Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: A) YAP1 mutants used in the rescue experiment in (B). B) Viability (Cell Titer Glo) of CTRL cells or PC-9 YAP1 KO cells transduced with YAP1 mutants (A) following 72h treatment with OT. C) Top: the structure of MYF-01–37. Bottom: MYF-01–37 binding to the palmitoylation pocket in TEAD1 based on molecular docking. The cysteine 359 targeted by MYF-01–37 is indicated. D) Effect of MYF-01–37 or the corresponding reversible control on YAP/TEAD interaction measured using Split Gaussia Luciferase Assay. E) Left: Western bot analysis of the expression of myc-tagged TEAD1 in PC-9 cells transduced as indicated. Right: QPCR analysis of CTGF expression after 24h treatment with XAV939 or MYF-01–37 in the transduced PC-9 cells. F) YAP activity in PC-9 YAP reporter cells after 72h treatment with OT or OT in combination with XAV939 (XAV) or MYF-01–37 (MYF). G) QPCR analysis of BMF expression in cells in (E) following 24h treatment as indicated. H) Apoptosis in PC-9 and HCC4006 cells treated as indicated. G) Regrowth of PC-9 and HCC4006 cells after drug washout following a two-week treatment as indicated. Mean ± SEM are shown in all plots except (E), where mean ± SD is shown. ANOVA was used for statistical analyses. ***, P<0.001; **, P<0.01. See also Figure S8.

Article Snippet: The TEAD1 C359C mutation was generated into pRK5-myc- TEAD1 backbone (a gift from Kunliang Guan, Addgene plasmid #33109) by PCR using primers 5’-TCCCCAATGAGTGAATATATGATCAAC-3’ and 5’-GCGGTTTATTCGGTATACAAATCG-3’.

Techniques: Transduction, Binding Assay, Luciferase, Western Blot, Expressing, Activity Assay

Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The TEAD1 C359C mutation was generated into pRK5-myc- TEAD1 backbone (a gift from Kunliang Guan, Addgene plasmid #33109) by PCR using primers 5’-TCCCCAATGAGTGAATATATGATCAAC-3’ and 5’-GCGGTTTATTCGGTATACAAATCG-3’.

Techniques: Recombinant, In Vivo, In Vitro, Synthesized, Expressing, Staining, Cell Viability Assay, TaqMan Assay, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins

doi: 10.1101/2023.09.03.556116

Figure Lengend Snippet: A) Immunofluorescent labeling of endogenous CaMKIIα and the GLuc-SERCaMP in rat primay neurons transduced with AAV1 CaMKIIα GLuc-SERCaMP. B) Extracellular (n=5-6/group) and C, intracellular levels of GLuc-SERCaMP following treatment with following thapsigargin (Tg) (100 nM) (n=4/group). D) Intracellular levels of GLuc-SERCaMP 24 h following treatment with glutamate (n=3/group). E) Correlation between GLuc-SERCaMP secretion and viability glutamate treatment (n=3/group, Pearson correlation). F) Intracellular levels of GLuc-SERCaMP 24 h following treatment with kainic acid (n=3/group). G) Correlation between GLuc-SERCaMP secretion and viability kainic acid treatment (n=3/group, Pearson correlation). Data are shown as mean + SEM. *p<.05, **p<.01 vs. vehicle. *p<.05, ***p<.0005, ****p<.0001 vs. vehicle. 40X magnification, scale bars = 75 µm.

Article Snippet: All plasmids were constructed using ligation-independent cloning (In-Fusion, Clontech) into the backbone of pAAV CaMKII iRFP-FLAG (Addgene 149513) after digestion with NheI and AscI restriction enzymes (New England Biolabs) and insert amplification by polymerase chain reaction.

Techniques: Labeling, Transduction

Journal: Frontiers in Cell and Developmental Biology

Article Title: Inhibition of GSK3 Represses the Expression of Retinoic Acid Synthetic Enzyme ALDH1A2 via Wnt/β-Catenin Signaling in WiT49 Cells

doi: 10.3389/fcell.2020.00094

Figure Lengend Snippet: Wnts affect ALDH1A2 expression in WiT49 cells. (A) Effects of Wnts on TCF reporter activity in WiT49 cells. 400 ng TOPFlash or FOPFlash constructs with 100 ng pRK5-mWnts expression vectors or pRK-5 empty vector, as well as 25 ng of the pRL-TK renilla control vector, was transiently transfected into WiT49 cells for 48 h. The pRK5-mWnts include pRK5-mWnt1, pRK5-mWnt2b, pRK5-mWnt3a, pRK5-mWnt4, pRK5-mWnt5a, pRK5-mWnt6, pRK5-mWnt7a, pRK5-mWnt7b, pRK5-mWnt9b, and pRK5-mWnt11. Luciferase reporter activities relative to renilla (pRL-TK) are shown. Values are the mean ± SD of four determinations; representative results from at least three separate experiments are shown. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 compared with their respective controls, calculated using the Student’s t -test. (B,C) Effect of Wnts on ALDH1A2 expression in WiT49 cells. WiT49 cells were transiently transfected with pRK5-mWnt1, pRK5-mWnt3a, pRK5-mWnt4, pRK5-mWnt9b, or the empty vector pRK5 for 48 h. The ALDH1A2 expression relative to TBP was measured by real-time RT-PCR (B) . Values are the mean ± SD of three determinations; representative results from at least three separate experiments. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 compared with the empty vector pRK5 control group. The ALDH1A2 protein expression was measured by Western blot (C) . pRM-67-ALDH1A2, positive control of ALDH1A2 protein made by transfection of mammalian expression vector pReceiver-M67-ALDH1A2 to WiT49 cells. pRM-67, negative control of ALDH1A2 protein generated by transfection of pReceiver-M67 empty vector. Per lane, 50 μg of the sample was loaded, and 2 μg ALDH1A2 protein-positive control was loaded. Mock represents samples of cells treated with transfection reagent and without plasmid DNA. Values under bands were calculated by the density of bands of ALDH1A2 divided by the density of bands of GAPDH.

Article Snippet: Expression plasmids of multiple Wnts, including pRK5-mWnt1 (Addgene #42273), pRK5-mWnt2b (Addgene #42275), pRK5-mWnt3a (Addgene #42277), pRK5-mWnt4 (Addgene #42278), pRK5-mWnt5a (Addgene #42279), pRK5-mWnt6 (Addgene #42281), pRK5-mWnt7a (Addgene #42282), pRK5-mWnt7b (Addgene #42283), pRK5-mWnt9b (Addgene #42287) and pRK5-mWnt11 (Addgene #42290), were gifts from Chris Garcia and Jeremy Nathans ( ). pRK5 empty vector was made by re-ligation of the backbone of pRK5-mWnt4 (Addgene #42278) digested by Pst I. ALDH1A2 promoter or intron1G was cloned using the primers shown in .

Techniques: Expressing, Activity Assay, Construct, Plasmid Preparation, Transfection, Luciferase, Quantitative RT-PCR, Western Blot, Positive Control, Negative Control, Generated