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(A) Mitochondrial ATP (mitoATP) production rate as measured by Seahorse metabolic analyzer in control and MED13L variant <t>fibroblasts</t> 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).

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1) Product Images from "Cyclin C nuclear release and mitochondrial dysfunction define molecular signatures of MED13L Syndrome"

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

Journal: bioRxiv

doi: 10.64898/2026.06.01.729270

(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).

Figure Legend 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).

Techniques Used: Control, Variant Assay

(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.

Figure Legend 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.

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

(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).

Figure Legend 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).

Techniques Used: Immunofluorescence, Control, Variant Assay, Fluorescence

(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).

Figure Legend 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).

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

(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).

Figure Legend 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).

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

(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.

Figure Legend 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.

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

(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.

Figure Legend 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.

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

Image Search Results

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

$A - Human fibroblasts were treated with mitochondrial toxins (CCCP, 50µM; valinomycin, 1µM; oligomycin 10µM + antimycin, 4µM) or DMSO for 0, 4, 8 and 24 hrs. Top: Representative immunofluorescence images from 24 hr timepoint showing distribution of pS65Ub (green). Bottom: Quantification shows mean +/- SEM pS65Ub intensity in cytoplasm, nucleus and nucleus:cytoplasm relative to 0 hrs timepoint (separated with vertical dashed line) from 1 experiment with 3 technical repeats. The horizontal line shows the 0hr-normalized baseline. B - Fibroblasts were treated with valinomycin (1µM, 24 hrs) before pS65Ub (green) was visualized by immunofluorescence using multiple specific antibodies. Nuclei (blue) are annotated with green asterisks. C - Confocal Z-stack max projections showing human induced dopaminergic neurons treated with oligomycin + antimycin (OA, 1µM, 24 hrs) and stained for pS65Ub (green), the dopaminergic neuron marker tyrosine hydroxylase (TH; magneta), MAP2 (white) and Hoechst (blue). Dashed boxes indicate areas magnified in pS65Ub channel inset and dashed circles delineate nuclei. D - Wild type (WT) and PINK1 knockout (KO) HeLa cells were treated with CCCP (20µM, 4 hrs) as indicated, then subcellular fractionations were analysed by Western blot. E - HeLa were treated with CCCP (20µM, 4 hrs) or OA (1µM, 24 hrs) before immunoblot analysis of whole-cell lysates using two pS65Ub antibodies. F - HeLa, HEK293T, NCI-H226, murine melanoma, murine PDAC or human skin fibroblast cells were treated with OA, then pS65Ub levels were assessed by Western blot. G - NGN2-induced iPSC-derived neurons were treated with OA (1µM, 6 or 24 hrs) before subcellular fractionation and Western blot analysis. H - HeLa were treated with CCCP (20µM, 4 hrs), rotenone (Rot - 20µM, 20 hrs), paraquat (P3/P6 - 3/6mM, 20 hrs) or CCCP+i (PINK1 inhibitor/PRT062607, 2.5µM, 4 hrs). Quantifications show mean +/-SEM from three independent repeats, *** p<0.0001 (CCCP+i treatment performed twice only so excluded from quantification). I - HeLa cells were treated with 10µM Gamitrinib-TPP (GTPP) for 0, 3 or 20hrs before analysis of whole-cell lysates by Western blot. Red asterisks mark the pS65Ub-histone band. Scale bars 10µm (50µM for panel D). Dashed lines in panels E and F indicate where identical samples were run on separate blots.$

Journal: bioRxiv

Article Title: Phosphorylated ubiquitin is a secondary messenger and an epigenetic mark mediating mitochondria to nucleus signaling

doi: 10.64898/2026.04.24.719390

Figure Lengend Snippet: A - Human fibroblasts were treated with mitochondrial toxins (CCCP, 50µM; valinomycin, 1µM; oligomycin 10µM + antimycin, 4µM) or DMSO for 0, 4, 8 and 24 hrs. Top: Representative immunofluorescence images from 24 hr timepoint showing distribution of pS65Ub (green). Bottom: Quantification shows mean +/- SEM pS65Ub intensity in cytoplasm, nucleus and nucleus:cytoplasm relative to 0 hrs timepoint (separated with vertical dashed line) from 1 experiment with 3 technical repeats. The horizontal line shows the 0hr-normalized baseline. B - Fibroblasts were treated with valinomycin (1µM, 24 hrs) before pS65Ub (green) was visualized by immunofluorescence using multiple specific antibodies. Nuclei (blue) are annotated with green asterisks. C - Confocal Z-stack max projections showing human induced dopaminergic neurons treated with oligomycin + antimycin (OA, 1µM, 24 hrs) and stained for pS65Ub (green), the dopaminergic neuron marker tyrosine hydroxylase (TH; magneta), MAP2 (white) and Hoechst (blue). Dashed boxes indicate areas magnified in pS65Ub channel inset and dashed circles delineate nuclei. D - Wild type (WT) and PINK1 knockout (KO) HeLa cells were treated with CCCP (20µM, 4 hrs) as indicated, then subcellular fractionations were analysed by Western blot. E - HeLa were treated with CCCP (20µM, 4 hrs) or OA (1µM, 24 hrs) before immunoblot analysis of whole-cell lysates using two pS65Ub antibodies. F - HeLa, HEK293T, NCI-H226, murine melanoma, murine PDAC or human skin fibroblast cells were treated with OA, then pS65Ub levels were assessed by Western blot. G - NGN2-induced iPSC-derived neurons were treated with OA (1µM, 6 or 24 hrs) before subcellular fractionation and Western blot analysis. H - HeLa were treated with CCCP (20µM, 4 hrs), rotenone (Rot - 20µM, 20 hrs), paraquat (P3/P6 - 3/6mM, 20 hrs) or CCCP+i (PINK1 inhibitor/PRT062607, 2.5µM, 4 hrs). Quantifications show mean +/-SEM from three independent repeats, *** p<0.0001 (CCCP+i treatment performed twice only so excluded from quantification). I - HeLa cells were treated with 10µM Gamitrinib-TPP (GTPP) for 0, 3 or 20hrs before analysis of whole-cell lysates by Western blot. Red asterisks mark the pS65Ub-histone band. Scale bars 10µm (50µM for panel D). Dashed lines in panels E and F indicate where identical samples were run on separate blots.

Article Snippet: Control primary fibroblasts (#106-05A) were from Cell Applications, Inc., PINK1 (Q456X/Q456X, #sc1028) and Parkin (Ex4-7 del / c.203_204 del AG, #sc1064) mutant fibroblasts are available from the NINDS stem cell repository.

Techniques: Immunofluorescence, Staining, Marker, Knock-Out, Western Blot, Derivative Assay, Fractionation

$A - Schematic depiction of subcellular fraction experiment performed in . Detergent buffers used for fractionation are given in italics. CIB and MIB buffers are from Cell Fractionation Kit (Cell Signaling Technologies, #9038), while HNTE was made in house. B - Cells were treated with OA (1µM, 18 hrs), MG132 (20µM, 4 hrs) or DMSO before subcellular fractionation and Western blotting. Protein subcellular localizations are annotated (IMM = inner mitochondrial membrane, OMM = outer mitochondrial membrane) and arrows indicate both full length and cleaved PINK1 bands. For the pS65Ub blot, identical samples were analyzed on a separate gel (separated by dashed lines). C - PINK1-HA was transiently expressed in HeLa cells before treatment with OA, MG132 or DMSO as before. The distributions of pS65Ub (green), HA (red) and DNA (blue) were assessed by immunofluorescence. Magnified regions of interest are indicated by dashed boxes. Red dashed lines indicate regions captured by intensity profile (performed in the red/HA channel), graphed below. D - WT or PINK1 KO HeLa were transiently transfected with empty vector (EV), PINK1-FKBP (P) or FIS1-FRB (F) in combinations indicated before 18 hrs treatment with OA (1μM) or rapalog (500nM). A dashed line reveals where identical samples were analyzed on a separate gel. E - Schematic depiction of experiments performed in Extended Data Figures 4F, G. F - WT or PINK1 KO HeLa cells were transiently transfected with EV, PINK1 WT, PINK1-NES or PINK1-NLS before treatment with CCCP and Western blot analysis. Two plasmid amounts were used for transfection to achieve high and low relative PINK1 expression, captured by high and low exposures (HiExp/LoExp respectively). G - PINK1-myc tagged with NES/NLS signals were transiently expressed in HeLa cells before treatment with CCCP (20µM, 4 hrs) or MG132 (20µM, 4 hrs). Cells were fixed and stained for myc (green), pS65Ub (blue, grayscale in the far right column) or mitochondrial marker HSP60 (red). Nuclear exclusion (NES) and sequestration (NLS) is observed after MG132 treatment. H - Quantification of nuclear vs cytoplasmic pS65Ub signal density (arbitrary units/µm 2 ) in healthy control, PRKN (left) and PINK1 (right) mutant iPSC-derived dopaminergic neurons after treatment with CCCP (10µM, 6 hrs). The horizontal line shows the signal on DMSO-treatment, considered background due to negligible PINK1 activation/pS65Ub levels. Mean +/- SEM from three independent cell lines per genotype. I - iPSC-derived dopaminergic neurons from PD patients with mutations in PINK1 were treated with CCCP (10µM, 6 hrs). Dashed boxes annotate the area magnified in the inset and dashed circles show nuclear borders. Inset zoom in of pS65Ub channel. See for Control and PRKN mutant conditions. J - Skin fibroblasts from PINK1 / PRKN mutant PD donors and healthy controls were treated with valinomycin (1µM, 8 hours) before Western blot analysis. Maximal Parkin activity (revealed by MFN2 ubiquitination, see band shift) and PINK1 activity (substrate pS65Ubiquitination) is only detected in WT cells treated with valinomycin. K - Fibroblasts were treated with valinomycin (1µM, 0-24 hrs) before fixation and immunofluorescence analysis of pS65Ub (green, grayscale in the left hand column), TOM20 (orange) and DNA (blue). Nuclear:cytoplasmic pS65Ub signal intensity from three experiments was quantified, with mean +/-SEM, 2-way ANOVA shown. Red asterisks identify the pS65Ub-histone band *p<0.05, **p<0.01, ***p<0.001. Scale bars 10µm.$

Journal: bioRxiv

Article Title: Phosphorylated ubiquitin is a secondary messenger and an epigenetic mark mediating mitochondria to nucleus signaling

doi: 10.64898/2026.04.24.719390

Figure Lengend Snippet: A - Schematic depiction of subcellular fraction experiment performed in . Detergent buffers used for fractionation are given in italics. CIB and MIB buffers are from Cell Fractionation Kit (Cell Signaling Technologies, #9038), while HNTE was made in house. B - Cells were treated with OA (1µM, 18 hrs), MG132 (20µM, 4 hrs) or DMSO before subcellular fractionation and Western blotting. Protein subcellular localizations are annotated (IMM = inner mitochondrial membrane, OMM = outer mitochondrial membrane) and arrows indicate both full length and cleaved PINK1 bands. For the pS65Ub blot, identical samples were analyzed on a separate gel (separated by dashed lines). C - PINK1-HA was transiently expressed in HeLa cells before treatment with OA, MG132 or DMSO as before. The distributions of pS65Ub (green), HA (red) and DNA (blue) were assessed by immunofluorescence. Magnified regions of interest are indicated by dashed boxes. Red dashed lines indicate regions captured by intensity profile (performed in the red/HA channel), graphed below. D - WT or PINK1 KO HeLa were transiently transfected with empty vector (EV), PINK1-FKBP (P) or FIS1-FRB (F) in combinations indicated before 18 hrs treatment with OA (1μM) or rapalog (500nM). A dashed line reveals where identical samples were analyzed on a separate gel. E - Schematic depiction of experiments performed in Extended Data Figures 4F, G. F - WT or PINK1 KO HeLa cells were transiently transfected with EV, PINK1 WT, PINK1-NES or PINK1-NLS before treatment with CCCP and Western blot analysis. Two plasmid amounts were used for transfection to achieve high and low relative PINK1 expression, captured by high and low exposures (HiExp/LoExp respectively). G - PINK1-myc tagged with NES/NLS signals were transiently expressed in HeLa cells before treatment with CCCP (20µM, 4 hrs) or MG132 (20µM, 4 hrs). Cells were fixed and stained for myc (green), pS65Ub (blue, grayscale in the far right column) or mitochondrial marker HSP60 (red). Nuclear exclusion (NES) and sequestration (NLS) is observed after MG132 treatment. H - Quantification of nuclear vs cytoplasmic pS65Ub signal density (arbitrary units/µm 2 ) in healthy control, PRKN (left) and PINK1 (right) mutant iPSC-derived dopaminergic neurons after treatment with CCCP (10µM, 6 hrs). The horizontal line shows the signal on DMSO-treatment, considered background due to negligible PINK1 activation/pS65Ub levels. Mean +/- SEM from three independent cell lines per genotype. I - iPSC-derived dopaminergic neurons from PD patients with mutations in PINK1 were treated with CCCP (10µM, 6 hrs). Dashed boxes annotate the area magnified in the inset and dashed circles show nuclear borders. Inset zoom in of pS65Ub channel. See for Control and PRKN mutant conditions. J - Skin fibroblasts from PINK1 / PRKN mutant PD donors and healthy controls were treated with valinomycin (1µM, 8 hours) before Western blot analysis. Maximal Parkin activity (revealed by MFN2 ubiquitination, see band shift) and PINK1 activity (substrate pS65Ubiquitination) is only detected in WT cells treated with valinomycin. K - Fibroblasts were treated with valinomycin (1µM, 0-24 hrs) before fixation and immunofluorescence analysis of pS65Ub (green, grayscale in the left hand column), TOM20 (orange) and DNA (blue). Nuclear:cytoplasmic pS65Ub signal intensity from three experiments was quantified, with mean +/-SEM, 2-way ANOVA shown. Red asterisks identify the pS65Ub-histone band *p<0.05, **p<0.01, ***p<0.001. Scale bars 10µm.

Techniques: Fractionation, Cell Fractionation, Western Blot, Membrane, Immunofluorescence, Transfection, Plasmid Preparation, Expressing, Staining, Marker, Control, Mutagenesis, Derivative Assay, Activation Assay, Activity Assay, Ubiquitin Proteomics, Electrophoretic Mobility Shift Assay

Relative mRNA expression levels of Axl, Tyro3, and Gas6 genes in IPF FBs and HPFs quantified by RT-qPCR. No Mer expression was detected in both fibroblast types. TBP gene was used as housekeeping gene. N = 4.

Journal: Medicina

Article Title: Evaluation of TAM Receptor Targeting in Pathophysiology of Idiopathic Pulmonary Fibrosis

doi: 10.3390/medicina61101837

Figure Lengend Snippet: Relative mRNA expression levels of Axl, Tyro3, and Gas6 genes in IPF FBs and HPFs quantified by RT-qPCR. No Mer expression was detected in both fibroblast types. TBP gene was used as housekeeping gene. N = 4.

Article Snippet: Control human pulmonary fibroblasts (HPFs, C12360 , PromoCell, St. Louis, MO, USA) were cultured in 25 mM glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin solution, and cells were used between passages 7 and 15.

Techniques: Expressing, Quantitative RT-PCR

Generation and characterization of iPSC-derived astrocytes. A Schematic overview of astrocyte differentiation from patient-derived iPSCs. Key compounds used to drive differentiation toward a mature astrocyte phenotype are indicated: LIF (leukemia inhibitory factor), CHIR99021, SB431542, CoE, FGF2 (fibroblast growth factor 2), EGF (epidermal growth factor), and CNTF (ciliary neurotrophic factor). B qPCR analysis of astrocyte-specific markers (ALDHL1, GLAST, S100b, GFAP, Vimentin) in iPSC-derived cells. Expression of MAP2 (neuron marker) and OLIG2 (oligodendrocyte marker) was assessed to evaluate cell population purity. C Phase-contrast images and immunocytochemical validation of astrocyte marker expression. Vimentin and GFAP (green), S100β (red), and DAPI-stained nuclei (blue) are shown. D Quantification of CLN3 protein levels in control and CLN3 patient-derived iPSC and astrocytes via targeted mass spectrometry

Journal: Journal of Biomedical Science

Article Title: Modeling CLN3 Batten disease in astrocytes reveals alterations in mitochondria homeostasis, fatty acid metabolism and oxidative stress response

doi: 10.1186/s12929-026-01253-y

Figure Lengend Snippet: Generation and characterization of iPSC-derived astrocytes. A Schematic overview of astrocyte differentiation from patient-derived iPSCs. Key compounds used to drive differentiation toward a mature astrocyte phenotype are indicated: LIF (leukemia inhibitory factor), CHIR99021, SB431542, CoE, FGF2 (fibroblast growth factor 2), EGF (epidermal growth factor), and CNTF (ciliary neurotrophic factor). B qPCR analysis of astrocyte-specific markers (ALDHL1, GLAST, S100b, GFAP, Vimentin) in iPSC-derived cells. Expression of MAP2 (neuron marker) and OLIG2 (oligodendrocyte marker) was assessed to evaluate cell population purity. C Phase-contrast images and immunocytochemical validation of astrocyte marker expression. Vimentin and GFAP (green), S100β (red), and DAPI-stained nuclei (blue) are shown. D Quantification of CLN3 protein levels in control and CLN3 patient-derived iPSC and astrocytes via targeted mass spectrometry

Article Snippet: Healthy control fibroblast lines (two cell lines) were obtained from ATCC (cat. number PCS-201—012) and the Coriell Institute (cat. number AG05836).

Techniques: Derivative Assay, Expressing, Marker, Biomarker Discovery, Staining, Control, Mass Spectrometry

Mitochondrial respiration is impaired in fibroblasts derived from patients with DLD deficiency. Mitochondrial oxygen consumption was assessed in controls (Ctrl1 and Ctrl2) and patient (Pt1–Pt6) fibroblasts using high-resolution respirometry (Oroboros O2k). ( A ) Routine respiration; ( B ) maximal respiration calculated as the difference between FCCP-stimulated and α-chaconine–permeabilized rates; ( C ) complex I-linked respiration (NADH-linked respiration, N-pathway), calculated as the difference between ADP and α-chaconine; ( D ) complex II-linked respiration (NS-pathway) calculated as the difference between respiration after rotenone and α-chaconine addition; ( E ) effect of complex I inhibition, calculated as the difference between FCCP-stimulated and rotenone-inhibited respiration; and ( F ) complex I/complex II respiration ratio (complex I-linked activity divided by complex II-linked activity). Each open circle represents an independent experimental run (N = 4–8 repeats per sample). All data were normalized to cell number. Statistical analysis was performed using the Mann–Whitney U test. * p < 0.05 vs. Ctrl1; numerical p values (0.05 < p < 0.1) are indicated on the plots. Abbreviations: Ctrl, control; Pt, patient.

Journal: Antioxidants

Article Title: Bioenergetic Signatures of DLD Deficiency: Dissecting PDHc- and α-KGDHc-Linked Defects

doi: 10.3390/antiox15010019

Figure Lengend Snippet: Mitochondrial respiration is impaired in fibroblasts derived from patients with DLD deficiency. Mitochondrial oxygen consumption was assessed in controls (Ctrl1 and Ctrl2) and patient (Pt1–Pt6) fibroblasts using high-resolution respirometry (Oroboros O2k). ( A ) Routine respiration; ( B ) maximal respiration calculated as the difference between FCCP-stimulated and α-chaconine–permeabilized rates; ( C ) complex I-linked respiration (NADH-linked respiration, N-pathway), calculated as the difference between ADP and α-chaconine; ( D ) complex II-linked respiration (NS-pathway) calculated as the difference between respiration after rotenone and α-chaconine addition; ( E ) effect of complex I inhibition, calculated as the difference between FCCP-stimulated and rotenone-inhibited respiration; and ( F ) complex I/complex II respiration ratio (complex I-linked activity divided by complex II-linked activity). Each open circle represents an independent experimental run (N = 4–8 repeats per sample). All data were normalized to cell number. Statistical analysis was performed using the Mann–Whitney U test. * p < 0.05 vs. Ctrl1; numerical p values (0.05 < p < 0.1) are indicated on the plots. Abbreviations: Ctrl, control; Pt, patient.

Article Snippet: Dermal fibroblast primary cell lines from six genetically confirmed patients with DLD deficiency were obtained from the Pediatric Metabolic Disease Unit, Sheba Medical Center (IRB# SMC-21-8644, Figure 1, Table 1, and ), as well as two control cell lines: a control human dermal fibroblast cell line was purchased from ATCC (PCS-201-012; Ctrl 1, Manassas, VA, USA), and a primary cell line from a 39-year-old healthy male (Ctrl 2).

Techniques: Derivative Assay, Inhibition, Activity Assay, MANN-WHITNEY, Control

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