alk4  (R&D Systems)

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: a MDA-MB-231 cells stably expressing a control shRNA (shNTC), shRNA targeting ALK4 (shALK4), or shRNA-resistant ALK4 (rALK4) were injected into the tail vein of nude mice ( n = 10 per group). Pulmonary lesions were detected by bioluminescent imaging. Total bioluminescence at the end of the experiment (week 7) is presented with representative images. Statistical analysis was performed using nonparametric one-way ANOVA (Kruskal–Wallis test) followed by Dunn’s multiple comparisons test. Data are presented as mean values ± SEM. b–f LM2 cells expressing pBABE or pBABE-ALK4 vectors were injected into the tail vein of nude mice ( n = 15 per group). Pulmonary lesions were imaged by bioluminescent imaging at the indicated times. b Representative images of LM2 pBABE- and pBABE-ALK4-injected mice. c Normalized bioluminescence of pulmonary lesions at the indicated times. Data are presented as mean values ± SD. Data were analyzed by ordinary two-way ANOVA with Tukey’s multiple comparisons test. d Average number of palpable pulmonary lesions with representative images of the lungs. Data are presented as mean values ± SEM. e Average lung weight at week 7 of LM2 pBABE- and pBABE-ALK4-injected mice. Data are presented as mean values ± SEM. f Kaplan–Meier survival curves for LM2 pBABE- and pBABE-ALK4-injected mice. g–k HPNE pancreatic cells stably expressing shNTC or shALK4 were orthotopically injected into the tail of mouse pancreata ( n = 8 per group). g Total bioluminescence and representative images of the mice 7 weeks after injection. h Primary pancreatic tumors from mice injected with HPNE cells expressing shNTC or shALK4 were dissected and weighed. i H&E staining of primary pancreatic tumors from mice bearing HPNE cells expressing shNTC or shALK4.Scale bar = 150 μm. j Representative images of spontaneous metastases observed in mice bearing orthotopic HPNE pancreatic cells expressing shALK4. Scale bar = 50 μm. k Quantification of spontaneous metastases in mice bearing orthotopic HPNE cells expressing shNTC or shALK4, data were analyzed by a two-sided Chi-square test. Other data comparing two groups were analyzed using a nonparametric two-tailed Mann–Whitney test.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Stable Transfection, Expressing, Control, shRNA, Injection, Imaging, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: a MDA-MB-231 and b PANC-1 CRISPR non-targeting control (NTC) cells and two CRISPR ALK4 KO clones for each cell line were analyzed for expression of EMT markers and β-actin by Western blotting. One representative out of three independent replicates is shown. c Soft agar colony formation assay of PANC-1 NTC cells and two ALK4 KOs clones ( n = 4 independent experiments), scale bar = 200 nm. Data were analyzed using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. Cell migration was assessed by transwell migration assay in PANC-1 ( d ) and HPNE ( e ) cells expressing shNTC or shALK4 ( n = 3 biological replicates). f MDA-MB-231 cells expressing shNTC, shALK4, or shALK4 and shRNA-resistant ALK4 ( n = 5 biological replicates) were analyzed using transwell migration assays. Cell invasion was assessed using Matrigel transwell assays in PANC-1 ( n = 10 biological replicates) ( g ) and MDA-MB-231( n = 4 biological replicates) ( h ) cells expressing shNTC or shALK4. LM2 cells expressing pBABE or pBABE-ALK4 ( n = 3 biological replicates) were analyzed for migration ( i ) and invasion ( j ). k MDA-MB-231 cells expressing shNTC or shALK4 were sparsely seeded and subjected to live cell imaging to track migration. Data from 23 cells for each line are shown, with directional persistence calculated in ( l ). Pancreatic (PAAD) and breast (BRCA) cancer patient data from TCGA, CPTAC-PAAD, QCMG-PAAD, and METABRIC-BRCA were stratified based on ALK4 expression. Patients in the bottom quartile (low ALK4) and top quartile (high ALK4) of ALK4 expression from these cohorts were selected. Gene expression data were used for gene set enrichment analysis (GSEA) with cancer-associated gene signatures related to EMT ( m ) or invasive cancer phenotypes ( n ) that were significantly enriched in the ALK4-low group. Normalized enrichment scores (NES) with p -values < 0.05 are indicated by the colored symbols, as defined in the plot legends. The soft agar, migration, and invasion assays were quantified in a blinded manner. Data comparing two groups were analyzed using two-tailed Student’s t -tests. Data are presented as mean values ± SEM.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: CRISPR, Control, Clone Assay, Expressing, Western Blot, Soft Agar Assay, Migration, Transwell Migration Assay, shRNA, Live Cell Imaging, Gene Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: HPNE ( a ) and PANC-1 ( b ) cells expressing shNTC or shALK4 were serum-starved for 3 h and then treated with vehicle, 100 pM activin A, 100 pM TGF-β1, 100 ng/ml Nodal, 1 nM GDF5, or 5% serum for 30 min. Protein lysates were analyzed by Western blotting. One representative out of three independent replicates is shown. c MDA-MB-231 NTC cells and two isogenic ALK4 KO lines were serum-starved for 3 h and then treated with vehicle or 100 pM TGF-β1 for 30 min. Protein lysates were analyzed by Western blotting. One representative out of three independent replicates is shown. d Nuclear localization of pSmad2 in NTC-TGF-β1 ( n = 165 cells), NTC + TGF-β1 ( n = 324 cells), ALK4 KO-TGF-β1 ( n = 93 cells), and ALK4 KO + TGF-β1 ( n = 194 cells) groups was assessed by immunofluorescent microscopy. Scale bar = 25 µm. Nuclear blob counts were quantified using BlobFinder v3.2. The central line marks the median, the box extends from the 25th to the 75th percentiles. The whiskers go from min to max. e Pancreatic (PAAD) and breast (BRCA) cancer patients from the TCGA, CPTAC-PAAD, QCMG-PAAD, and METABRIC-BRCA cohorts were stratified into bottom quartile (low ALK4) and top quartile (high ALK4) groups. Gene set enrichment analysis (GSEA) of TGF-β pathway-related gene signatures was performed, with normalized enrichment scores (NES, p < 0.05) indicated by the colored symbols, as defined in the plot legend. f Five out of seven TGF-β target genes are significantly negatively correlated with ACVR1B in the TCGA breast cancer expression dataset ( n = 960). g Seven TGF-β target genes are significantly and negatively correlated with ACVR1B in the TCGA pancreatic cancer expression dataset ( n = 149). h–j MDA-MB-231 CRISPR NTC and ALK4 KO cells, with or without dominant-negative TβRII (DN-TβRII), were serum-starved for 3 h and treated with vehicle or 100 pM TGF-β1 for 30 min. h Protein lysates were analyzed by Western blotting. i SERPINE1 expression was measured by qRT-PCR ( n = 3 biological replicates). j Cell migration was assessed using transwell migration assays ( n = 4 biological replicates). For experiments with two independent variables, statistical analyses were performed using ordinary two-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean values ± SEM.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Expressing, Western Blot, Microscopy, CRISPR, Dominant Negative Mutation, Quantitative RT-PCR, Migration

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: a–c Cell surface levels of TGF-β receptors were assessed using I 125 -TGF-β binding and crosslinking assays. MDA-MB-231 ( a ) and PANC-1 ( b ) cells expressing shNTC control or shALK4 were serum-starved for 3 h and treated with 100 pM TGF-β1 for the indicated times. c 293T cells were transfected with expression plasmids for TβRII and/or TβRI, along with wild-type (WT), constitutively active (CA), or dominant-negative (DN) ALK4. ALK4 and β-actin were analyzed. d LM2 cells were transfected with expression plasmids for TβRII, along with wild-type (WT) or dominant-negative (DN) ALK4. Biotinylated cell surface proteins were analyzed for TβRII, and whole cell lysates were analyzed for ALK4 and β-actin using Western blotting. e LM2 cells expressing pBABE control or pBABE-ALK4 were serum-starved for 3 h and treated with 100 pM TGF-β1 for the indicated times. Cell surface levels of TGF-β receptors were assessed using I 125 -TGF-β binding and crosslinking assays. f 293T cells were transfected with WT ALK4 as indicated, and total cell lysates were processed with PNGase F and assessed by Western blotting. g PANC-1 and MDA-MB-231 cells expressing shNTC or shALK4 were lysed. Total cell lysates were processed with PNGase F or EndoH and assessed by Western blotting. h MDA-MB-231 cells stably expressing shNTC or shALK4 were treated with tunicamycin at the indicated concentrations. Cell surface levels of TGF-β receptors were assessed using an I 125 -TGF-β binding and crosslinking assay (top). Total TβRII and β-actin were assessed by Western blotting. i MDA-MB-231 cells expressing shNTC or shALK4 were treated with 10 nM cycloheximide (CHX) for the indicated times to block synthesis of new proteins. Cells were incubated with 50 ng/ml tunicamycin for 4 h prior to harvesting. Whole-cell lysates were assessed for TβRII and β-actin by Western blotting. Quantification was performed using ImageJ. For in vitro analysis, each experiment was done with at least three independent biological replicates.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Binding Assay, Expressing, Control, Transfection, Dominant Negative Mutation, Western Blot, Stable Transfection, Blocking Assay, Incubation, In Vitro

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: a Quantitative proteomic analysis of whole-cell lysates from PANC-1 control (NTC) and two ALK4 CRISPR knockout (ALK4 KO) lines. Heatmap showing the expression of differentially expressed proteins (DEPs, p < 0.05) between ALK4 KO and control samples. Colors represent relative expression values in log fold change (logFC). b Functional annotation and pathway enrichment analysis of the top 125 upregulated DEPs in ALK4 KO samples compared to controls using DAVID Bioinformatics. Significant gene sets are shown with circle size indicating the number of genes and color indicating the transformed p-value. Fold enrichment is represented on the x -axis. c–f Gene set enrichment analysis (GSEA) plots for DEPs (KO vs. WT) showing significant enrichment in indicated gene signatures. NES: normalized enrichment score. g Protein–protein interaction network of the top-regulated proteins in ALK4 KO cells, generated using STRING analysis. Interactions with a score > 0.5 are depicted, with line color indicating the type of interaction (cyan-curated databases; pink-experimentally determined; blue-gene co-occurrence; khaki-text mining; black-co-expression; light blue-protein homology). Dots represent the pathways/biology as indicated.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Control, CRISPR, Knock-Out, Expressing, Functional Assay, Transformation Assay, Generated

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: a Western blot (left) and qRT-PCR (right) analysis of protein and LGALS3 mRNA expression in PANC-1 NTC and ALK4 KO cells ( n = 4 per group). b Flow cytometric analysis of MGAT5-modified glycans labeled with PHA-L lectin in PANC-1 NTC ( n = 14), ALK4 KO1 ( n = 12), ALK4 KO2 ( N = 14), and ALK4 KO3 ( n = 13) cells. c qRT-PCR analysis of MGAT5 expression in PANC-1 NTC and ALK4 KO cells ( n = 4 per group). d Magnetic beads were coated with PHA-L lectin and incubated with whole-cell lysates from PANC-1 NTC or ALK4 KO cells. Eluted samples were analyzed for TβRII. Whole-cell lysates (WCL) were analyzed for TβRII and β-actin. e PANC-1 control and ALK4 KO cells transfected with control or MGAT5-specific siRNA. After 2 days, cells were transfected again for an additional 3 days, serum-starved for 3 h, and treated with 100 pM TGF-β1 for 30 min before harvesting. Protein lysates were assessed for expression of the indicated proteins using western blotting. f Western blot analysis of protein expression in PANC-1 NTC and ALK4 KO cells transfected with control or galectin-3-specific siRNA for 5 days. Cells were treated with 100 pM TGF-β1 for 30 min before harvesting. g Internalization of TβRII in PANC-1 NTC ( n = 3) and ALK4 KO cells ( n = 5). Quantification of internalized TβRII under the indicated condition is shown at the bottom. h Resullts of soft agar colony formation assay of indicated groups ( n = 6 per group), scale bar = 200 nm. i–k In vivo pulmonary lesions in NSG mice injected via tail vein with PANC-1 NTC, PANC-1 NTC crMGAT5 KO, ALK4 KO, or ALK4 KO crMGAT5 KO cells ( n = 10 per group). Pulmonary lesions were imaged via bioluminescent imaging in a blinded manner ( i and j ). k Bioluminescent imaging of lungs from the top three mice (highest bioluminescence) from each group at week 7. For in vitro analysis, each experiment was done with at least 3 independent biological replicates. For multiple comparisons with one independent variable, ordinary one-way ANOVA was used, followed by Dunnett’s multiple comparisons test. For experiments with two independent variables, ordinary 2-way ANOVA was used, followed by Tukey’s multiple comparisons test. Data are presented as mean values ± SEM.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Modification, Labeling, Magnetic Beads, Incubation, Control, Transfection, Soft Agar Assay, In Vivo, Injection, Imaging, In Vitro

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: Dot plots of the correlation between ACVR1B and ETS1 expression (left) and between MGAT5 and ETS1 expression (right) using the TCGA pancreatic ( a ) and breast ( b ) cancer databases. c Spearman correlation coefficients between the indicated genes in the TCGA pancreatic and breast cancer databases. NS not significant. d qRT-PCR analysis of ETS1 mRNA levels in PANC-1 NTC and ALK4 KO cells. qRT-PCR analysis of ETS1 ( e ) and MGAT5 ( f ) PANC-1 NTC and ALK4 KO cells transfected with control or ETS1-specific siRNA for 48 h. Results were repeated with three independent biological replicates. For experiments with two independent variables, data were analyzed using ordinary two-way ANOVA followed by Tukey’s multiple comparisons test. For comparison between two groups, data were analyzed using two-tailed Student’s t tests. Data are presented as mean values ± SEM.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: A Model for the Diversity Explosion Fundamental to Metastasis

doi: 10.64898/2025.12.13.694153

Figure Lengend Snippet: Chromosome squashes of ALK4 KO cells showing examples of fluorescently labeled (DAPI) chromosomes prepared after blockage in metaphase. The histograms show the number of times a particular number of chromosome pairs were found (n-77). The same data is binned as 5 (B) or 1 (C) chromosome/cell.

Article Snippet: The ALK4 WT (Cat# 80879) and ALK4 CA (Cat# 27223) DNA plasmids were purchased from Addgene.

Techniques: Labeling

Journal: bioRxiv

Article Title: A Model for the Diversity Explosion Fundamental to Metastasis

doi: 10.64898/2025.12.13.694153

Figure Lengend Snippet: When entering mitosis cells have maximum volumes, have rounded up, slightly detached from the substrate and therefore can be modeled as spherical. Cells cultures of equal confluence (60-80%) were imaged at 4 min intervals for 24 hr. Videos of cell cultures were observed both in forward and reverse to determine the last frame before cytokinesis. The diameter of each was then measured using ImageJ and volumes calculated as 4/3p(d/2) 3 . Consistent with the distributions of nuclear sizes, we find that the KO cell population were skewed toward the very small. Both normally passaged KO cultures and those regrown from cells gently rocked off from that culture showed very similar distributions. N=50 (EV), 144 (ALK4 KO), 86 (Rocked Off). A-C: Data plotted for individual cultures, D: Same data plotted together.

Article Snippet: The ALK4 WT (Cat# 80879) and ALK4 CA (Cat# 27223) DNA plasmids were purchased from Addgene.

Techniques:

Journal: Biology

Article Title: BMP-2-Driven Osteogenesis: A Comparative Analysis of Porcine BMSCs and ASCs and the Role of TGF-β and FGF Signaling.

doi: 10.3390/biology14060610

Figure Lengend Snippet: Figure 6. Receptor expressions of ALK2, ALK4, ALK6, ALK5, ALK7, BMPR-II in pASC. Grouped representation of the respective receptor expression in the course of osteogenic differentiation (OM +/−BMP-2). From day 19, there is a significant induction of ALK 2, ALK 6, and ALK 5 with the addition of BMP-2. BMPR-II expression in the OM group decreased in OM and tended to stay increased under BMP-2 supplementation from day 19, but was not considered significant (* p ≤0.05, ** p ≤0.01; n = 6, BMP-2 450 ng/mL).

Article Snippet: The respective conjugated antibodies were used for the expressions of ALK3 (Cat. No.: AF436), ALK 5 (Cat. No.: FAB5871), ALK6 (Cat. No.: FAB5051A), TGF-β2-RII (Cat. No.: FAB532P), ALK7 (Cat. No.: FAB77491A), ALK2 (Cat. No.: AF637), ALK4 (Cat. No.: MAB2221), and BMPR-II (Cat. No.: AF811) (by R&D Systems, Minneapolis, MN, USA), and the pASCs and pBMSCs were compared for their expressions of the specific surface antigens CD45 (Cat. No.: MCA1568GA, BioRad, Hercules, CA, USA), HLA-DR (human leukocyte antigen–antigen D-related surface molecule) (Cat. No.: MCA2314F, Bio-Rad, Hercules, CA, USA), CD29 (Cat. No.: 561,496, BD Pharmingen, Franklin Lakes, NJ, USA), CD79alpha (Bio-Rad, Cat. No.: MCA2538GA), CD14 (Cat. No.: MCA1568GA, Bio-Rad, Hercules, CA, USA), CD31 (Cat. No.: AF3387, R&D Systems, Minneapolis, MN, USA), CD105 (Cat. No.: NB110-58718APC, Novus Biologicals, Minneapolis, MN, USA), CD26 (, Cat. No.: NB600-552APC, Novus Biologicals, Minneapolis, MN, USA), CD73 (, Cat. No.: AF4488, R&D Systems, Minneapolis, MN, USA), CD90 (Cat. No.: 559,869, BD Pharmingen, Franklin Lakes, NJ, USA), CD34 (Cat. No.: 81289, abcam, Cambridge, UK), and CD44 (Cat. No.: 5531, BD Pharmingen, Franklin Lakes, NJ, USA).

Techniques: Expressing