Journal: Signal Transduction and Targeted Therapy

Article Title: Anti-lymphangiogenesis for boosting drug accumulation in tumors

doi: 10.1038/s41392-024-01794-4

Figure Lengend Snippet: Anti-lymphangiogenesis activity of anlotinib and SAR131675 both in vitro and in vivo. a – c Anlotinib inhibited the proliferation ( a ), migration ( b ), and tubule network formation ( c ) of hLECs ( n = 4). Scale bar, 200 μm in ( b ) and 1000 μm in ( c ). d – f SAR131675 inhibited the proliferation ( d ), migration ( e ), and tubule network formation ( f ) of hLECs ( n = 4). Scale bar, 200 μm in ( e ) and 1000 μm in ( f ). g Quantification of the recovered area by hLECs migration as shown in ( b ) ( n = 3). h Quantification of the recovered area by hLECs migration as shown in and ( e ) ( n = 3). i Quantification of tube lengths as shown in ( c ) ( n = 3). j Quantification of tube lengths as shown in ( f ) ( n = 3). k Immunohistochemistry staining for LVs (LYVE-1, brown) in 4T1 tumor sections from mice treated with saline, anlotinib or SAR131675. Scale bar, 40 μm. l Quantification of tumor LV density ( n = 9; images were from three mice per group). m Typical images of inguinal LNs from different groups. Scale bar, 0.5 cm. The data are presented as the mean ± s.d. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The human lymphatic endothelial cell line (hLECs) was purchased from iCell, China.

Techniques: Activity Assay, In Vitro, In Vivo, Migration, Immunohistochemistry, Staining, Saline

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet: (A and B) OSS and Wnt/β-catenin signaling do not enhance FOXC2 and GATA2 expression in human umbilical vein endothelial cells (HUVECs). Primary human lymphatic endothelial cells (HLECs) or HUVECs were cultured under OSS for 48 hr (A) or in the presence of recombinant Wnt3a (rWnt3a) for 24 hr (B). Subsequently, RNA was extracted and analyzed by real-time qPCR for AXIN2, FOXC2 , and GATA2 . The expression levels were normalized to that of GAPDH. (C and D) PROX1 provides competence to HUVECs to respond to OSS and Wnt/β-catenin signaling and enhance the expression of FOXC2 and GATA2. HUVECs were infected with lentiviral particles expressing GFP or human PROX1 for 48 hr. Subsequently, the cells were exposed to (C) OSS for 48 hr or to (D) the Wnt agonist BIO for 12 hr. (C) PROX1-expressing HUVECs were cultured under static or OSS conditions with or without the Wnt antagonist iCRT3. Western blot was performed to quantify FOXC2 expression. (D) PROX1-expressing HUVECs were cultured with BIO or DMSO for 24 hr, and the expression of FOXC2 and GATA2 was quantified by real-time qPCR. (E and F) PROX1 is necessary for OSS- and Wnt/β-catenin signaling-mediated expression of FOXC2 and GATA2 in HLECs. HLECs were infected with lentiviral particles expressing shRNAs that target GFP (sh-Con) or PROX1 (sh-P1#1 and sh-P1#2) for 48 hr. Subsequently, HLECs were additionally cultured under OSS for 48 hr (E) or with rWnt3a for 24 hr (F). Real-time qPCR was performed to quantify the expression of FOXC2 and GATA2 . Statistics: n = 3 for all experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars in graphs represent ± SEM.

Article Snippet: Briefly, 1.0 X 10 7 HLECs (Lonza) at 90% confluency were treated with DMSO or Bio (0.5 μM) for 3 hours.

Techniques: Expressing, Cell Culture, Recombinant, Infection, Western Blot

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet: (A) PROX1 enhances the expression of Wnt/β-catenin signaling target genes in HLECs. HLECs were infected with lentiviral particles expressing shRNAs that target GFP (sh-GFP) or PROX1 (shPROX1#1 and sh-PROX1#2) for 72 hr. Subsequently, RNA was extracted, and real-time qPCR was performed for the expression of Wnt/β-catenin target genes. (B–D) PROX1 synergizes with β-catenin to enhance Wnt/β-catenin signaling. (B) 293T cells were co-transfected with TOPFlashor FOPFlash luciferase reporters with PROX1- and or β-catenin-expressing vectors. The TCF/LEF binding sites of TOPFLASH are inactivated to generate FOPFlash as a negative control for Wnt/β-catenin signaling. (C and D) 293T cells were co-transfected with TOPFlash- and PROX1-expressing vectors together with AXIN-expressing (C) or ΔN-TCF7L2expressing (D) plasmids. DN-TCF7L2 cannot interact with β-catenin and functions as a dominant-negative mutant. A Renilla luciferase-expressing plasmid was used as an internal control, and luciferase activities were measured 36 hr after transfection. (E–G) PROX1 associates with β-catenin. (E) 293T cells were transfected with Myc-tagged β-catenin and FLAG-tagged PROX1 plasmids. 48 hr later, the cell lysate was subjected to a co-immunoprecipitation assay using anti-FLAG antibody or anti-Myc antibody. The precipitates were probed by western blot using the indicated antibodies. (F) HLEC lysate was immunoprecipitated using anti-PROX1 antibody and probed by western blot using anti-PROX1 or anti-β-catenin antibodies. (G) HLECs were treated with BIO for the indicated number of hours. The lysates were analyzed as in (F). (H) HLECs were treated with DMSO or BIO (0.5 μM) for 3 hr, and ChIP was performed using anti-PROX1 antibody. qPCR was performed using primers flanking the TCF/LEF-binding site in the 3.5 kb location. As a negative control, primers flanking a TCF/LEF-binding site that is located at a more upstream location (5.5 kb) were used. Statistics: n = 3 for all experiments. **p < 0.01, ***p < 0.001. Error bars in graphs represent ± SEM.

Article Snippet: Briefly, 1.0 X 10 7 HLECs (Lonza) at 90% confluency were treated with DMSO or Bio (0.5 μM) for 3 hours.

Techniques: Expressing, Infection, Transfection, Luciferase, Binding Assay, Negative Control, Dominant Negative Mutation, Plasmid Preparation, Control, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet:

Article Snippet: Briefly, 1.0 X 10 7 HLECs (Lonza) at 90% confluency were treated with DMSO or Bio (0.5 μM) for 3 hours.

Techniques: Virus, Recombinant, Electron Microscopy, Transfection, SYBR Green Assay, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Software