neonatal  (ATCC)

Journal: bioRxiv

Article Title: Cyclin C nuclear release and mitochondrial dysfunction define molecular signatures of MED13L Syndrome

doi: 10.64898/2026.06.01.729270

Figure Lengend Snippet: (A) Mitochondrial ATP (mitoATP) production rate as measured by Seahorse metabolic analyzer in control and MED13L variant fibroblasts as indicated. Open box indicates cell line with similar mitoATP production to control. (n ≥ 5 biological replicates; technical duplicates) (B) Glycolytic ATP (glycoATP) production rate measured as in (A) using proton efflux rate (n ≥ 5 biological replicates; technical duplicates). Blue boxes indicate cell lines exhibiting reduced glycoATP production, the red box indicates higher production. (C) Combined ATP production (glycoATP + mitoATP) was calculated from panels (A) and (B). Open box indicates cell lines with no difference in ATP production from control, the red box indicates higher production. The remaining cell lines exhibited reduced total ATP production. (n ≥ 5 biological replicates; technical duplicates) (D) Bioenergetic balance expressed as the ratio of mitochondrial to glycolytic ATP production (mitoATP:glycoATP) for the cell lines indicated. Values <1 indicate mitochondrial-dominant energy metabolism, whereas values approaching 1 reflect increasing reliance on glycolysis. (n ≥ 4 biological replicates; technical duplicates). In all graphs, control cell line is represented in black, missense variant in red, medPIWI/IDR mutations in shades of purple (W1359*, Q1537*, T1663C fs* ), C-terminal variants in shades of blue (N1824M fs , W2178*), and exon deletions (single or multi) in shades of green (Ex2del-1, Ex2del-2, Ex3-4del, Ex3-25del). Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc correction for variant-to-control comparisons. All data represented as mean ± IQR. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Control fibroblasts were purchased from ATCC (ATCC: Primary Dermal Fibroblasts Normal; Human, Neonatal (HDFn): PCS-201-010).

Techniques: Control, Variant Assay

Journal: bioRxiv

Article Title: Cyclin C nuclear release and mitochondrial dysfunction define molecular signatures of MED13L Syndrome

doi: 10.64898/2026.06.01.729270

Figure Lengend Snippet: (A) Representative immunofluorescence images of mitochondrial morphology (red) and CCNC (cyan) in control and MED13L -variant fibroblasts as indicated. For each genotype, merged and 4x magnified images (indicated by boxes) are shown. White arrows indicate CCNC-mitochondrial overlap. See Figure S1-S2 for additional, full-field images. (B) Percentages of MED13L cells exhibiting >50% fragmented mitochondria are shown. Boxes highlight cell lines not displaying elevated mitochondrial fission. (n ≥ 100 cells analyzed). Control cells are represented in black, missense variant in red (P866L), medPIWI/IDR variants in shades of purple (L971*, W1359*, Q1537*, T1663C fs* ), C-terminal variants in shades of blue (N1824M fs *, W2178*), and N-terminal or deletion variants in shades of green (R148*, Ex2del-1, Ex2del-2, Ex3-4del, Ex3-25del). Examples of mitochondrial fragmentation can be found in Figure S1. (C) UniProt-derived domain map and mutation locations of MED13L syndrome variants. Globular (yellow) and unstructured (blue) protein domains are indicated under the amino acid numbering. Frameshift, missense, and nonsense mutations are shown above the numbering, deletions (solid line) are indicated below. Dotted lines indicate predicted coding region eliminated by frameshift mutation in deletion alleles. The mutations were grouped based on position with N-terminal (green) mutations including one frameshift and three deletion alleles (R148*, Ex2del, Ex3-4del, Ex3-25del). The single missense variant (P866L) is represented by red box (variant type) with purple border (functional domain classification = IDR). IDR missense and truncation mutations, including those in the MID medPIWI domain, are represented with purple (L971*, W1359*, Q1537*, T1663C fs* ). C-terminal mutations are represented in blue (N1823M fs *, W2178*). (D) Fragmented mitochondria phenotype was grouped by MED13L mutations as described in (C). All data represent mean ± SEM or distributions as indicated. Statistical comparisons were performed using one-way ANOVA with Dunnett’s post-hoc test comparing genotype or group to controls (*p < 0.05, **p < 0.01, *p < 0.001). See Table S1 for variant details.

Article Snippet: Control fibroblasts were purchased from ATCC (ATCC: Primary Dermal Fibroblasts Normal; Human, Neonatal (HDFn): PCS-201-010).

Techniques: Immunofluorescence, Control, Variant Assay, Derivative Assay, Mutagenesis, Functional Assay

Journal: bioRxiv

Article Title: Cyclin C nuclear release and mitochondrial dysfunction define molecular signatures of MED13L Syndrome

doi: 10.64898/2026.06.01.729270

Figure Lengend Snippet: (A) Representative immunofluorescence images showing double-stranded DNA (dsDNA; blue) and mitochondria (red) in control and two MED13L variant fibroblasts ( P866L and N1824* ). Images depict mitochondrial network architecture and the distribution of mitochondrial nucleoids across genotypes. (B) Relative mitochondrial DNA (mtDNA) copy number in control and MED13L variant fibroblasts quantified by qPCR and normalized to the nuclear DNA standard (see methods) (n ≥ 3 biological replicates; technical triplicates). (C) mtDNA levels in control and mutations groups described in . (D) Cytosolic ROS levels measured using dihydroethidium (DHE) and presented as background-subtracted fluorescence values for control, missense, and PTV fibroblast lines (n ≥ 4 biological replicates; technical duplicates). ( E) Quantification of mitochondrial ROS using MitoSOX fluorescence in control and MED13L variant fibroblasts classes described in . Relative fluorescence intensity (normalized to background) is shown (n ≥ 6 biological replicates; technical duplicate). All values represent mean ± SEM. Statistical comparisons were performed using one-way ANOVA with Dunnett’s post-hoc test. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Control fibroblasts were purchased from ATCC (ATCC: Primary Dermal Fibroblasts Normal; Human, Neonatal (HDFn): PCS-201-010).

Techniques: Immunofluorescence, Control, Variant Assay, Fluorescence

Journal: bioRxiv

Article Title: Cyclin C nuclear release and mitochondrial dysfunction define molecular signatures of MED13L Syndrome

doi: 10.64898/2026.06.01.729270

Figure Lengend Snippet: (A) Heatmap of relative mRNA expression (log 2 FC vs. pooled controls) for genes involved in mitochondrial biogenesis ( PGC1ɑ, SIRT1 ), mitochondrial transcription ( HSPA9, TFB1M ), electron transport chain and oxidative phosphorylation ( ND1, NDUFV3, SDHB, CYTB ), TCA cycle ( MDH1 ), mitochondrial dynamics ( OPA1, MFN1, MFN2 ), and Mediator Kinase Module components ( MED13, CCNC, MED13L ). Columns represent individual fibroblast lines stratified by mutation class as described in , and color-coordinated. Values are normalized to pooled control fibroblasts. Blue indicates relative downregulation and red indicates relative upregulation (n ≥ 4 biological replicates; technical triplicates). (B-F) Transcript RT-qPCR quantification for MED13L , MED13 , CCNC , TFB1M and PGC1ɑ as indicated. All graphs are presented as log 2 fold change relative to control fibroblasts. All RT-qPCR data represent median ± interquartile range; individual fibroblast lines plotted in heatmap. All data were assessed by one-way ANOVA with Dunnett’s post-hoc test comparing each mutation class to controls (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Control fibroblasts were purchased from ATCC (ATCC: Primary Dermal Fibroblasts Normal; Human, Neonatal (HDFn): PCS-201-010).

Techniques: Expressing, Phospho-proteomics, Mutagenesis, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Cyclin C nuclear release and mitochondrial dysfunction define molecular signatures of MED13L Syndrome

doi: 10.64898/2026.06.01.729270

Figure Lengend Snippet: (A) Percentage of mitotically active cells determined by BrdU incorporation assays in control and representative MED13L variant fibroblasts. Reduced BrdU positivity indicates diminished proliferative capacity (n ≥ 5 biological replicates). (B) Relative expression of senescence marker p16 INK4a measured by RT-qPCR and normalized to GAPDH . Data are shown as Log 2 fold change relative to control fibroblasts (n ≥ 3 biological replicates; technical triplicates). (C) Senescence-associated β-galactosidase (SA-β-gal) activity quantified by flow cytometry using a fluorogenic β-gal substrate. Background-subtracted fluorescence intensity is shown for control and select MED13L variant fibroblasts (n ≥ 4 biological replicates). (D, E) MitoATP production (D) or total ATP production (E) in extended cell cultures for control and representative MED13L variant fibroblast lines. Timepoints taken every 10 generations. Simple linear regression and best fit analysis performed, as shown by indicated corresponding dotted lines. Data represent individual biological replicates with summary statistics displayed as median and interquartile range. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc correction for variant-to-control comparisons. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Control fibroblasts were purchased from ATCC (ATCC: Primary Dermal Fibroblasts Normal; Human, Neonatal (HDFn): PCS-201-010).

Techniques: BrdU Incorporation Assay, Control, Variant Assay, Expressing, Marker, Quantitative RT-PCR, Activity Assay, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Cyclin C nuclear release and mitochondrial dysfunction define molecular signatures of MED13L Syndrome

doi: 10.64898/2026.06.01.729270

Figure Lengend Snippet: (A) Mitochondrial ATP production (mitoATP) measured in control and MED13L patient-derived fibroblasts grouped by variant location (IDR, N-terminal, MedPIWI or C-terminal). Each point represents an independent biological replicate (n ≥ 6). (B) Glycolytic ATP production (glycoATP), calculated from proton efflux rates, shown by variant class as in (A) (n ≥ 6). (C) Relationship between mitoATP production (mean values from ) and MED13L amino acid position of each variant. Each point represents a fibroblast line carrying a variant at the indicated residue position within the MED13L protein (R = 0.515). (D) Relationship between total ATP production (mitoATP + glycoATP) and MED13L amino acid position of mutation. Each point represents an individual fibroblast line carrying variants at the indicated residue position. (R = 0.597) (E) Relationship between mitoATP production and participant age at time of sample collection, in months (R = 0.697). (F) Relationship between total ATP production (mitoATP + glycoATP) and participant age at time of sample collection, in months (R = 0.775). Lines in (C-F) represent line of best fit following simple linear regression. Corresponding equations are represented on each graph. For (C-F), color of the symbol corresponds to type and/or location of mutation and are as follows: green = N-terminal deletions, red = missense, purple = IDR/medPIWI nonsense and frameshift, blue = C-terminal nonsense and frameshift.

Article Snippet: Control fibroblasts were purchased from ATCC (ATCC: Primary Dermal Fibroblasts Normal; Human, Neonatal (HDFn): PCS-201-010).

Techniques: Control, Derivative Assay, Variant Assay, Residue, Mutagenesis

Journal: bioRxiv

Article Title: Cyclin C nuclear release and mitochondrial dysfunction define molecular signatures of MED13L Syndrome

doi: 10.64898/2026.06.01.729270

Figure Lengend Snippet: (A) Vineland motor standard scores (combined gross and fine motor domains) plotted against MED13L amino acid position. Each point represents individual participant harboring indicated MED13L variant. Linear regression performed, best fit line shown (R = 0.659). (B) Dual axis comparison of Vineland motor severity scores (R = 0.465; magenta) with mitochondrial ATP (R = 0.669; mitoATP; green) and amino acid location of the variant. Independent linear regression analyses were performed for each variable relative to motor severity. (C) Composite functional severity scores generated from integrated adaptive, communication, and motor assessments (see Methods) plotted against MED13L amino acid position. Linear regression and line of best fit are shown (R = 0.671, p = 0.06) (D) Dual-axis comparison of composite functional severity scores (R = 0.635; magenta line) with mitochondrial ATP production (R = 0.709; mitoATP; green) and MED13L amino acid position. Independent linear regression analyses were performed for each variable relative to composite severity score. (E) Autism/social trait severity scores generated from harmonized caregiver-reported and clinical behavioral datasets plotted against MED13L amino acid position. Linear regression and line of best fit are shown (R = 0.905, p = 0.0008). For all panels, amino acid position corresponds to the predicted location of the MED13L protein alteration. Each point represents an individual participant-derived fibroblast line. For (A, C, and E), color of the symbol corresponds to type and/or location of mutation and are as follows: green = N-terminal deletions, red = missense, purple = IDR/medPIWI nonsense and frameshift, blue = C-terminal nonsense and frameshift.

Article Snippet: Control fibroblasts were purchased from ATCC (ATCC: Primary Dermal Fibroblasts Normal; Human, Neonatal (HDFn): PCS-201-010).

Techniques: Variant Assay, Comparison, Functional Assay, Generated, Derivative Assay, Mutagenesis