recombinant gdf11  (R&D Systems)

Journal: bioRxiv

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1101/2025.03.03.641323

Figure Lengend Snippet: 293T cells co-expressing PCSK5 M452I secrete mature ectopic GDF11 with intermediate efficiency. A, Catalytically active PCSK5 promotes ectopic GDF11 secretion. Cells were lipofected with 3 ng of the indicated PCSK5 allele (or EGFP overexpression control) plus 100 ng of GDF11, and conditioned medium was collected after 24 hours to measure GDF11 release by ELISA. PCSK5 T288P was included as a catalytically dead control . B, GDF11 release by PCSK5 M452I plus wildtype PCSK5 (PCSK5 WT ) is additive. Cells were treated as in ( A ) and compared with 1.5 ng PCSK5 WT plus 1.5 ng PCSK5 M452I . C, Cotransfection with oncogenic HRAS G12V approximates the level of prenylated HRAS (HRAS prenyl ) in MCF10DCIS.com . 293T cells were lipofected with 2 ng of HRAS G12V and 3 ng of the indicated PCSK5 allele (or EGFP overexpression control) and immunoblotted for HRAS with vinculin and p38 used as loading controls. MCF10A-5E cells are a negative control for HRAS overexpression. D, HRAS G12V cotransfection does not alter the relative GDF11 secretion efficiencies of wildtype PCSK5, PCSK5 M452I , and PCSK5 T288P . Cells were treated as in ( C ) and measured for GDF11 release by ELISA. For ( A ), ( B ), and ( D ), GDF11 ELISA results are normalized to the GDF11-only condition [gray dashed in ( A )] and shown as the mean ± SEM of N = 4 biological replicates. Differences among +GDF11 groups were analyzed by multiway ANOVA with PCSK5 genotype as a fixed effect. Significant factors were followed up pairwise by Tukey-Kramer post hoc analysis.

Article Snippet: GDF11 secretion assay—pLX302 GDF11-V5 puro (Addgene, 83097), pLX304 (wildtype) PCSK5-V5 blast (Addgene, 83100), and pLX304 PCSK5 (T288P)-V5 blast (Addgene, 83101) were described previously ( ). pDONR223 PCSK5 (M452I) (Addgene, 232445) was prepared by QuikChange II XL site-directed mutagenesis (Agilent, 200521) of pDONR223 (wildtype) PCSK5 from the human ORFeome v5.1 and recombined into pLX304 (Addgene, 25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (Addgene, 232446). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (Addgene, 141348) or pLX304 EGFP-V5 blast (Addgene, 232447) was used when diluting GDF11 or PCSK5 plasmid dosage and for negative controls. pcDNA3.1 HRAS (G12V) was kindly provided by David Kashatus.

Techniques: Expressing, Over Expression, Control, Enzyme-linked Immunosorbent Assay, Cotransfection, Negative Control

Journal: bioRxiv

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1101/2025.03.03.641323

Figure Lengend Snippet: Inducible reconstitution of PCSK5 alleles in PCSK5 −/− MCF10DCIS.com cells. A, Approach to MCF10DCIS.com engineering. Cells were transduced with a destabilizing domain (DD)-containing Cas9-P2A-Venus and a single-guide RNA targeting Exon 4 of PCSK5 (sgPCSK5). Transduced cells were treated with 200 nM Shield-1 to stabilize Cas9-P2A-Venus and 2% matrigel to promote PCSK5 expression before sorting single Venus-positive cells into 10 ng/ml GDF11 (to aid recovery upon PCSK5 loss) and screening genomic DNA (gDNA) of expanded clones for knockout. One confirmed PCSK5 −/− clone was then transduced with sgPCSK5-resistant, tetracycline (tet)-regulated, V5-tagged alleles of PCSK5 and selected polyclonally for hygromycin resistance. B, Sequence-confirmed knockout alleles of MCF10DCIS.com clone 3D8. The PCSK5 coding sequence (CDS) is shown with annotations for the signal peptide (SP, purple), proprotein sequence (Pro, green), and peptidase domain (blue) including its catalytic triad (yellow stars). The protospacer adjacent motif (PAM) of sgPCSK5 is just upstream of the first triad amino acid, and deletions (white, Allele 1) or insertions (pink, Allele 2) induce frameshift mutations (gray) removing the first amino acid in the catalytic triad (black outlined stars) and producing premature stop codons (red). C, Quantification of reconstituted PCSK5 alleles by calibrating against recombinant V5-containing Multitag protein at the indicated copies per cell ( , ). Cells were treated with 1 µg/ml doxycycline for 24 hours, and total protein from counted cells was immunoblotted for V5 with tubulin and p38 used as loading controls for cells. Copy number estimates are: PCSK5 WT , 136,000 ± 11,000 copies per cell; PCSK5 M452I , 164,000 ± 6,000 copies per cell; PCSK5 T288P , 176,000 ± 15,000 copies per cell ( N = 4 independent samples).

Article Snippet: GDF11 secretion assay—pLX302 GDF11-V5 puro (Addgene, 83097), pLX304 (wildtype) PCSK5-V5 blast (Addgene, 83100), and pLX304 PCSK5 (T288P)-V5 blast (Addgene, 83101) were described previously ( ). pDONR223 PCSK5 (M452I) (Addgene, 232445) was prepared by QuikChange II XL site-directed mutagenesis (Agilent, 200521) of pDONR223 (wildtype) PCSK5 from the human ORFeome v5.1 and recombined into pLX304 (Addgene, 25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (Addgene, 232446). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (Addgene, 141348) or pLX304 EGFP-V5 blast (Addgene, 232447) was used when diluting GDF11 or PCSK5 plasmid dosage and for negative controls. pcDNA3.1 HRAS (G12V) was kindly provided by David Kashatus.

Techniques: Transduction, Expressing, Clone Assay, Knock-Out, Sequencing, Recombinant

Journal: bioRxiv

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1101/2025.03.03.641323

Figure Lengend Snippet: PCSK5 activity promotes rounded multi-cell organization in 3D matrigel cultures of MCF10DCIS.com . A, Spheroid growth rates for the indicated PCSK5 addback lines estimated by nonlinear least-squares regression of cross-sectional area at 4, 8, and 12 days from N = 8 biological replicates (gray dashed). B and C, Reduced multi-cell circularity of MCF10DCIS.com upon loss of PCSK5. For ( B ), the scale bar is 100 µm. For ( C ), circularities were calculated from N = 1819 (DCIS.com) and 2028 (PCSK5 −/− ) segmented spheroids collected from 4 biological replicates at 16 days. Arcsine-transformed circularities were analyzed by two-sample homoscedastic t test. D and E, Multi-cell circularity of PCSK5 −/− cells is restored by wildtype PCSK5 or addition of recombinant GDF11, but not PCSK5 M452I or PCSK5 T288P . For ( D ), the scale bar is 100 µm. For ( E ), circularities were calculated from N = 5503 (wildtype PCSK5), 5016 (PCSK5 M452I ), 3495 (PCSK5 T288P ), and 3815 (PCSK5 T288P +GDF11) segmented spheroids collected from 8 biological replicates at 8 days, and arcsine-transformed circularities were analyzed by multiway ANOVA with PCSK5 genotype as a fixed effect. Significant factors were followed up pairwise by Tukey-Kramer post hoc analysis. F and G, PCSK5 alleles do not alter the differentiation phenotypes of MCF10DCIS.com cells in 3D matrigel culture. Cultures in ( A ) plus PCSK5 T288P +GDF11 cultures were lysed and immunoblotted for CDH1, TP63, and VIM with vinculin, tubulin, ERK1/2, and p38 used as loading controls. For ( G ), data from N = 4 biological replicates were normalized to the mean of wildtype PCSK5 cultures, and the three unstimulated genotypes were Box-Cox-transformed and compared by multiway ANOVA with PCSK5 genotype as a fixed effect.

Article Snippet: GDF11 secretion assay—pLX302 GDF11-V5 puro (Addgene, 83097), pLX304 (wildtype) PCSK5-V5 blast (Addgene, 83100), and pLX304 PCSK5 (T288P)-V5 blast (Addgene, 83101) were described previously ( ). pDONR223 PCSK5 (M452I) (Addgene, 232445) was prepared by QuikChange II XL site-directed mutagenesis (Agilent, 200521) of pDONR223 (wildtype) PCSK5 from the human ORFeome v5.1 and recombined into pLX304 (Addgene, 25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (Addgene, 232446). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (Addgene, 141348) or pLX304 EGFP-V5 blast (Addgene, 232447) was used when diluting GDF11 or PCSK5 plasmid dosage and for negative controls. pcDNA3.1 HRAS (G12V) was kindly provided by David Kashatus.

Techniques: Activity Assay, Transformation Assay, Recombinant