Journal: Nature

Article Title: Hypothalamic Programming of Systemic Aging Involving IKKβ/NF-κB and GnRH

doi: 10.1038/nature12143

Figure Lengend Snippet: a–c Hypothalamic GnRH mRNA of indicated mice. d–g . GT1–7 cells were transfected with CA IKKβ, RelA or DN IκBα vs. control (Con) plasmid (d,e,g) , co-transfected with GnRH -promoter luciferase plasmid (e&f) , or together with RelA shRNA (sh-RelA) vs. control shRNA (sh-Con) plasmid (f) , and were measure for GnRH release (d) , GnRH promoter (e&f) , and c-Fos , c-Jun , PKCα and PKCδ mRNA levels (g). h . GnRH promoter activities were measured for GT1–7 cells transfected with GnRH -promoter luciferase plasmid, co-transfected with c-Jun or c-Fos plasmid vs. control plasmid (Con), or treated with TPA vs. vehicle (Veh). i . GnRH promoter activities were measured for GT1–7 cells transfected with GnRH -promoter luciferase plasmid, co-transfected with CA IKKβ vs. control (Con) plasmid, and with c-Fos /c- Jun shRNA plasmids (sh-c-Fos/sh-c-Jun) vs. scramble shRNA control (sh-Con). j . Summarized schematic model. *P < 0.05, **P < 0.01, ***P < 0.001; n = 12 (a&e) and 3 (f–i) per group, and n = 6 (b) , 8 (c) and 4 (d) in Con, n = 8 (b) and 6 (d) in IKKβ, and n = 8 (c) and 6 (d) in IκBα. Error bars reflect mean ± SEM.

Article Snippet: RelA shRNA and control (GFP) shRNA vectors were obtained from Addgene (# 22507, 22508, and 31848), as studied in the literature (RelA shRNA: GCA-TGC-GAT-TCC-GCT-ATA-A, control shRNA: ACA-GCC-ACA-ACG-TCT-ATA-T).

Techniques: Transfection, Control, Plasmid Preparation, Luciferase, shRNA

Journal: Nature Communications

Article Title: CDK12 promotes tumorigenesis but induces vulnerability to therapies inhibiting folate one-carbon metabolism in breast cancer

doi: 10.1038/s41467-022-30375-8

Figure Lengend Snippet: a Two CDK12 HIGH (Pt#1 and #2) and two CDK12 LOW (Pt#3 and #4) breast cancer patient-derived xenografts (PDXs) were silenced with a lentiviral CDK12 shRNA or with a control shRNA (Ctr), and injected orthotopically into the mammary fat pads of NGS mice. Tumor volume was measured at the endpoint (4–6 weeks post-injection). Bars represent the mean ± SD (6 mice per experimental group). *** P < 0.001 vs. Ctr-KD cells, two-sided unpaired t -test. b Kaplan–Meier survival analysis of mice injected intracardiacally with CDK12 HIGH Pt#1 and Pt#2, or CDK12 LOW Pt#3 PDX cells treated as in ( a ). Individual mice were sacrificed when signs of distress or severe debilitation became manifest. Number of mice/experimental group: PT#1, 14 for Ctr-KD and 13 for CDK12-KD; Pt#2, 23 for Ctr-KD and 12 for CDK12-KD; Pt#3, 14 for Ctr-KD and 14 for CDK12-KD. P, P- values, by log-rank test. c RT-qPCR analysis of the indicated SGOC genes in CDK12 HIGH PDX cells (Pt#1 and Pt#2) silenced or not for CDK12 as in ( a ). Data are expressed as relative to control shRNA (Ctr-KD = 1) for each condition. Bars represent the means ± SEM ( n = 5, Pt#1; n = 3, Pt#2). * P < 0.05; ** P < 0.01; *** P < 0.001, vs. Ctr-KD, two-sided unpaired t -test. d Relative abundance of the indicated folate cycle metabolites in CDK12 HIGH PDX-derived cells control- (Ctr-KD) or CDK12-silenced (CDK12-KD), identified by LC-MS metabolomics. Data are expressed as relative to an internal standard (reserpine) and are means ± SD ( n = 3). ** P = 0.001; *** P < 0.001, vs. Ctr-KD, two-sided unpaired t -test. e Three-day in vitro proliferation of CDK12 HIGH (Pt #1 and #2) vs. CDK12 LOW (Pt #3) cells, control silenced (Ctr-KD) or silenced for CDK12 (CDK12-KD), PSAT1 (PSAT1-KD) and MTHFD1 (MTHFD1-KD), or cultured in the presence of THZ531 (100 nM). Data are expressed as relative to day 3 in each condition and are the mean ± SD ( n = 3). *** P < 0.001 relative to Ctr-KD in each condition, two-sided unpaired t -test. Source data are provided as Source Data file.

Article Snippet: PsicoR-shRNA lentiviral vectors were used to abrogate human CDK12 expression.

Techniques: Derivative Assay, shRNA, Control, Injection, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy, In Vitro, Cell Culture

Journal: PLoS ONE

Article Title: Disruption of microRNA Biogenesis Confers Resistance to ER Stress-Induced Cell Death Upstream of the Mitochondrion

doi: 10.1371/journal.pone.0073870

Figure Lengend Snippet: A) QRT-PCR for pri-miR-17 in HCT116 WT and Exn5/Exn5 cells. B) QRT-PCR for miRNAs of the miR-17-92 cluster and others in HCT116 WT and Exn5/Exn5 cells. C) QRT-PCR for Dicer mRNA in HCT116 EV and DICER shRNA cells. D) QRT-PCR for Pri-miRNA-17 in HCT116 EV and DICER shRNA cells. E) QRT-PCR for selected miRNAs in HCT116 EV and DICER shRNA cells.

Article Snippet: HCT116 WT cells were virally transduced with pSicoR- empty vector (EV) -puromycin and a pSicoR- puromycin DICER targeting shRNA vector lentivirus (Addgene 14763_Tyler Jacks lab).

Techniques: Quantitative RT-PCR, shRNA

Journal: PLoS ONE

Article Title: Disruption of microRNA Biogenesis Confers Resistance to ER Stress-Induced Cell Death Upstream of the Mitochondrion

doi: 10.1371/journal.pone.0073870

Figure Lengend Snippet: A) HCT116 WT and Exn5/Exn5 cells were treated with a range of concentrations of Bfa for 24 h (LHS) or of Tm for 48 h (RHS). Flow cytometry based measurement of Annexin V/PI positive cells was used to estimate % cell death. B) HCT116 WT and Exn5/Exn5 cells were treated with 500 ng/ml of Tm, 500 ng/ml of Bfa, 100 µM of Etop and 250 nM of Sts for 24 or 48 h. Flow cytometry based measurement, of Annexin V/PI positive cells was used to estimate % cell death. C) Western blots for apoptotic caspases-3 and 9 as well as downstream substrate PARP following treatment of HCT116 WT and Exn5/Exn5 cells with 500 ng/ml of Bfa for 12 h and 24 h. D) HCT116 EV and DICER shRNA cells were treated with 500 ng/ml of Tm and 500 ng/ml of Bfa for 24 h and % cell death was estimated by flow cytometry based measurement of Annexin V/PI.

Article Snippet: HCT116 WT cells were virally transduced with pSicoR- empty vector (EV) -puromycin and a pSicoR- puromycin DICER targeting shRNA vector lentivirus (Addgene 14763_Tyler Jacks lab).

Techniques: Flow Cytometry, Western Blot, shRNA

Journal: PLoS ONE

Article Title: Disruption of microRNA Biogenesis Confers Resistance to ER Stress-Induced Cell Death Upstream of the Mitochondrion

doi: 10.1371/journal.pone.0073870

Figure Lengend Snippet: A) QRT-PCR for Drosha in HCT116 WT, and DROSHA shRNA cells, with or without 500 ng/ml of Doxycycline. B) QRT-PCR for Pri-miRNA-17 WT and DROSHA shRNA cells with Doxycycline for three days. C) QRT-PCR for miRNA in WT and DROSHA shRNA cells with or without Doxycycline. D) Western blot for cleaved caspase-3 following 500 ng/ml of Bfa for 24 h and 48 h in the presence of doxycycline in HCT116 WT and DROSHA shRNA cells. E) HCT116 WT and DROSHA shRNA cells exposed to Doxycycline for 3 days were treated with 500 ng/ml of Tm and 500 ng/ml of Bfa for 24 and 48 h. Flow cytometry based measurement of the percentage of cells that lose TMRE (% TMRE negative cells) was used to indicate the % of cell death.

Article Snippet: HCT116 WT cells were virally transduced with pSicoR- empty vector (EV) -puromycin and a pSicoR- puromycin DICER targeting shRNA vector lentivirus (Addgene 14763_Tyler Jacks lab).

Techniques: Quantitative RT-PCR, shRNA, Western Blot, Flow Cytometry