Journal: Integrative Cancer Therapies

Article Title: Analyses of BMAL1 and PER2 Oscillations in a Model of Breast Cancer Progression Reveal Changes With Malignancy

doi: 10.1177/1534735419836494

Figure Lengend Snippet: Endogenous mRNA expression of BMAL1 and PER2 in synchronized cell lines belonging to the MCF10 series. Relative mRNA expression levels were determined via RT-PCR, normalized to GAPDH , using 3 biological replicates with 3 technical replicates each. Error bars represent relative error.

Article Snippet: For generation of PER2- luciferase ( PER2:Luc ) plasmid, a 159-bp EcoRI/NotI fragment was isolated from a pGL3 basic PER2 construct, obtained from Addgene (plasmid #48747, deposited by Dr Joseph Takahashi).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Integrative Cancer Therapies

Article Title: Analyses of BMAL1 and PER2 Oscillations in a Model of Breast Cancer Progression Reveal Changes With Malignancy

doi: 10.1177/1534735419836494

Figure Lengend Snippet: Circadian oscillations of (A) BMAL1:Luc and (B) PER2:Luc in the MCF10 series of cells. Shown for each cell line are the 21 time series for BMAL1 and 20 for PER2 obtained, de-trended and de-noised using a discrete wavelet transform. Both reporters show stable oscillations across cellular transformations from nonmalignant MCF10A (blue) to premalignant MCF10AT.Cl2 (orange) to malignant MCF10Ca1h (yellow) and MCF10Ca1a cells (purple). However, BMAL1 has more time-series out of phase with the majority (gray; N = 4 for MCF10A, N = 2 for MCF10A.Cl2, N = 4 for MCF10Ca1h, and N = 6 for MCF10Ca1a) versus PER2 (gray; N = 5 for MCF10A).

Article Snippet: For generation of PER2- luciferase ( PER2:Luc ) plasmid, a 159-bp EcoRI/NotI fragment was isolated from a pGL3 basic PER2 construct, obtained from Addgene (plasmid #48747, deposited by Dr Joseph Takahashi).

Techniques:

Journal: Integrative Cancer Therapies

Article Title: Analyses of BMAL1 and PER2 Oscillations in a Model of Breast Cancer Progression Reveal Changes With Malignancy

doi: 10.1177/1534735419836494

Figure Lengend Snippet: Peaks and troughs of normalized (A) BMAL1:Luc and (B) PER2:Luc show that the first cycles of most oscillations are in phase and that PER2 and BMAL1 are antiphase. Heat maps are included, representing low (blue) to high (yellow) bioluminescence with one row per time series, organized by cell line (color bar on right indicating blue for MCF10A, orange for MCF10AT.Cl2, yellow for MCF10Ca1h, and purple for MCF10Ca1a cells). Black bars indicate recordings where no data were recorded at that time. Peaks (black triangles) and troughs (white triangles with black outline) are indicated for each recording and are used to determine outliers (for BMAL1:Luc , outliers trough after t = 48 hours, for PER2:Luc , outliers peak before t = 35 hours). Outliers are indicated by a gray entry in the first column, versus white for non-outliers.

Article Snippet: For generation of PER2- luciferase ( PER2:Luc ) plasmid, a 159-bp EcoRI/NotI fragment was isolated from a pGL3 basic PER2 construct, obtained from Addgene (plasmid #48747, deposited by Dr Joseph Takahashi).

Techniques:

Journal: Integrative Cancer Therapies

Article Title: Analyses of BMAL1 and PER2 Oscillations in a Model of Breast Cancer Progression Reveal Changes With Malignancy

doi: 10.1177/1534735419836494

Figure Lengend Snippet: Continuous wavelet transforms (CWT) estimate circadian periods for (A) BMAL1 and (B) PER2 over time. Shown are Morlet-wavelet estimates of the period of oscillation for each cell line and reporter (blue for MCF10A, orange for MCF10AT.Cl2, yellow for MCF10Ca1h, and purple for MCF10Ca1a). The periods of outlier time series are shown in gray.

Article Snippet: For generation of PER2- luciferase ( PER2:Luc ) plasmid, a 159-bp EcoRI/NotI fragment was isolated from a pGL3 basic PER2 construct, obtained from Addgene (plasmid #48747, deposited by Dr Joseph Takahashi).

Techniques:

Journal: Nature Communications

Article Title: Concurrent BMP7 and FGF9 signalling governs AP-1 function to promote self-renewal of nephron progenitor cells

doi: 10.1038/ncomms10027

Figure Lengend Snippet: ( a , b ) CCNE1 (G1-phase) and PCNA (S-phase) immunostaining of E14.5 and E17.5 NPCs treated for 24 h with BMP7, TAK1 and JNK inhibitors. Graphs show the percentage of G1, G1-S and S-phase cells per condition. Three biological replicates analysed per condition ( n =3). Error bars represent s.d. ** P <0.005 and ** P <0.001, Student's t -test. ( c , d ) RT–qPCR of JUN and MYC targets in E14.5 and E17.5 NPCs treated with vehicle, BMP7, TAK1 and JNK inhibitors for 2 h. Error bars indicate s.d. Three biological replicates analysed per condition ( n =3). ( e , f ) RT–qPCR of JUN and MYC targets on NPCs isolated from E14.5 Jun het , Jun NP C and E17.5 Tak1 het , Tak1 NPC kidneys. Error bars represent s.d. Two biological replicates analysed per genotype ( n =2). ( g , h ) CCND1 and CCNE1 immunostaining in E14.5 Jun het , Jun NPC and E17.5 Tak1 C-WT , Tak1 C-NPC kidneys. Scale bars, 100 and 150 μM. Quantitation of CCND1+ and CCNE1+ cells per cap mesenchyme (yellow arrows) was calculated by scoring at least 30 random cap mesenchymes per kidney section per genotype for a total of 5 sections from each experimental group. Error bars indicate mean (s.d.). ** P <0.005 and ** P <0.001, Student's t -test. ( i ) Model for BMP7-TAK1-JNK-JUN pathway regulation of NPC self-renewal in early (E14.5) and later (E17.5) phases of nephrogenesis. CD, collecting duct; CM, cap mesenchyme.

Article Snippet: 3 × AP1-pGL3 was a gift from Dr Alexander Dent (Addgene # 40342) . pGL3Basic-962 CCND1 promoter luciferase), pGL3Basic-962 CCND1 promoter AP-1 site mutant (Addgene #32727 and # 32728) were gifts from Dr Frank McCormick . pRL-CMV (Renilla-Luciferase) was obtained from Promega.

Techniques: Immunostaining, Quantitative RT-PCR, Isolation, Quantitation Assay

Journal: Nature Communications

Article Title: Concurrent BMP7 and FGF9 signalling governs AP-1 function to promote self-renewal of nephron progenitor cells

doi: 10.1038/ncomms10027

Figure Lengend Snippet: ( a ) Immunostaining of EdU (S-phase) and pHH3 (M-phase), ( b ) CCNE1 (G1-phase) and PCNA (S-phase) in E17.5 NPCs stimulated with vehicle, BMP7, FGF9 or BMP7+FGF9 for 24 h. Scale bars, 50 μM. Graphs show the percentage of EdU+,pHH3+ cells and G1, G1-S and S-phase cells in each condition. Error bars represent mean (s.d.). ** P <0.005 and P <0.001 (Student's t -test). Two to three biological replicates analysed per condition ( n =2 in a ) and ( n =3 in b ) ( c , d ) RT–qPCR of cell cycle genes ( Ccnd1 and Myc ), Jun and Fos in NPCs stimulated with vehicle, BMP7, FGF9 or BMP7+FGF9 for 2 h. Error bars represent s.d. Three biological replicates analysed per condition, n =3. ( e , f ) pJUN and pFOS immunoblot of NPCs stimulated with vehicle, BMP7, FGF9 or BMP7+FGF9 for 20 min. Graph shows the relative density of pJUN and pFOS normalized to β-tubulin in each condition . ( g – i ) Bars in the graphs represent the average fold change in luciferase activity of 3xAP1-Luc, CCND1-Luc and CCND1 ΔAP-1 -Luc in NPCs stimulated with BMP7 and FGF9 relative to vehicle treatment for 24 h. Three biological replicates analysed per condition ( n =3). Error bars represent s.d. ** P <0.005, NS, not significant P >0.05, Student's t -test.

Article Snippet: 3 × AP1-pGL3 was a gift from Dr Alexander Dent (Addgene # 40342) . pGL3Basic-962 CCND1 promoter luciferase), pGL3Basic-962 CCND1 promoter AP-1 site mutant (Addgene #32727 and # 32728) were gifts from Dr Frank McCormick . pRL-CMV (Renilla-Luciferase) was obtained from Promega.

Techniques: Immunostaining, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A CRISPR-Cas9 knockout screening identifies IRF2 as a key driver of OAS3/RNase L-mediated RNA decay during viral infection

doi: 10.1073/pnas.2412725121

Figure Lengend Snippet: IRF2 promotes OAS3 expression in unstressed cells. ( A and B ) The levels of OAS3, IRF2, RNase L, and GAPDH were analyzed in the indicated U2OS ( A ) or A549 ( B ) cell lines by western blot. ( C ) The OAS3 mRNA levels were monitored by RT-qPCR in indicated U2OS ( Left ) or A549 ( Right ) cell lines. Mean values ± SD (n = 3). **** P < 0.0001 (two-tailed t test). ( D ) The levels of OAS3 were analyzed by western blot using indicated antibodies in A549 IRF2 KO cells expressing either wild-type IRF2 or DNA binding mutant IRF2K78R. ( E and F ) Total RNAs were isolated from A549 WT or IRF2 KO cells expressing wild-type IRF2, IRF2K78R, or OAS3 and monitored for integrity by a bioanalyzer after transfection with poly(I:C) (10 ng/mL, 4 h). The red arrows indicated ribosomal RNA cleavage products. ( G ) IgG and IRF2 ChIP-sequencing in A549 cells. Analysis of ChIP-sequencing data focuses on the promoter region of OAS3. (H) IRF2 ChIP was performed in A549 WT and IRF2 KO cells. IRF2 binding on the OAS3 promoter was determined by qPCR. Mean values ± SD. *** P < 0.001 (two-tailed t test). ( I ) Schematic of pGL3-OAS3-luciferase constructs harboring OAS3 WT promotor region (−1 to −900 bp upstream of transcription start site [TSS]) and IRF2 binding motif region deleted (Δ−1 to −200 bp). ( J ) The relative luciferase activity (ratio of Firefly:Renilla) was measured in U2OS cells transiently transfected with the empty pGL3 and pGL3 vector harboring either WT and Δ200 bp OAS3 promoter region. ( K ) The relative luciferase activity was monitored U2OS WT or IRF2 KO cells 24 h following transfection with the pGL3-OAS3 vector.

Article Snippet: The OAS3 luciferase reporter plasmid was generated by cloning the OAS3 promoter region (TSS −1 to −900) into pGL3 basic luciferase reporter (Addgene #128046).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test, Binding Assay, Mutagenesis, Isolation, Transfection, ChIP-sequencing, Luciferase, Construct, Activity Assay, Plasmid Preparation