Journal: Frontiers in Pharmacology

Article Title: Apigenin promotes melanogenesis and melanosome transport through the c-KIT/Raf-1/MAPK/CREB pathway in HEMCs

doi: 10.3389/fphar.2025.1572878

Figure Lengend Snippet: Apigenin promotes pigmentation independent of the classic MC1R/cAMP/PKA-pigmentation pathway in HEMCs. (A) HEMCs were treated with apigenin (10 μM) for the indicated time periods (0–180 min), and the phosphorylation of PKA and CREB was assessed by Western blotting. (B) HEMCs were pretreated with or without 10 μM DDA (an AC inhibitor) for 1 h before the addition of apigenin for 48 h. Melanin content, as well as the expression levels of Tyrosinase, MITF, Cdc42, and Rab27a, were measured as described previously. (C) HEMCs were pretreated with or without 10 μM N-1A (an MC1R inhibitor) for 1 h before apigenin treatment for 48 h. Melanin content and the expression levels of Tyrosinase, Cdc42, and Rab27a were measured as described previously. Data are expressed as the mean ± SEM (n = 3). *p < 0.05 vs. untreated cells. MC1R, melanocortin-1 receptor; cAMP, Cyclic Adenosine Monophosphate; PKA, protein kinase A; HEMCs, human epidermal melanocytes; CREB, cAMP response element-binding protein; AC, adenylate cyclase; MITF, Melanocytes inducing transcription factor; Cdc42, cell division cycle 42; Rab27a, Ras-related protein Rab-27a.

Article Snippet: Anti-Myosin Va (sc-365986, 1:200), KIF5b (sc-133184, 1:200), Cdc42 (sc-8401, 1:200), Rab27a (sc-74586, 1:200), p-c-KIT (sc-365504, 1:200), c-KIT (sc-13508, 1:200), p-Raf-1 (sc-271929, 1:200), Raf-1 (sc-7267, 1:200), p-p38 (sc-166182, 1:200), p38 (sc-398546, 1:200), p-ERK (sc-7383, 1:200), ERK (sc-514302, 1:200), p-RSK (sc-377526, 1:200), RSK (sc-74575, 1:200), p-MEK (sc-81503, 1:200), MEK (sc-13159, 1:200), MSK1 (sc-518173, 1:200), p-PKA (sc-293036, 1:200), PKA (sc-390548, 1:200), p-CREB (sc-81486, 1:200), CREB (sc-240, 1:200), CRTC1 (sc-271333, 1:200), c-KIT siRNA (sc-29225), Rab 27a siRNA (sc-41834) and Cdc42 siRNA (sc-29256), siRNA Transfection Reagent (sc-29528), siRNA Transfection Medium (sc-36868) were obtained from Santa Cruz Biotechnology (Dallas, United States).

Techniques: Phospho-proteomics, Western Blot, Expressing, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Apigenin promotes melanogenesis and melanosome transport through the c-KIT/Raf-1/MAPK/CREB pathway in HEMCs

doi: 10.3389/fphar.2025.1572878

Figure Lengend Snippet: Apigenin promotes pigmentation through the c-KIT-CRTCs/CREB signaling pathway. (A,B) HEMCs were pretreated with or without 10 μM ISCK03 (c-KIT inhibitor) for 1 h before apigenin was added for an additional 1 h. Phosphorylated CREB (p-CREB) and nuclear levels of CRTC1 were measured by Western blotting. Histone H3 served as a reference for nuclear proteins, and β-actin served as a reference for cytosolic proteins. (C,D) HEMCs were pretreated with or without 10 μM ISCK03 for 1 h before apigenin was added for 48 h. Melanin content and the expression levels of Tyrosinase, MITF, Cdc42, and Rab27a were measured as described previously. (E,F) HEMCs were transfected with si-NC or si-c-KIT for 24 h, followed by apigenin (10 μM) treatment for 48 h. Melanin content and the expression levels of Tyrosinase, MITF, Cdc42, and Rab27a were measured as described previously. (G) Binding of c-KIT with apigenin was analyzed by microscale thermophoresis (MST). The binding curve represents data points from 3 measurements. The calculated Kd is 2.6 ± 0.14 µM. Data are expressed as means ± SEM (n = 3). *p < 0.05 vs. non-treated cells, # p < 0.05 vs. apigenin-treated cells. c-KIT, cellular-KIT; CREB, cAMP response element-binding protein; CRTC, CREB-regulated co-activator; HEMCs, human epidermal melanocytes; MITF, Melanocytes inducing transcription factor; Cdc42, cell division cycle 42; Rab27a, Ras-related protein Rab-27a.

Article Snippet: Anti-Myosin Va (sc-365986, 1:200), KIF5b (sc-133184, 1:200), Cdc42 (sc-8401, 1:200), Rab27a (sc-74586, 1:200), p-c-KIT (sc-365504, 1:200), c-KIT (sc-13508, 1:200), p-Raf-1 (sc-271929, 1:200), Raf-1 (sc-7267, 1:200), p-p38 (sc-166182, 1:200), p38 (sc-398546, 1:200), p-ERK (sc-7383, 1:200), ERK (sc-514302, 1:200), p-RSK (sc-377526, 1:200), RSK (sc-74575, 1:200), p-MEK (sc-81503, 1:200), MEK (sc-13159, 1:200), MSK1 (sc-518173, 1:200), p-PKA (sc-293036, 1:200), PKA (sc-390548, 1:200), p-CREB (sc-81486, 1:200), CREB (sc-240, 1:200), CRTC1 (sc-271333, 1:200), c-KIT siRNA (sc-29225), Rab 27a siRNA (sc-41834) and Cdc42 siRNA (sc-29256), siRNA Transfection Reagent (sc-29528), siRNA Transfection Medium (sc-36868) were obtained from Santa Cruz Biotechnology (Dallas, United States).

Techniques: Western Blot, Expressing, Transfection, Binding Assay, Microscale Thermophoresis

Journal: Frontiers in Pharmacology

Article Title: Apigenin promotes melanogenesis and melanosome transport through the c-KIT/Raf-1/MAPK/CREB pathway in HEMCs

doi: 10.3389/fphar.2025.1572878

Figure Lengend Snippet: A proposed model showing that apigenin promotes pigmentation through the c-KIT/Raf-1/MAPK/CREB signaling pathway. Apigenin activates the c-KIT receptor, leading to phosphorylation of Raf-1. Once activated, Raf-1 phosphorylates and activates MAPK, resulting in CREB phosphorylation and subsequent nuclear translocation of CRTCs. Phosphorylated CREB, in association with CRTCs, promotes MITF transcription, which in turn induces the expression of Tyrosinase, Rab27a, and Cdc42. This cascade of events drives melanosome maturation and transport. Tyrosinase, TRP-1, and TRP-2 are key regulators of melanosome maturation. Cdc42 facilitates dendrite extension and filopodia formation, while Rab27a interacts with its effectors, Mlph and Myosin Va, to regulate actin-dependent melanosome transport and anchoring to the plasma membrane. c-KIT, cellular-KIT; Raf-1, rapidly accelerated fibrosarcoma-1; MAPK, mitogen-activated protein kinase; CRTC, CREB-regulated co-activator; CREB, cAMP response element-binding protein; MITF, Melanocytes inducing transcription factor; Cdc42, cell division cycle 42; Rab27a, Ras-related protein Rab-27a; TRP-1, tyrosinase-related protein-1; TRP-2, tyrosinase-related protein-2.

Article Snippet: Anti-Myosin Va (sc-365986, 1:200), KIF5b (sc-133184, 1:200), Cdc42 (sc-8401, 1:200), Rab27a (sc-74586, 1:200), p-c-KIT (sc-365504, 1:200), c-KIT (sc-13508, 1:200), p-Raf-1 (sc-271929, 1:200), Raf-1 (sc-7267, 1:200), p-p38 (sc-166182, 1:200), p38 (sc-398546, 1:200), p-ERK (sc-7383, 1:200), ERK (sc-514302, 1:200), p-RSK (sc-377526, 1:200), RSK (sc-74575, 1:200), p-MEK (sc-81503, 1:200), MEK (sc-13159, 1:200), MSK1 (sc-518173, 1:200), p-PKA (sc-293036, 1:200), PKA (sc-390548, 1:200), p-CREB (sc-81486, 1:200), CREB (sc-240, 1:200), CRTC1 (sc-271333, 1:200), c-KIT siRNA (sc-29225), Rab 27a siRNA (sc-41834) and Cdc42 siRNA (sc-29256), siRNA Transfection Reagent (sc-29528), siRNA Transfection Medium (sc-36868) were obtained from Santa Cruz Biotechnology (Dallas, United States).

Techniques: Phospho-proteomics, Translocation Assay, Expressing, Clinical Proteomics, Membrane, Binding Assay

Journal: Journal of extracellular vesicles

Article Title: Extracellular Vesicles Secreted by Cancer-Associated Fibroblasts Drive Non-Invasive Cancer Cell Progression to Metastasis via TGF-β Signalling Hyperactivation.

doi: 10.1002/jev2.70055

Figure Lengend Snippet: FIGURE 5 CAFs require intact sEV secretion to hyperactivate the TGF-β signalling in poorly metastatic breast cancer cells in vitro. (a–c) TGF-β/SMAD signalling reporter (Ad-CAGA-Fluc) activity in MCF7 cells ± 19TT cells treated as indicated. MCF7 cells were challenged with (b) SMAD7 overexpression or (c) SB431542 treatment. Ad-CMV-GFP: control adenovirus. DMSO: vehicle for SB431542. (d) Rab27a expression in parental (WT) and Rab27a knockdown (KD) 19TT cells. (e) Total sEV secretion quantified by BCA assay in 19TT-sEVs. (f) Particle size distribution evaluated by nanoparticle tracking analysis (NTA) in 19TT-sEVs. (g) Quantification of 19TT-sEV secretion by NTA. (h) TGF-β/SMAD signalling reporter (Ad-CAGA- Fluc) activity quantified as in (A) in MCF7 cells co-cultured with 19TT (Rab27a WT or Rab27a KD) cells. (i) Total sEV secretion quantified by BCA assay in 19TT-sEVs treated with DMA for 2 h. (j) Particle size distribution evaluated by NTA in 19TT-sEVs treated as in (i). (k) Quantification of sEV secretion by NTA in 19TT cells treated as in (i). (l-n) TGF-β/SMAD signalling reporter (Ad-CAGA-Fluc) activity analysed as in (A) in MCF7 cells in cell cultures treated with (l) DMA, (m) Heparin, or (n) PNP-Xyl for 24 h. Results represent mean ± SD (n ≥3). DMSO: vehicle for DMA and PNP-Xyl. One-way ANOVA test followed by Dunn’s Multiple Comparison test were used to analyse data in (a–c, h & l–n). Unpaired Student’s t-test was used to analyse data in (e, g, i & k). ns: statistically non-significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Stable Rab27a knockdown was established in 19TT CAFs (19TT.Rab27a.shRNA) infection with Rab27a shRNA lentivirus (Santa Cruz Biotechnology, sc41834-v) using Polybrene (sc-134220, Santa Cruz Biotechnology).

Techniques: In Vitro, Activity Assay, Over Expression, Control, Expressing, Knockdown, BIA-KA, Cell Culture, Comparison

Journal: Journal of extracellular vesicles

Article Title: Extracellular Vesicles Secreted by Cancer-Associated Fibroblasts Drive Non-Invasive Cancer Cell Progression to Metastasis via TGF-β Signalling Hyperactivation.

doi: 10.1002/jev2.70055

Figure Lengend Snippet: FIGURE 6 CAF-induced MCF7 cell aggressiveness depends on EV secretion and TGF-β signalling pathway activation. (a) Schematic illustration for the quantification of cancer cell migration in transwell inserts using Gaussia luciferase-labelled MCF7 cells co-cultured ± 19TT CAFs. Alternatively, transwell insert membranes were coated with Matrigel to evaluate cancer cell invasion. (b) Titration of Gaussia luciferase activity by luciferase assay using increasing numbers of Gaussia luciferase-labelled MCF7 (MCF7.Gluc) cells. (c) Migration and (d) invasion of Gaussia luciferase- labelled MCF7 (MCF7.Gluc) cells cultured ± 19TT cells and treated ± recombinant human (rh)TGF-β1. (e) Wound healing assay for GFP-labelled MCF7 cells cultured with 19TT (Rab27a WT or Rab27a KD) and treated as indicated. (f) Wound healing assay for GFP-labelled MCF7 cells treated with vehicle (DMSO) or DMA. Results represent mean ± SD (n ≥3). One-way ANOVA test followed by Dunn’s Multiple Comparison test were used to analyse the presented data. ns: statistically non-significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Stable Rab27a knockdown was established in 19TT CAFs (19TT.Rab27a.shRNA) infection with Rab27a shRNA lentivirus (Santa Cruz Biotechnology, sc41834-v) using Polybrene (sc-134220, Santa Cruz Biotechnology).

Techniques: Activation Assay, Migration, Luciferase, Cell Culture, Titration, Activity Assay, Recombinant, Wound Healing Assay, Comparison

Journal: Journal of extracellular vesicles

Article Title: Extracellular Vesicles Secreted by Cancer-Associated Fibroblasts Drive Non-Invasive Cancer Cell Progression to Metastasis via TGF-β Signalling Hyperactivation.

doi: 10.1002/jev2.70055

Figure Lengend Snippet: FIGURE 9 CAF-induced TGF-β signalling hyperactivation and breast cancer progression is impaired by genetically and pharmaco- logically targeting EV trafficking. (a) Schematic illustration and timeline for NOD-SCID mice implanted with Gaussia luciferase-labelled MCF7 (MCF7.Gluc) cells ±19TT (Rab27a wild type/WT or knockdown/KD). (b) Quantification of TGF-β/SMAD3 signalling reporter (Ad-CAGA-Fluc) activity in MCF7.Gluc cells by In Vivo Imaging System (n = 2 tumour/mice; two mice/group). (c) Schematic illustration and timeline for NOD-SCID mice implanted with unlabelled MCF7 cells and Gaussia luciferase-labelled MCF7 (MCF7.Gluc) cells ±19TT (Rab27a WT or KD). (d-h) Gaussia luciferase activity in (d) blood, (e) liver, (f) bone, (g) unlabelled MCF7 tumours, and (h) lung samples (n = 6 mice). Animals are color-coded. Results represent mean ± SEM. One-Way ANOVA followed by Tukey’s Multiple Comparison Test. *p < 0.05, **p < 0.01, ***p < 0.001, ns: statistically non-significant.

Article Snippet: Stable Rab27a knockdown was established in 19TT CAFs (19TT.Rab27a.shRNA) infection with Rab27a shRNA lentivirus (Santa Cruz Biotechnology, sc41834-v) using Polybrene (sc-134220, Santa Cruz Biotechnology).

Techniques: Luciferase, Knockdown, Activity Assay, In Vivo Imaging, Comparison

Journal: Journal of extracellular vesicles

Article Title: Extracellular Vesicles Secreted by Cancer-Associated Fibroblasts Drive Non-Invasive Cancer Cell Progression to Metastasis via TGF-β Signalling Hyperactivation.

doi: 10.1002/jev2.70055

Figure Lengend Snippet: FIGURE 10 Working model. Human breast CAFs secrete elevated levels of vesicular TGF-β. Uptake of CAF-sEVs by breast cancer cells drives TGF-β signalling hyperactivation to ultimately increase multiorgan metastasis and tumour self-seeding. Consequently, decreasing Rab27a levels in CAFs reduces sEV secretion and prevents the amplification of TGF-β signalling levels in breast cancer cells to ameliorate cancer progression. Accordingly, sEV trafficking disruption by treatment with DMA, heparin, or PNP-Xyl is a novel therapeutic strategy that normalizes the TGF-β signalling activity in breast cancer cells and efficiently blocks metastatic progression. In this illustration, arrow thickness represents the intensity of a given step. Red and white boxes highlight therapeutic strategies used in this work to normalize TGF-β signalling levels by decreasing CAF-sEV secretion and uptake. MVB: multivesicular body (also termed late endosome). ILVs, intraluminal vesicles.

Article Snippet: Stable Rab27a knockdown was established in 19TT CAFs (19TT.Rab27a.shRNA) infection with Rab27a shRNA lentivirus (Santa Cruz Biotechnology, sc41834-v) using Polybrene (sc-134220, Santa Cruz Biotechnology).

Techniques: Amplification, Disruption, Activity Assay

Journal: Signal transduction and targeted therapy

Article Title: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis.

doi: 10.1038/s41392-023-01711-1

Figure Lengend Snippet: Fig. 3 Rab27a knockdown decreases sEV secretion and inhibits the TGF-β signaling activity in breast cancer cells in vitro. sEVs were isolated from parental (P) MDA231 and MDA.Rab27a.shRNA (RAB27A knockdown (KD)) cell culture conditioned medium. a sEV secretion was quantified by BCA assay. b, c Particle size distribution and concentration evaluated by NTA. d Alix and TSG101 expression assessed by western blot in (P) and (KD) whole cell lysates (WCL) and sEVs. e, f pSMAD2 levels evaluated in (P) and (KD) cells treated (e) with 2 ng/mL rhTGF-β1 (increasing time intervals) or (f) with indicated rhTGF-β1 concentrations (8 h). Total (t)SMAD2 and β-Actin were used as loading controls. g, h The TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) activity was quantified in parental and RAB27A KD cells treated ± rhTGF-β1 for (g) 24 h or (h) 48 h. Results normalized by Gaussia luciferase (Ad-CMV-Gluc) activity. Results represent mean ± SD (n ≥3). Unpaired Student’s t-test used for comparison. **p < 0.01, ***p < 0.001

Article Snippet: Transient Rab27a knockdown in MDA231 cells was established by Rab27a siRNA (sc-41834, Santa Cruz Biotechnology, Bio-Strategy Pty Limited, Australia) transfection using LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Australia PTY LTD).

Techniques: Knockdown, Activity Assay, In Vitro, Isolation, shRNA, Cell Culture, BIA-KA, Concentration Assay, Expressing, Western Blot, Luciferase, Comparison

Journal: Signal transduction and targeted therapy

Article Title: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis.

doi: 10.1038/s41392-023-01711-1

Figure Lengend Snippet: Fig. 4 TGF-β signaling is inhibited by DMA, Heparin and PNP-Xyl in vitro. a–d sEVs were isolated from MDA231 cell culture conditioned medium after treatment ± 100 µM DMA (2 h). a sEV secretion quantified by BCA assay. b, c Particle size distribution and concentration evaluated by NTA. d pSMAD2 levels were assessed by western blot in MDA231 cells treated ± DMA (100 µM) and rhTGF-β1 (5 ng/mL). Total (t) SMAD2 and β-actin were used as loading controls. e, f TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) activity quantified in (e) MDA231 and (g) MCF7 cells treated ± rhTGF-β1 (5 ng/mL) ± DMA (24 h). Luciferase assay results normalized by Gaussia luciferase (Ad-CMV-Gluc) activity. g pSMAD2 levels assessed in MDA231 cells treated with rhTGF-β1 or MDA231-sEVs after treatment ± heparin. h, i TGF-β/SMAD3 signaling activity quantified in (h) MDA231 and (i) MCF7 cells treated with MDA231-sEVs ± heparin as in (e, f). j pSMAD2 levels assessed in MDA231 cells treated with rhTGF-β1 or MDA231-sEVs after treatment ± PNP-Xyl as in (d, g). k, l TGF-β/SMAD3 signaling activity quantified in (k) MDA231 and (l) MCF7 cells treated with MDA231-sEVs after treatment ± PNP-Xyl (24 h). Results represent mean ± SD (n ≥3). Unpaired Student’s t-test was used to analyze data in (a, c). One-way ANOVA test followed by Dunn’s Multiple Comparison test was used to analyze data in (e–f, h–i, k–l). ns: statistically non-significant, **p < 0.01, ***p < 0.001

Article Snippet: Transient Rab27a knockdown in MDA231 cells was established by Rab27a siRNA (sc-41834, Santa Cruz Biotechnology, Bio-Strategy Pty Limited, Australia) transfection using LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Australia PTY LTD).

Techniques: In Vitro, Isolation, Cell Culture, BIA-KA, Concentration Assay, Western Blot, Activity Assay, Luciferase, Comparison

Journal: Signal transduction and targeted therapy

Article Title: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis.

doi: 10.1038/s41392-023-01711-1

Figure Lengend Snippet: Fig. 5 sEVs induce cancer cell EMT, migration, and invasion in vitro. a MCF7 cell morphology evaluated by phase contrast after treatment ± rhTGF-β1 or MDA231-sEVs (5 days). Red arrows indicate elongated cells. Scale bar is equal to 100 µm. b ZO-1 and E-cadherin localization analyzed by immunofluorescence staining after single treatment ± rhTGF-β1 or MDA231-sEVs (5 days). Scale bar is equal to 50 µm (c) ZO-1 and E-cadherin expression assessed by western blot in MCF7 cells treated once ± rhTGF-β1 or MDA231-sEVs (5 days). β-actin used as loading control. d MCF7 cell invasion quantified in cells seeded in Matrigel-coated transwell inserts and treated ± rhTGF-β1 or MDA231-sEVs (48 h). Scale bar is equal to 100 µm. e, f Cell migration quantified by wound healing assay in (e) MDA231 and (f) MCF7 cell cultures infected ± Ad-CMV-Flag-SMAD7 and treated as indicated. Ad-CMV-GFP: control adenovirus. Results represent mean ± SD (n ≥3). One-Way ANOVA followed by Dunn’s Multiple Comparison Test. *p < 0.05, **p < 0.01, ***p < 0.001, ns: statistically non-significant

Article Snippet: Transient Rab27a knockdown in MDA231 cells was established by Rab27a siRNA (sc-41834, Santa Cruz Biotechnology, Bio-Strategy Pty Limited, Australia) transfection using LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Australia PTY LTD).

Techniques: Migration, In Vitro, Staining, Expressing, Western Blot, Control, Wound Healing Assay, Infection, Comparison

Journal: Signal transduction and targeted therapy

Article Title: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis.

doi: 10.1038/s41392-023-01711-1

Figure Lengend Snippet: Fig. 6 sEVs increase TGF-β signaling and enhance MDA231 breast cancer progression in vivo. a Illustration of breast cancer mouse model showing the orthotopic implantation of MDA231 cells. Unlabeled MDA231 and Gaussia luciferase-labeled MDA.Gluc cells were implanted contralaterally. b Experiment timeline for (a). c TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) activity quantified in MDA.Gluc tumors (3 animals/group) by IVIS. d–f Gaussia luciferase activity quantified by luciferase assay in (d) blood, (e) lung, and (f) bone samples (5–6 mice/ group). Animals are color-coded. Black dashed lines indicate the background activity for the Gaussia luciferase quantified in samples from non-implanted mice (n = 2). Results represent mean ± SEM. One-Way ANOVA followed by Tukey’s Multiple Comparison Test. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Transient Rab27a knockdown in MDA231 cells was established by Rab27a siRNA (sc-41834, Santa Cruz Biotechnology, Bio-Strategy Pty Limited, Australia) transfection using LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Australia PTY LTD).

Techniques: In Vivo, Luciferase, Labeling, Activity Assay, Comparison

Journal: Signal transduction and targeted therapy

Article Title: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis.

doi: 10.1038/s41392-023-01711-1

Figure Lengend Snippet: Fig. 8 In vivo TGF-β signaling activity and breast cancer progression are impaired by combined treatment with DMA and SB431542 at suboptimal doses. a Illustration of breast cancer mouse model showing MDA231 orthotopic implantation. Unlabeled MDA231 and Gaussia luciferase-labeled MDA.Gluc cells were implanted contralaterally. b Experiment timeline for (a). c TGF-β/SMAD3 signaling reporter (Ad-CAGA- Fluc) activity quantified in MDA.Gluc tumors (5–6 animals/group). d–g Gaussia luciferase activity quantified by luciferase assay in (d) blood, (e) lung, (f) bone, and (g) unlabeled MDA231 tumor samples (6 animals/group). Animals are color-coded. Red dashed lines indicate the background activity for the Gaussia luciferase quantified in samples from non-implanted mice (n = 2). One-Way ANOVA followed by Tukey’s Multiple Comparison Test. Unpaired Student’s t-test used for comparison of drug-treated groups. *p < 0.05, **p < 0.01, ***p < 0.001, ns: statistically non-significant

Article Snippet: Transient Rab27a knockdown in MDA231 cells was established by Rab27a siRNA (sc-41834, Santa Cruz Biotechnology, Bio-Strategy Pty Limited, Australia) transfection using LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Australia PTY LTD).

Techniques: In Vivo, Activity Assay, Luciferase, Labeling, Comparison

Journal: Signal transduction and targeted therapy

Article Title: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis.

doi: 10.1038/s41392-023-01711-1

Figure Lengend Snippet: Fig. 3 Rab27a knockdown decreases sEV secretion and inhibits the TGF-β signaling activity in breast cancer cells in vitro. sEVs were isolated from parental (P) MDA231 and MDA.Rab27a.shRNA (RAB27A knockdown (KD)) cell culture conditioned medium. a sEV secretion was quantified by BCA assay. b, c Particle size distribution and concentration evaluated by NTA. d Alix and TSG101 expression assessed by western blot in (P) and (KD) whole cell lysates (WCL) and sEVs. e, f pSMAD2 levels evaluated in (P) and (KD) cells treated (e) with 2 ng/mL rhTGF-β1 (increasing time intervals) or (f) with indicated rhTGF-β1 concentrations (8 h). Total (t)SMAD2 and β-Actin were used as loading controls. g, h The TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) activity was quantified in parental and RAB27A KD cells treated ± rhTGF-β1 for (g) 24 h or (h) 48 h. Results normalized by Gaussia luciferase (Ad-CMV-Gluc) activity. Results represent mean ± SD (n ≥3). Unpaired Student’s t-test used for comparison. **p < 0.01, ***p < 0.001

Article Snippet: Transient Rab27a knockdown in MDA231 cells was established by Rab27a siRNA (sc-41834, Santa Cruz Biotechnology, Bio-Strategy Pty Limited, Australia) transfection using LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Australia PTY LTD).

Techniques: Knockdown, Activity Assay, In Vitro, Isolation, shRNA, Cell Culture, BIA-KA, Concentration Assay, Expressing, Western Blot, Luciferase, Comparison

Journal: Signal transduction and targeted therapy

Article Title: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis.

doi: 10.1038/s41392-023-01711-1

Figure Lengend Snippet: Fig. 9 Working model. Normal and non-invasive breast cancer cells show low TGF-β signaling activity required for homeostasis. The TGF-β signaling in these cells activated downstream ligand-receptor binding is shortly terminated following endocytosis of the ligand-receptor complex and its degradation in endolysosomes. However, invasive breast cancer cells exhibit TGF-β signaling hyperactivation that induces metastasis. In these cells, degradation of ligand-receptor complex is potently antagonized by increased exosome secretion. Uptake of exosomes containing TGF-β activity drives TGF-β signaling hyperactivation which is prolonged in highly-metastatic breast cancer cells by sustaining cycles of exosome secretion and uptake. Targeting exosome secretion by reducing Rab27a levels or DMA treatment reduces TGF-β signaling levels in invasive breast cancer cells, an effect similarly observed by targeting exosome uptake via heparin or PNP-Xyl treatment. Simultaneously targeting exosome trafficking (DMA, heparin, or PNP-Xyl) and TβRI kinase activity (SB431542) is a novel therapeutic strategy to normalize TGF-β signaling levels and efficiently impair cancer progression and metastasis. Here, fading colors and arrow thickness represent the intensity of a given step. Red arrows indicate TGF-β signaling termination. Green arrows highlight TGF-β signaling hyperactivation. Red and white boxes show strategies to inhibit the TGF-β signaling

Article Snippet: Transient Rab27a knockdown in MDA231 cells was established by Rab27a siRNA (sc-41834, Santa Cruz Biotechnology, Bio-Strategy Pty Limited, Australia) transfection using LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Australia PTY LTD).

Techniques: Activity Assay, Binding Assay

Journal: Signal transduction and targeted therapy

Article Title: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis.

doi: 10.1038/s41392-023-01711-1

Figure Lengend Snippet: Fig. 3 Rab27a knockdown decreases sEV secretion and inhibits the TGF-β signaling activity in breast cancer cells in vitro. sEVs were isolated from parental (P) MDA231 and MDA.Rab27a.shRNA (RAB27A knockdown (KD)) cell culture conditioned medium. a sEV secretion was quantified by BCA assay. b, c Particle size distribution and concentration evaluated by NTA. d Alix and TSG101 expression assessed by western blot in (P) and (KD) whole cell lysates (WCL) and sEVs. e, f pSMAD2 levels evaluated in (P) and (KD) cells treated (e) with 2 ng/mL rhTGF-β1 (increasing time intervals) or (f) with indicated rhTGF-β1 concentrations (8 h). Total (t)SMAD2 and β-Actin were used as loading controls. g, h The TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) activity was quantified in parental and RAB27A KD cells treated ± rhTGF-β1 for (g) 24 h or (h) 48 h. Results normalized by Gaussia luciferase (Ad-CMV-Gluc) activity. Results represent mean ± SD (n ≥3). Unpaired Student’s t-test used for comparison. **p < 0.01, ***p < 0.001

Article Snippet: The stable Rab27a knockdown cell line (MDA.Rab27a.shRNA) was established by Rab27a shRNA lentivirus (sc-41834-v, Santa Cruz Biotechnology, BioStrategy Pty Limited, Australia) transduction using Polybrene® (sc-134220, Santa Cruz Biotechnology, Bio-Strategy Pty Limited, Australia) in puromycin resistant cells.

Techniques: Knockdown, Activity Assay, In Vitro, Isolation, shRNA, Cell Culture, BIA-KA, Concentration Assay, Expressing, Western Blot, Luciferase, Comparison

Journal: Signal transduction and targeted therapy

Article Title: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis.

doi: 10.1038/s41392-023-01711-1

Figure Lengend Snippet: Fig. 9 Working model. Normal and non-invasive breast cancer cells show low TGF-β signaling activity required for homeostasis. The TGF-β signaling in these cells activated downstream ligand-receptor binding is shortly terminated following endocytosis of the ligand-receptor complex and its degradation in endolysosomes. However, invasive breast cancer cells exhibit TGF-β signaling hyperactivation that induces metastasis. In these cells, degradation of ligand-receptor complex is potently antagonized by increased exosome secretion. Uptake of exosomes containing TGF-β activity drives TGF-β signaling hyperactivation which is prolonged in highly-metastatic breast cancer cells by sustaining cycles of exosome secretion and uptake. Targeting exosome secretion by reducing Rab27a levels or DMA treatment reduces TGF-β signaling levels in invasive breast cancer cells, an effect similarly observed by targeting exosome uptake via heparin or PNP-Xyl treatment. Simultaneously targeting exosome trafficking (DMA, heparin, or PNP-Xyl) and TβRI kinase activity (SB431542) is a novel therapeutic strategy to normalize TGF-β signaling levels and efficiently impair cancer progression and metastasis. Here, fading colors and arrow thickness represent the intensity of a given step. Red arrows indicate TGF-β signaling termination. Green arrows highlight TGF-β signaling hyperactivation. Red and white boxes show strategies to inhibit the TGF-β signaling

Article Snippet: The stable Rab27a knockdown cell line (MDA.Rab27a.shRNA) was established by Rab27a shRNA lentivirus (sc-41834-v, Santa Cruz Biotechnology, BioStrategy Pty Limited, Australia) transduction using Polybrene® (sc-134220, Santa Cruz Biotechnology, Bio-Strategy Pty Limited, Australia) in puromycin resistant cells.

Techniques: Activity Assay, Binding Assay

Journal: Signal transduction and targeted therapy

Article Title: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis.

doi: 10.1038/s41392-023-01711-1

Figure Lengend Snippet: Fig. 3 Rab27a knockdown decreases sEV secretion and inhibits the TGF-β signaling activity in breast cancer cells in vitro. sEVs were isolated from parental (P) MDA231 and MDA.Rab27a.shRNA (RAB27A knockdown (KD)) cell culture conditioned medium. a sEV secretion was quantified by BCA assay. b, c Particle size distribution and concentration evaluated by NTA. d Alix and TSG101 expression assessed by western blot in (P) and (KD) whole cell lysates (WCL) and sEVs. e, f pSMAD2 levels evaluated in (P) and (KD) cells treated (e) with 2 ng/mL rhTGF-β1 (increasing time intervals) or (f) with indicated rhTGF-β1 concentrations (8 h). Total (t)SMAD2 and β-Actin were used as loading controls. g, h The TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) activity was quantified in parental and RAB27A KD cells treated ± rhTGF-β1 for (g) 24 h or (h) 48 h. Results normalized by Gaussia luciferase (Ad-CMV-Gluc) activity. Results represent mean ± SD (n ≥3). Unpaired Student’s t-test used for comparison. **p < 0.01, ***p < 0.001

Article Snippet: Transient Rab27a knockdown in MDA231 cells was established by Rab27a siRNA (sc-41834, Santa Cruz Biotechnology, Bio-Strategy Pty Limited, Australia) transfection using LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Australia PTY LTD).

Techniques: Knockdown, Activity Assay, In Vitro, Isolation, shRNA, Cell Culture, BIA-KA, Concentration Assay, Expressing, Western Blot, Luciferase, Comparison

Journal: Signal transduction and targeted therapy

Article Title: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis.

doi: 10.1038/s41392-023-01711-1

Figure Lengend Snippet: Fig. 9 Working model. Normal and non-invasive breast cancer cells show low TGF-β signaling activity required for homeostasis. The TGF-β signaling in these cells activated downstream ligand-receptor binding is shortly terminated following endocytosis of the ligand-receptor complex and its degradation in endolysosomes. However, invasive breast cancer cells exhibit TGF-β signaling hyperactivation that induces metastasis. In these cells, degradation of ligand-receptor complex is potently antagonized by increased exosome secretion. Uptake of exosomes containing TGF-β activity drives TGF-β signaling hyperactivation which is prolonged in highly-metastatic breast cancer cells by sustaining cycles of exosome secretion and uptake. Targeting exosome secretion by reducing Rab27a levels or DMA treatment reduces TGF-β signaling levels in invasive breast cancer cells, an effect similarly observed by targeting exosome uptake via heparin or PNP-Xyl treatment. Simultaneously targeting exosome trafficking (DMA, heparin, or PNP-Xyl) and TβRI kinase activity (SB431542) is a novel therapeutic strategy to normalize TGF-β signaling levels and efficiently impair cancer progression and metastasis. Here, fading colors and arrow thickness represent the intensity of a given step. Red arrows indicate TGF-β signaling termination. Green arrows highlight TGF-β signaling hyperactivation. Red and white boxes show strategies to inhibit the TGF-β signaling

Article Snippet: Transient Rab27a knockdown in MDA231 cells was established by Rab27a siRNA (sc-41834, Santa Cruz Biotechnology, Bio-Strategy Pty Limited, Australia) transfection using LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Australia PTY LTD).

Techniques: Activity Assay, Binding Assay