Journal: bioRxiv

Article Title: ALK R1275Q mutation drives expansion of SCP-like cells during sympathoadrenal commitment and primes neuroblastoma initiation

doi: 10.64898/2026.01.27.701690

Figure Lengend Snippet: a, UMAP of all samples (as in ), colored by ALK expression. b, Violin plots of ALK expression (top) and ALK activity scores based on Reactome pathways (bottom) across time points in Ctrl and patient lines (computed as detailed in Methods). Comparisons were performed using the Wilcoxon test. ALK expression, Ctrl vs NB in SAP, D8, and D12, P < 0.0001 = ****; ALK Reactome score, Ctrl and NB in tNCC, D8, and D12, P < 0.0001 = ****, in SAP, P = 0.036 = *. Black triangles indicate mean values. c, Immunoblot analysis of protein lysates collected at NCC, SAP, day 8 (D8), and day 12 (D12) from two Ctrl lines and two patient-derived lines. Total and phosphorylated ALK, AKT, and ERK1/2 were probed. GAPDH was used as a loading control. P.C., positive control: NB-1 neuroblastoma cell line (CVCL_1440) treated with ALKAL2 . d, Quantification of EdU+ cells at the SAP stage after Lorlatinib treatment of both Ctrl (Ctrl 7, Ctrl10, and Ctrl14 in blue) and patient-derived (NB1, NB2, and NB4 in red) lines. Quantification of EdU⁺ cell percentages is shown as mean ± s.d. from independent biological replicates. Statistical significance was assessed by one-way ANOVA followed by Šídák’s multiple comparisons test. In DMSO, Ctrl vs NB, P < 0.0001 = ****; in NB, lorlatinib (20nM) vs DMSO, P < 0.0001 = ****; Lorlatinib (30nM) vs DMSO, P < 0.0001 = ****. e, Schematic of in vivo experimental design. iPSCs were stably transfected with a vector encoding constitutively expressed nuclear mCherry and doxycycline-inducible MYCN . Differentiated tNCCs were injected subcutaneously or orthotopically into the adrenal gland. MYCN was activated by mice receiving doxycycline (200 μg/ml) in drinking water. f, Kaplan–Meier survival curves from the subcutaneous injection model. Solid line, mice treated with doxycycline; dashed line, untreated controls. Significance was assessed by Cox proportional hazards test. g, Table summarizing results of the orthotopic injection experiment. Tumor size is reported as the longest axis of the tumor. h, Histological analysis of tumors from orthotopically injected mice. Gross tumor morphology is shown (with ruler indicating size, top). Representative sections were stained for PHOX2B, KI67, and hematoxylin and eosin (H&E) in the same tumor region. Scale bar, 100 µm.

Article Snippet: Membranes were blocked in 5% BSA in PBST (0.05% Tween-20) for 1h, incubated overnight (4 °C) with primary antibodies—phospho-: pALK (CST 6941 1:2000), ALK (CST 3633, 1:2000), AKT (proteintech 10176-2-AP, 1:2000), phospho-AKT (CST 4060, 1:2000), ERK1/2 (BD Bioscience 610124, 1:3000), phospho-ERK1/2 (CST 4370, 1:2000), MYCN (Proteintech 10159-2-AP), GAPDH (Santa Cruz sc-32233, 1:1000 or sc-44724, 1:5000)—and then with HRP-conjugated goat anti-rabbit (Thermo Fisher 65-6120) or goat anti-mouse (Dako P0447) secondary antibodies.

Techniques: Expressing, Activity Assay, Western Blot, Derivative Assay, Control, Positive Control, In Vivo, Stable Transfection, Transfection, Plasmid Preparation, Injection, Staining

Journal: Advanced Science

Article Title: Skeletal Muscle HSF1 Alleviates Age‐Associated Sarcopenia and Mitochondrial Function Decline via SIRT3‐PGC1α Axis

doi: 10.1002/advs.202510368

Figure Lengend Snippet: Skeletal muscle HSF1 levels are reduced during aging and correlate with genes related to muscle atrophic and mitochondrial genes in mice and humans. A) Hsf1 mRNA levels in the hind limb muscle of young and aged mice from GSE75523 (left, n = 16) and Hsf1 mRNA levels (qRT‐PCR) in GAS muscles of young and aged mice (right, n = 6). B) Pearson correlation analysis between Hsf1 and Mstn or Tfam mRNA levels (qRT‐PCR) in GAS of mice (n = 30). C) HSF1 mRNA levels (qRT‐PCR) in vastus lateralis muscle biopsies from young and old individuals (n = 10‐11). D) Pearson correlation analysis between HSF1 mRNA levels (qRT‐PCR) in human muscles and age (n = 38). E) Pearson correlation analysis between HSF1 and MSTN or TFAM mRNA levels obtained from GTEx human muscles (right, n = 818). F) Pearson correlation analysis of Hsf1 mRNA levels with muscle mass, grip strength, and running endurance in mice (n = 30). G) Immunoblotting analysis and quantification of HSF1 phosphorylation at Ser326 (p‐HSF1) and total HSF1 in GAS muscles from young or aged mice (n = 3). H) Immunoblotting analysis of HSF1 in nuclear (Nuc) and cytoplasmic (Cyto) fractions from young or aged GAS muscle, with quantification of nuclear HSF1 levels and Nuc/Cyto ratio. GAPDH and Histone H3 served as cytoplasmic and nuclear controls, respectively. Statistical significance was assessed by unpaired Student's t ‐test (A, C, and F‐I) and Pearson's r correlation coefficient (B, D and E). Data are presented as mean ± SEM and * p < 0.05; ** p < 0.01; ns, not significant compared to control group.

Article Snippet: Primary antibodies against HSF1 (GB11931‐100, Servicebio), p‐HSF1‐Ser326 (bsm‐52166R, Bioss), MAFbx (sc‐166806, Santa Cruz), MuRF‐1 (sc‐398608, Santa Cruz), MSTN (sc‐393335, Santa Cruz), IGF1 (sc‐74116, Santa Cruz), p‐mTOR‐Ser2448 (2971S, CST), t‐mTOR (AM832, Beyotime), p‐S6K1‐Thr389 (9205S, CST), t‐S6K1 (9202S, CST), PGC1α (sc‐518025, Santa Cruz), p‐FAK‐Tyr397 (AF5821, Beyotime), t‐FAK (AF1108, Beyotime), p‐AKT‐Thr308 (AF5734, Beyotime), t‐AKT (AA326, Beyotime), p‐CREB‐Ser133 (AG1680, Beyotime), t‐CREB (AF1018, Beyotime), OXPHOS Rodent WB Antibody Cocktail (ab110413, Abcam), CS (A5713, ABclonal), SIRT3 (A20805, ABclonal), β‐actin (sc‐8432, Santa Cruz) and GAPDH (GB15002‐100, Servicebio) were applied, followed by incubation with appropriate secondary antibodies.

Techniques: Quantitative RT-PCR, Muscles, Western Blot, Phospho-proteomics, 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: 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