITNEY, Trypan Blue Exclusion Assay, MTT Assay

Journal: Science signaling

Article Title: The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

doi: 10.1126/scisignal.aaj1784

Figure Lengend Snippet: Fig. 2. TSP1 promotes cell and tissue senescence in vitro and oxidative stress in wild-type middle-aged mice. Data for TSP1−/−samples are plotted in purple; data for cell experiments are in red; data in gray represent the corresponding controls. (A and B) Abundance of the S phase–associated proliferation marker PCNA in middle-aged TSP1−/−mice (purple bar) compared to age-matched wild-type controls (gray bar) as analyzed by Western blotting (A)and in HPAECs exposed to TSP1 as analyzed by immunofluorescence (B). Data are means ± SEM [n = 6 animals per group (A); n = 3 cell populations per treatment, 10 to 20 images per immunodetection (scale bar, 20 mm)] (B); *P < 0.05 compared to wild-type or vehicle control by Student’s t test. (C to F) Endothelial cell senescence as detected by SA-b-Gal labeling [C; HPAECs and human aortic endothelial cells (HuAoECs)] and increases in cell size (D; HPAECs). SA-b-Gal (E, left) and trichrome staining (E, right) were used on en face tissue to visualize senescence, gross lung vessel morphology, and collagen deposition (blue) in middle- aged wild-type and TSP1−/−lungs. The abundance of SASP factors [MCP-1, p19Arf, and interleukin-6 (IL-6)] was assessed by Western blotting in middle-aged TSP1−/−and age-matched wild-type controls (F; purple compared to gray bars). Data are means ± SEM (n = 3 independent experiments or n = 3 animals; 12 images per group). Scale bar, 100 mm. *P < 0.05 for TSP1 challenge compared to vehicle control or null compared to age-matched wild-type mice by Student’s t test. (G and H) NADPH-driven O2 •−(G; cytochrome c assay) and H2O2 (H; Amplex Red assay) production in HPAEC lysates. Data are means ± SEM (n = 4 biological replicates per treatment). *P < 0.05 compared to vehicle controls by Student’s t test. (I and J) En face cell-permeant O2 •−scavenger (Tiron)–inhibitable dihydroethidium (DHE)–ROS, as measured by fluorescence microscopy (I), and NADPH-driven homogenate O2 •−production, as measured by cytochrome c reduction (J). Data are means ± SEM (n = 3 to 6 mice per group). Scale bar, 50 mm. *P < 0.05 compared to wild-type controls by Student’s t test. a.u., arbitrary units.

Article Snippet: In these experiments, cells were pretreated with peptides for 1 hour before TSP1 (2.2 nM) challenge. siRNA gene silencing and Nox1 plasmid transfection HPAECs were seeded at a density to achieve a 75 to 80% confluence overnight and were subjected to either p53 or Nox1 siRNA transfection (Santa Cruz Biotechnology) using the Lipofectamine 2000 transfection reagent (Invitrogen) according to themanufacturer’s protocol, as documented previously (51).

Techniques: In Vitro, Marker, Western Blot, Immunofluorescence, Immunodetection, Control, Labeling, Staining, Amplex Red Assay, Fluorescence, Microscopy

Journal: Science signaling

Article Title: The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

doi: 10.1126/scisignal.aaj1784

Figure Lengend Snippet: Fig. 3. TSP1 induces DDR-p53-p21cip–mediated senescence. Data for TSP1−/−samples are depicted in purple; data for cell experiments are in red; data in gray are the corresponding controls. (A) Abundance of Rb, total and phospho-Ser15 p53, and p16INK4A after 24 hours of TSP1 challenge in HPAECs, as measured by Western blot. Data are means ± SEM (n = 3 biological replicates per treatment); *P < 0.05 compared to vehicle by Student’s t test. (B) Time-dependent changes in p53 abundance in HPAECs stimulated with TSP1, as measured by Western blot. Graphical data are means ± SEM (n = 3 biological replicates per treatment), by repeated-measures analysis. (C and D) Activation of the p53 pathway, as assessed by nuclear localization of p53 (C), and p21cip abundance (D), as measured by immunofluorescence, compared to vehicle controls. Data are means ± SEM (n = 3 biological replicates per treatment; 12 to 20 images per sample). Scale bar, 40 mm. *P < 0.05 for TSP1 challenge compared to vehicle control by Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole. (E) Effect of p53 knockdown by small interfering RNA (siRNA) on TSP1-induced senescence in HPAECs. Graphical data are means ± SEM (n = 3 biological replicates per treatment). Scale bar, 40 mm. *P < 0.05 for TSP1 challenge compared to vehicle control; #P < 0.05 for p53 siRNA-TSP1 compared to scrambled siRNA (SCR)–TSP1 by one-way analysis of variance (ANOVA). (F) mRNA expression of p53 and p21cip in middle-aged TSP1−/−animals. Data are means ± SEM (n = 3 individual animals for each group). *P < 0.05 compared to wild-type by Student’s t test. (G) Abundance of p21cip and total and phospho-p53 in middle-aged TSP1−/−lungs compared to wild-type, age-matched controls. Graphical data are means ± SEM (n = 6 animals per group). *P < 0.05 for TSP1−/−compared to wild-type by Student’s t test.

Article Snippet: In these experiments, cells were pretreated with peptides for 1 hour before TSP1 (2.2 nM) challenge. siRNA gene silencing and Nox1 plasmid transfection HPAECs were seeded at a density to achieve a 75 to 80% confluence overnight and were subjected to either p53 or Nox1 siRNA transfection (Santa Cruz Biotechnology) using the Lipofectamine 2000 transfection reagent (Invitrogen) according to themanufacturer’s protocol, as documented previously (51).

Techniques: Western Blot, Activation Assay, Immunofluorescence, Control, Knockdown, Small Interfering RNA, Expressing

Journal: Science signaling

Article Title: The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

doi: 10.1126/scisignal.aaj1784

Figure Lengend Snippet: Fig. 4. TSP1 induces Nox1-dependent O2 •−production in HPAECs through its receptor CD47. Data for TSP1−/−

Article Snippet: In these experiments, cells were pretreated with peptides for 1 hour before TSP1 (2.2 nM) challenge. siRNA gene silencing and Nox1 plasmid transfection HPAECs were seeded at a density to achieve a 75 to 80% confluence overnight and were subjected to either p53 or Nox1 siRNA transfection (Santa Cruz Biotechnology) using the Lipofectamine 2000 transfection reagent (Invitrogen) according to themanufacturer’s protocol, as documented previously (51).

Techniques:

Journal: Science signaling

Article Title: The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

doi: 10.1126/scisignal.aaj1784

Figure Lengend Snippet: Fig. 5. Nox1 as a potential therapeutic target to inhibit matricellular-mediated endothelial senescence. Data for Nox1OE are shown in blue; data for Nox1−/−samples are shown in orange; data for cell experiments are shown in red; data in gray are the corresponding controls. (A and B) Effect of human Nox1OE on SA-b-Gal staining and O2 •−production. Graph- icaldataare means ±SEM(n = 3 biological replicates per treatment). Scale bar, 40mm. *P < 0.05for Nox1 plasmid compared to empty plasmid control by Student’s t test. (C) NADPH-driven O2 •−production, as measured by cytochrome c reduction assay in middle-aged wild-type and Nox1−/−mouse lungs. Left: Representative cytochrome c kinetic curve. Right: Quan- tification of O2 ·−production. Bar graphs are means ± SEM (n = 6 mice per group). *P < 0.05 compared to wild-type by Student’s t test. (D) mRNA expression of p53 and p21cip in wild-type and Nox1−/−mouse lungs. Data are the means ± SEM (n = 3 mice per group); *P < 0.05 compared to wild type by Student’s t test. (E to G) Effect of Nox1 inhibition (using NoxA1ds) on TSP1-induced cell cycle arrest in HPAECs, as measured by MTTassay(E), trypan blue exclusionassay (F), and cell cycle profile analysis (G). Graphical data are means ± SEM (n = 3 to 4 biological replicates per treatment). *P < 0.05 for TSP1 challenge compared to scrambled vehicle control (SCRAMB) by one-way ANOVA. (H) Activation of p53 (red), as assessed bynuclear localization (DAPI-labeled, blue) and p21cip immunofluorescence (green) in HPAECs challenged with TSP1 in the presence or absence of the Nox1 inhibitor NoxA1ds. Graphical data are means ± SEM (n = 3 biological replicates per treatment, 12 to 20 images per group). Scale bar, 50 mm. *P < 0.05 for TSP1 challenge compared to scrambled vehicle control by one-way ANOVA.

Article Snippet: In these experiments, cells were pretreated with peptides for 1 hour before TSP1 (2.2 nM) challenge. siRNA gene silencing and Nox1 plasmid transfection HPAECs were seeded at a density to achieve a 75 to 80% confluence overnight and were subjected to either p53 or Nox1 siRNA transfection (Santa Cruz Biotechnology) using the Lipofectamine 2000 transfection reagent (Invitrogen) according to themanufacturer’s protocol, as documented previously (51).

Techniques: Staining, Plasmid Preparation, Control, Expressing, Inhibition, Activation Assay, Labeling, Immunofluorescence

Journal: Science signaling

Article Title: The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

doi: 10.1126/scisignal.aaj1784

Figure Lengend Snippet: Fig. 6. Potential clinical relevance of Nox1-mediated endothelial senescence in human pulmonary vascular tissue. Data points for human tissue are shown in green; data for cell experiments are shown in red; data in gray are the corresponding controls. Data for (A) to (E) are plotted as linear regression (n = 8 samples); equation, r 2, and P values are indicated in the corresponding graph. (A and B) Correlation between NADPH-driven O2 ·−production, as measured by cytochrome c reduction (A), or H2O2 production, as measured by Amplex Red fluorescence (B), and age in human lung homogenates. (C and D) Abundance of Nox1 (C) and p53 and p21cip (D), as measured by Western blot of total homoge- nates of aging human lung. (E) Intimal immunofluorescence for Nox1 (top, green) and p21cip (bottom, red) in aged human lung sections. Scale bar, 50 mm. Graphs show linear regression analyses. (F) HPAEC senescence induced by TSP1 in the presence or absence of NoxA1ds, as measured by SA-b-Gal staining. Graphical data are means ± SEM (n = 3 biological replicates per treatment). Scale bar, 40 mm. *P < 0.05 for TSP1 challenge compared to scrambled vehicle control by Student’s t test.

Article Snippet: In these experiments, cells were pretreated with peptides for 1 hour before TSP1 (2.2 nM) challenge. siRNA gene silencing and Nox1 plasmid transfection HPAECs were seeded at a density to achieve a 75 to 80% confluence overnight and were subjected to either p53 or Nox1 siRNA transfection (Santa Cruz Biotechnology) using the Lipofectamine 2000 transfection reagent (Invitrogen) according to themanufacturer’s protocol, as documented previously (51).

Techniques: Fluorescence, Western Blot, Immunofluorescence, Staining, Control

Journal: Science signaling

Article Title: The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

doi: 10.1126/scisignal.aaj1784

Figure Lengend Snippet: Fig. 7. A signaling model for TSP1-induced senescence. Upon binding to CD47, TSP1 activates the Nox1 complex, which generates robust and sustained accumulation of ROS. In turn, this triggers committed senescence through a pathway involving p53-p21cip–induced DNA damage response and decreased Rb phosphorylation. This pathway can be disrupted using the selective Nox1 in- hibitor NoxA1ds.

Article Snippet: In these experiments, cells were pretreated with peptides for 1 hour before TSP1 (2.2 nM) challenge. siRNA gene silencing and Nox1 plasmid transfection HPAECs were seeded at a density to achieve a 75 to 80% confluence overnight and were subjected to either p53 or Nox1 siRNA transfection (Santa Cruz Biotechnology) using the Lipofectamine 2000 transfection reagent (Invitrogen) according to themanufacturer’s protocol, as documented previously (51).

Techniques: Binding Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: Immunoblots confirming the absence of SHIP2 protein in independent ATDC5 (a) and Saos-2 clones (b). Wild-type cells plus two clones of ATDC5 (c) and SaOs-2 (d) genetically-depleted for SHIP2 differentiated and then stained with Alizarin Red S. ATDC5 (e) or SaOs-2 (f) cells differentiated in presence of DMSO (vehicle) or SHIP2 inhibitor AS1949490 (0-10 μM) and stained for mineralization with Alizarin Red S. Histograms represent the quantification of the cell staining after extraction. Data is representative of 3 individual experiments (3 wells per experiment). * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Western Blot, Clone Assay, Staining, Extraction

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Differentiated ATDC5 were stained for proteoglycans using Alcian Blue. Histograms represent the quantification of the cell staining after extraction. (b) Relative expression of hypertrophy and terminal differentiation markers (c) and matrix related genes in ATDC5-neg#1 cells. (d) Relative expression of osteoblast differentiation markers in SaOs-2 neg#1 cells. Histograms represent the difference in expression compared to WT cells (b, c, d). Data is representative of 3 individual experiments (3 wells per experiment). *p<0.05, **p<0.01, ***p<0.001. WT vs SHIP2-neg.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Staining, Extraction, Expressing

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Immuno-detection and quantification of MMP13 protein and (b) detection of MMP13 activity by collagen zymography in conditioned media of ATDC5 WT and SHIP2-negative ATDC5 cells. Data is representative of two individual experiments.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Activity Assay, Zymography

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) ATDC5 cells differentiated in presence of DMSO (VEH) or 1.5 μM of AS1949490 (top panels) and SHIP2-negative ATDC5 cells (bottom panels) were immunostained for PI(3,4,5)P3. Arrows indicate focal adhesion-like structures. (b) Immunodetection of Akt and pAKT (S473) protein in SHIP2-negative ATDC5 cells. Scale bars represent 0.01mm.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Immunodetection

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Wild-type and SHIP2-negative ATDC5 (top panels) and SaOs-2 (bottom panels) cells immunostained for vinculin. Arrows indicate focal adhesion-like structures. Table shows the number of focal adhesions per cell and their surface. (b) Cell adhesion assay showing increased ATDC5 and SaOs-2 attachment 30 min after seeding cells on fibronectin * p <0.05, ** p <0.01, *** p <0.001 for wild-type vs SHIP2-deleted cells. Scale bars represent 0.01 mm.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Cell Adhesion Assay

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: (a) Alizarin Red staining of ATDC5 and SaOs-2 WT cells vs SHIP2-negative cells differentiated in presence of 0.3μM of the actin polymerization inhibitor, cytochalasin D. (b) Alizarin Red staining of ATDC5 and SaOs-2 WT cells vs SHIP2-negative ATDC5 cells differentiated in presence of 15 μM of the ROCK pathway inhibitor, Y-27632. (c) Alcian Blue staining of ATDC5 WT cells vs SHIP2-negative cells differentiated in presence of 15 μM of the ROCK pathway inhibitor, Y-27632. Histograms represent the quantification of the cell staining after extraction. *,‡ p <0.05, **,‡‡ p <0.01, ***,‡‡‡ p <0.001. * WT vs SHIP2-neg. ‡ vehicle vs treatment.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Staining, Extraction

Journal: bioRxiv

Article Title: SHIP2 controls matrix mineralization by regulation of the RhoA/ROCK pathway and remodeling of the actin cytoskeleton

doi: 10.1101/2022.10.30.514432

Figure Lengend Snippet: In the absence of SHIP2, PI(3,4,5)P3 accumulates, activates ROCK which stimulates the production of ECM through the phosphorylation of Sox9 in chondrocytes. Increased ROCK activity also leads to stabilization of the actin cytoskeleton and inhibition of MV budding and ECM mineralization.

Article Snippet: ATDC5 and SaOS-2 cells were transfected with the mouse or human SHIP2 double nickase plasmid (Santa Cruz, sc-421138-NIC (mouse), sc-401622-NIC (human)) using the UltraCruz transfection reagent and the Plasmid transfection medium (Santa Cruz) following manufacturer’s instructions.

Techniques: Phospho-proteomics, Activity Assay, Inhibition