plx304 yap1 v5 addgene addgene  (Addgene inc)

Journal: Cell Reports Medicine

Article Title: Inhibiting KRAS with CD47 and immune checkpoint overcomes intrinsic resistance to combined KRAS and immune checkpoint inhibitor therapy

doi: 10.1016/j.xcrm.2025.102317

Figure Lengend Snippet: FOXA1 binds to the super-enhancer region of CD47 and regulates its expression (A) ATAC-seq signal for the CD47 gene in NCI-H358 cells. Arrows are pile-up reads in super-enhancer regions (peak A, blue; and peak B, green) and a promoter region (peak C, orange). (B) Flowchart identifying transcription factors that regulate CD47 is shown on the left. Fold change in expression of each candidate transcription factor in NCI-H358 cells transfected with constitutively active YAP (YAP S6A) or in NCI-H358 cells treated with 1 μM sotorasib for 72 h relative to NCI-H358 cells is shown on the right. (C) qPCR analysis of mRNA levels of FOXA1 normalized to ubiquitin following treatment with 1 μM sotorasib for 72 h in NCI-H358 and LU65 cells. Data are mean ± SD ( n = 3 independently treated cell cultures, two-sided Student’s t test, ∗ p < 0.05). (D) NCI-H358 and LU65 cells were treated with DMSO or 1 μM sotorasib at the indicated time points. Cell lysates were probed with the indicated antibodies. (E and F) NCI-H358 and LU65 cells transduced with control sgRNA or sgFOXA1 were treated with 1 μM sotorasib for 72 h. Cell lysates were probed with the indicated antibodies. Western blot was repeated 2 independent times with similar results (E). The MFI of CD47 on NCI-H358 and LU65 cells is shown (F). The data represent MFI ± SD of 3 wells, ∗ p < 0.05 by t test. (G) ChIP-qPCR analysis shows significantly more enrichment of FOXA1 at the super-enhancer regions of the CD47 gene. Data are mean ± SD ( n = 3 independently treated cell cultures, two-sided Student’s t test, ∗ p < 0.05).

Article Snippet: pLX304-YAP S6A , Addgene , Plasmid 42562.

Techniques: Expressing, Transfection, Ubiquitin Proteomics, Transduction, Control, Western Blot, ChIP-qPCR

Journal: Science Advances

Article Title: Regulation of feather length: FGF/IGF signaling and NOTCH/YAP modulation of progenitor cell topology

doi: 10.1126/sciadv.adw2382

Figure Lengend Snippet: ( A ) Examples of main sickle, lesser sickle, and tail feathers collected from a 1-year-old male Phoenix chicken. ( B ) Comparison of main sickle feathers between White Leghorn chicken and Phoenix chicken shows similar growth rate. For White Leghorn chicken, n = 13. For Phoenix chicken, n = 22. n.s., not significant. ( C ) Comparison of regenerated contour, lesser sickle, and main sickle feathers from Phoenix chickens. ( D ) LRC and TA double staining. ( D′ ) High magnification. Yellow arrows indicate the LRC/TA–double positive cells. ( E ) Calculation of percentage of LRC/TA–double positive cells. *** P < 0.001. ( F ) Schematic drawing to summarize the epidermal progenitor cell zone configuration difference between main sickle feathers in White Leghorn chickens and Phoenix chickens. ( G ) CellChat analysis shows the increased cell-cell interaction in superlong Phoenix main sickle feathers. ( H ) Comparison of NOTCH pathway cell-cell interactions in White Leghorn chicken long feathers and Phoenix chicken superlong feathers. ( I ) RNAscope analysis of YAP1/DLL1/NOTCH1 showing DLL1 expression domain are expanded (red arrow). In Phoenix chicken superlong feathers, the expression patterns of both NOTCH1 and YAP1 are expanded and curve around the follicle base, extending toward the DS (green arrow). ( J ) Summary of YAP1/DLL1/NOTCH1 expression in White Leghorn chicken and Phoenix chicken sickle feather follicles. ( K ) Summary of enhanced NOTCH, WNT, and HIPPO pathway activities in superlong Phoenix sickle feathers.

Article Snippet: For the RCAS–dominant negative form YAP1 (RCAS-dnYAP1) cloning, plasmid pLX304-YAP1 _60-89, encoding dominant-negative YAP1, was obtained from Addgene (W. Hahn’s group).

Techniques: Comparison, Double Staining, RNAscope, Expressing

Journal: Science Advances

Article Title: Regulation of feather length: FGF/IGF signaling and NOTCH/YAP modulation of progenitor cell topology

doi: 10.1126/sciadv.adw2382

Figure Lengend Snippet: ( A and B ) Disruption of Notch signaling by misexpression NICD shows short feather filaments. ( C to F′ ) Comparison of H&E, PCNA, and YAP1 staining between control and RCAS-NICD–infected samples. Note the precocious barb ridge formation (C′) and the lack of PCNA-positive cells in the epidermis (D′) (pink arrow). RCAS-NICD samples show the disappearance of YAP1 in the epidermis (E′) (red arrow) but a paradoxical increase in YAP1 in the PP. ( G and H ) Inhibition of YAP activity converts the tapering feather filament into short, stubby, cylindrical filaments. ( I to L′ ) Comparison of control and RCAS-dnYAP1–infected samples. Note the reduced thickness of TA cell zone (J′) (white arrow) and intermediate layer (K′) (green arrow). There are large numbers of NOTCH1-positive PP cells. The distal end fails to form barb branches. ( M and N ) Inhibition of WNT signaling by RCAS-WIF1 reduces feather length. ( O to R′ ) Comparison of control and RCAS-WIF1–infected samples. Note the reduced feather follicle width (O′), the reduction of PCNA-positive cells in the epidermis (P′) (yellow arrow), and reduced YAP1 expression (Q′) (blue arrow). ( S ) Table summarizing the findings using RCAS-mediated gene misexpression. ( T ) Model of YAP1 interacted with the DLL/NOTCH pathway regulating feather length by fine-tuning the progenitor cell topology. Compared to short feathers, long feathers have a bigger stem cell zone marked by DLL1 expression (red color). The Phoenix chicken main sickle feathers have an elongated stem cell zone and higher YAP1 activity that may contribute to the longer growth period. Left: Schematic drawing of a feather follicle. For [(B), (H), and (N)], five flight feather filaments from the right (experiment) and left (control) wings were measured. *** P < 0.001.

Article Snippet: For the RCAS–dominant negative form YAP1 (RCAS-dnYAP1) cloning, plasmid pLX304-YAP1 _60-89, encoding dominant-negative YAP1, was obtained from Addgene (W. Hahn’s group).

Techniques: Disruption, Comparison, Staining, Control, Infection, Inhibition, Activity Assay, Expressing