Journal: Molecular cancer research : MCR

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

doi: 10.1158/1541-7786.MCR-25-0211

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 ( 28 ). 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: Concentrated samples were stored at −80°C, volumed up to 205 μl with Reagent Diluent (R&D Systems, DY008B) after thawing, and measured with the GDF11 ELISA (R&D Systems, DY1958) after adding two extended serial dilutions of recombinant GDF11 (15.5 pg/ml and 7.75 pg/ml).

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

Journal: Molecular cancer research : MCR

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

doi: 10.1158/1541-7786.MCR-25-0211

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 ( 61 ) and a single-guide RNA targeting Exon 4 of PCSK5 (sgPCSK5). Transduced cells were treated with 200 nM Shield-1 ( 61 ) to stabilize Cas9-P2A-Venus and 2% matrigel to promote PCSK5 accessibility 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 ( 63 , 72 ). Cells were treated with 1 μg/ml doxycycline for 24 hours, and total protein from counted cells was immunoblotted by two-color fluorescence detection for V5 (800 channel) with tubulin and p38 (700 channel) 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 biological replicates).

Article Snippet: Concentrated samples were stored at −80°C, volumed up to 205 μl with Reagent Diluent (R&D Systems, DY008B) after thawing, and measured with the GDF11 ELISA (R&D Systems, DY1958) after adding two extended serial dilutions of recombinant GDF11 (15.5 pg/ml and 7.75 pg/ml).

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

Journal: Molecular cancer research : MCR

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

doi: 10.1158/1541-7786.MCR-25-0211

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 ( 50 ) 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 250 ng/ml 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: Concentrated samples were stored at −80°C, volumed up to 205 μl with Reagent Diluent (R&D Systems, DY008B) after thawing, and measured with the GDF11 ELISA (R&D Systems, DY1958) after adding two extended serial dilutions of recombinant GDF11 (15.5 pg/ml and 7.75 pg/ml).

Techniques: Activity Assay, Transformation Assay, Recombinant

Journal: Molecular cancer research : MCR

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

doi: 10.1158/1541-7786.MCR-25-0211

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 ( 28 ). 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 (RRID:Addgene_83097), pLX304 (wildtype) PCSK5-V5 blast (RRID:Addgene_83100), and pLX304 PCSK5 (T288P)-V5 blast (RRID:Addgene_83101) were described previously ( 28 ). pBabe GFP (RRID:Addgene_10668), pBabe puro HA PIK3CA H1047R (RRID:Addgene_12524), pHAGE GFP (RRID:Addgene_106281), and pHAGE PIK3CA H1047R (RRID:Addgene_116500) were commercially obtained. pDONR223 PCSK5 (M452I) (RRID: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 (RRID:Addgene_25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (RRID:Addgene_232446). pDONR223 BMP2 and pDONR223 BMP4 from the human ORFeome v5.1 were similarly recombined into pLX302 (RRID:Addgene_25896) with LR clonase II (Invitrogen, 11791020) to yield pLX302 BMP2-V5 puro (RRID:Addgene_246525) and pLX302 BMP4-V5 puro (RRID:Addgene_246526). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (RRID:Addgene_141348) or pLX304 EGFP-V5 blast (RRID: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: Molecular cancer research : MCR

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

doi: 10.1158/1541-7786.MCR-25-0211

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 ( 61 ) and a single-guide RNA targeting Exon 4 of PCSK5 (sgPCSK5). Transduced cells were treated with 200 nM Shield-1 ( 61 ) to stabilize Cas9-P2A-Venus and 2% matrigel to promote PCSK5 accessibility 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 ( 63 , 72 ). Cells were treated with 1 μg/ml doxycycline for 24 hours, and total protein from counted cells was immunoblotted by two-color fluorescence detection for V5 (800 channel) with tubulin and p38 (700 channel) 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 biological replicates).

Article Snippet: GDF11 secretion assay—pLX302 GDF11-V5 puro (RRID:Addgene_83097), pLX304 (wildtype) PCSK5-V5 blast (RRID:Addgene_83100), and pLX304 PCSK5 (T288P)-V5 blast (RRID:Addgene_83101) were described previously ( 28 ). pBabe GFP (RRID:Addgene_10668), pBabe puro HA PIK3CA H1047R (RRID:Addgene_12524), pHAGE GFP (RRID:Addgene_106281), and pHAGE PIK3CA H1047R (RRID:Addgene_116500) were commercially obtained. pDONR223 PCSK5 (M452I) (RRID: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 (RRID:Addgene_25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (RRID:Addgene_232446). pDONR223 BMP2 and pDONR223 BMP4 from the human ORFeome v5.1 were similarly recombined into pLX302 (RRID:Addgene_25896) with LR clonase II (Invitrogen, 11791020) to yield pLX302 BMP2-V5 puro (RRID:Addgene_246525) and pLX302 BMP4-V5 puro (RRID:Addgene_246526). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (RRID:Addgene_141348) or pLX304 EGFP-V5 blast (RRID: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, Clone Assay, Knock-Out, Sequencing, Recombinant, Fluorescence

Journal: Molecular cancer research : MCR

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

doi: 10.1158/1541-7786.MCR-25-0211

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 ( 50 ) 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 250 ng/ml 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 (RRID:Addgene_83097), pLX304 (wildtype) PCSK5-V5 blast (RRID:Addgene_83100), and pLX304 PCSK5 (T288P)-V5 blast (RRID:Addgene_83101) were described previously ( 28 ). pBabe GFP (RRID:Addgene_10668), pBabe puro HA PIK3CA H1047R (RRID:Addgene_12524), pHAGE GFP (RRID:Addgene_106281), and pHAGE PIK3CA H1047R (RRID:Addgene_116500) were commercially obtained. pDONR223 PCSK5 (M452I) (RRID: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 (RRID:Addgene_25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (RRID:Addgene_232446). pDONR223 BMP2 and pDONR223 BMP4 from the human ORFeome v5.1 were similarly recombined into pLX302 (RRID:Addgene_25896) with LR clonase II (Invitrogen, 11791020) to yield pLX302 BMP2-V5 puro (RRID:Addgene_246525) and pLX302 BMP4-V5 puro (RRID:Addgene_246526). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (RRID:Addgene_141348) or pLX304 EGFP-V5 blast (RRID: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

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: METTL3-Mediated m 6 A Regulation of GDF11 Promotes Socket Healing in Diabetic Rats

doi: 10.2147/DMSO.S536806

Figure Lengend Snippet: METTL3 regulates GDF11 through specific m 6 A modification sites. ( A – C ) HEK293T cells transduced with METTL3-overexpressing or control lentivirus, n = 3 independent experiments. ( A ) Relative expression of METTL3 mRNA, ( B ) GDF11 m 6 A modification levels and ( C ) GDF11 mRNA in HEK293T cells overexpressing METTL3. Data are presented as mean ± SD. p <0.001 indicates a statistically significant difference between two groups. ( D ) RIP assay showing the enrichment of GDF11 in METTL3 antibody pulldowns, n = 3 independent experiments. Data are presented as mean ± SD. p <0.001 indicates a statistically significant difference compared to the IgG control. ( E ) Prediction of m 6 A modification sites on GDF11 using the SRAMP database. ( F ) Four high-confidence sites were identified. ( G – J ) Dual-luciferase reporter assay showing the effect of METTL3 overexpression on luciferase activity at predicted m 6 A sites, n = 3 independent experiments. Data are presented as mean ± SD. p <0.001 indicates a statistically significant difference compared to the control. ( K ) GDF11 mRNA in HEK293T cells overexpressing METTL3 treated with Actinomycin D at 0, 4, 8, and 12 h, n = 3 independent experiments. Data are presented as mean ± SD. p <0.01 indicates a statistically significant difference compared to the control.

Article Snippet: After dental extraction, GK rats were administered lentiviral particles overexpressing METTL3 (LV-METTL3) or lentiviral particles downregulating GDF11 (LV-shGDF11) at the extraction sites (Vigene Biosciences).

Techniques: Modification, Transduction, Control, Expressing, Luciferase, Reporter Assay, Over Expression, Activity Assay

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: METTL3-Mediated m 6 A Regulation of GDF11 Promotes Socket Healing in Diabetic Rats

doi: 10.2147/DMSO.S536806

Figure Lengend Snippet: Knockdown of GDF11 partially reverses the tooth extraction socket healing effects of METTL3 overexpression. ( A ) Relative expression of GDF11 in the different groups, n = 6 sockets per group. Data are presented as mean ± SD. p <0.001 indicates a statistically significant difference between the LV-shGDF11 group and the LV-shNC group. ( B ) Representative images showing tooth extraction socket healing at the tooth extraction sites in Wistar control, GK, GK+LV-NC, GK+LV-METTL3, GK+LV-METTL3+LV-shNC, and GK+LV-METTL3+LV-shGDF11 groups, n = 6 sockets per group. ( C – F ) BV/TV, Tb.N, Tb.Th, and Tb.Sp in the extraction socket 21 days after tooth extraction, n = 6 sockets per group. Data are presented as mean ± SD. p <0.001 indicates a statistically significant difference between groups.

Article Snippet: After dental extraction, GK rats were administered lentiviral particles overexpressing METTL3 (LV-METTL3) or lentiviral particles downregulating GDF11 (LV-shGDF11) at the extraction sites (Vigene Biosciences).

Techniques: Knockdown, Extraction, Over Expression, Expressing, Control