mouse rab27a  (Addgene inc)

Journal: Communications Biology

Article Title: Tumor-secreted factors induce aberrant accumulation of vitamin A–enriched lipid droplets in the liver

doi: 10.1038/s42003-025-09404-x

Figure Lengend Snippet: A Schematic of the animal model. B Western blot analysis of Rab27a expression in MDA-MB-231 and MDA-MB-231/shRab27a #1 and #2 cells. C Nano-flow cytometer analysis of EV number from MDA-MB-231 and MDA-MB-231/shRab27a #2 cells. D SRS images of liver tissues from TB and Rab27a KD TB mice in the C–H stretching region. E Quantification of LD size in lipid-rich regions. F Quantification of lipid intensity at 2855 cm −1 for each LD. G Intensity ratio of 3015 cm −1 and 2855 cm −1 peaks, representing relative unsaturated lipid content in each LD. H Mean SRS spectra of lipid-rich and protein-rich regions in the fingerprint region. I SRS images of liver tissues from TB and Rab27a KD TB mice at 1595 cm −1 . J Quantification of vitamin A levels in each LD within liver tissues from TB and Rab27a KD TB mice, measured by the ratio of SRS intensity at 1595 cm −1 to 1455 cm −1 . Error bars represent SD. **** p < 0.0001. Scale bars: 20 μm. Part of the figure is created in BioRender. Lee, H. (2025) https://BioRender.com/jozm8oh .

Article Snippet: Primary antibodies list as follows: Rab27a (E-8) mouse monoclonal antibodies (Santa Cruz, sc-74586, 1:1000), FABP5 Rabbit pAb (ABclonal, A0947, 1:10000), Beta Actin Monoclonal antibody (Proteintech, 66009-1-Ig, 1:20000), and Alix Polyclonal antibody (Proteintech, 12422-1-AP, 1:5000).

Techniques: Animal Model, Western Blot, Expressing, Flow Cytometry

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) SYTL5-EGFP expression was induced for 24 hr with 100 ng/ml doxycycline in U2OS cells with stable inducible expression of SYTL5-EGFP and constitutive expression of mScarlet-RAB27A. Cells were co-stained with MitoTracker DR for 30 min before live imaging. Arrows indicate mitochondrion filaments. Scale bars: 10 μm, 2 μm (insets). ( B ) CLEM analysis of the cells described in A. Before imaging, cells growing in monolayer were fixed in warm (≈37 °C) 3.7% paraformaldehyde in 0.2 M HEPES (pH 7). After fixation, cells were imaged using a confocal microscope to acquire Z-stacks of optical sections and DIC images to locate the cells of interest. The cells were finally fixed using 2% glutaraldehyde in 0.2 M HEPES (pH 7.4) for 120 min before sample preparation for TEM. Arrows in panel 1 indicate mitochondrion filaments. Scale bars: 15 μm (left), 5 μm (middle) and 2 μm (right). ( C ) U2OS dKO cells were rescued with mScarlet-RAB27A and co-stained with 50 nM MitoTracker green for 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( D ) U2OS dKO cells were rescued with SYTL5-EGFP and co-stained with 50 nM MitoTracker red for 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( E ) Live confocal microscopy imaging of U2OS dKO cells rescued with SYTL5-EGFP and mScarlet-RAB27A (upper panel); SYTL5-EGFP and mScarlet-RAB27A-T23N (middle panel) or SYTL5-EGFP and mScarlet-RAB27A-Q78L (lower panel). All cells were co-stained with 50 nM MitoTracker DR for 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( F ) Lysates from U2OS cells stably expressing EGFP (control) and U2OS dKO cells expressing SYTL5-EGFP and/or mScarlet-RAB27A (wild-type, T23N, or Q78L mutants) were immunoprecipitated using GFP-Trap beads and analysed by western blot using RAB27A and EGFP antibodies. Figure 2—source data 1. Original image files for . Figure 2—source data 2. Original uncropped blots for . Figure 2—source data 3. Uncropped blots with the relevant bands clearly labelled for .

Article Snippet: Antibody , Mouse monoclonal RAB27A , Santa Cruz Biotechnology , #sc-74586 , 1:1000.

Techniques: Expressing, Staining, Imaging, Microscopy, Sample Prep, Confocal Microscopy, Stable Transfection, Control, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) Live confocal microscopy imaging of U2OS constitutively expressing mScarlet-RAB27A co-stained with MitoTracker green added 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( B ) Total cell lysate (TCL), cytosol, and a crude mitochondria fraction from U2OS control cells and cells with stable expression of mScarlet-RAB27A or SYTL5-EGFP were analysed by western blotting for EGFP, RAB27A, tubulin, TIM23, and COXIV. ( C ) Live confocal microscopy imaging of HeLa constitutively expressing SYTL5-EGFP co-stained with MitoTracker Red added 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). ( D ) Live confocal microscopy imaging of HeLa constitutively expressing mScarlet-RAB27A co-stained with MitoTracker green added 30 min before imaging. Scale bars: 10 μm, 2 μm (insets). Figure 2—figure supplement 1—source data 1. Original image files for , C, D. Figure 2—figure supplement 1—source data 2. Original uncropped blots for . Figure 2—figure supplement 1—source data 3. Uncropped blots with the relevant bands clearly labelled for .

Article Snippet: Antibody , Mouse monoclonal RAB27A , Santa Cruz Biotechnology , #sc-74586 , 1:1000.

Techniques: Confocal Microscopy, Imaging, Expressing, Staining, Control, Western Blot

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) RAB27A KO validation by genotyping. The WT RAB27A gDNA sequence was aligned with the RAB27A KO clone 21(c21) gDNA sequence. CRISPR/Cas9 editing produced an indel (single nucleotide deletion indicated by *) in a genomic location shared between all RAB27A isoforms. gRNA targeting region is underlined in the WT RAB27A gDNA sequence. ( B ) RAB27A KO validation by western blot. Cell lysates from U2OS control cells and RAB27A KO cells ( c21 ) were compared to cell lysates where RAB27A was depleted for 72 hr using two independent siRNAs and probed with RAB27 and actin antibodies. ( C ) SYTL5 CRISPR/Cas9 exon deletion was performed using two gRNAs simultaneously. These gRNAs target the 5´UTR or intronic regions of SYTL5 (transcript NM_138780 ) that are shared by all SYTL5 isoforms. Genomic deletion validation and individual clone genotyping were performed using a primer pair that flanked the region to be edited (about 1307 bp length between these primer binding sites). ( D ) SYTL5 KO validation was performed with conventional PCR reaction. A PCR product of 1307 bp is expected for wild-type U2OS gDNA, while a PCR product of about 618 bp is expected if a clone has a deletion in the targeted region. This method was used to validate the SYTL5 KO clone 2 and for dKO RAB27A/SYTL5 clone 57. The deleted sequence includes a coding exon from SYTL5, resulting in a non-functional SYTL5 protein. Figure 2—figure supplement 2—source data 1. Original uncropped blots and gel for . Figure 2—figure supplement 2—source data 2. Uncropped blots with the relevant bands clearly labelled for .

Article Snippet: Antibody , Mouse monoclonal RAB27A , Santa Cruz Biotechnology , #sc-74586 , 1:1000.

Techniques: Biomarker Discovery, Sequencing, CRISPR, Produced, Western Blot, Control, Binding Assay, Functional Assay

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) SYTL5/RAB27A dKO U2OS cells rescued with SYTL5-EGFP and mScarlet-RAB27A were untreated (upper panel) or treated with 1 mM DFP (2nd panel), hypoxia (1% O 2 ) (3rd panel), or 1 mM DMOG (4th panel) for 24 hr. All cells were stained with 50 nM MitoTracker DR 30 min prior to live confocal microscopy imaging. Scale bars: 10 μm, 2 μm (insets). ( B ) SYTL5/RAB27A dKO U2OS cells rescued with SYTL5-EGFP and mScarlet-RAB27A were treated or not with 1 mM DFP for 24 hr. Cells were stained with 50 nM MitoTracker DR prior to fixation. Fixed cells were stained with a LAMP1 antibody and imaged by confocal microscopy. Scale bars: 10 μm, 2 μm (insets). Arrowheads point to SYTL5/RAB27A-positive structures. ( C ) U2OS cells expressing pSu9-Halo-mGFP were transfected with 20 nM control siRNA, SYTL5 siRNA #1 or RAB27A siRNA #1 for 48 hr before 20 min incubation with the TMR-conjugated Halo ligand followed by 24 hr incubation with 1 mM DFP or 1 mM DMOG. Cell lysates were analysed by western blot for the Halo tag. Actin was used as a loading control. ( D ) Quantification of data in C. Error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. *=p < 0.05, **=p < 0.01, ***=p < 0.001. Figure 4—source data 1. Original image files for . Figure 4—source data 2. Original image files for . Figure 4—source data 3. Original uncropped blots for . Figure 4—source data 4. Uncropped blots with the relevant bands clearly labelled for and quantification of blots in 2D.

Article Snippet: Antibody , Mouse monoclonal RAB27A , Santa Cruz Biotechnology , #sc-74586 , 1:1000.

Techniques: Staining, Confocal Microscopy, Imaging, Expressing, Transfection, Control, Incubation, Western Blot, Standard Deviation, Comparison

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) SYTL5/RAB27A dKO U2OS cells rescued with SYTL5-EGFP and mScarlet-RAB27A were treated with 1 mM DFP for 24 hr. Fixed cells were stained with a LAMP1 antibody in addition to either p62 (upper panel) or LC3 (lower panels) antibodies and imaged by confocal microscopy. Arrowheads point to SYTL5/RAB27A-positive structures. Scale bars: 10 μm, 2 μm (insets). ( B ) SYTL5/RAB27A dKO U2OS cells rescued with SYTL5-EGFP and mScarlet-RAB27A were treated with 1 mM DFP for 24 hr and 100 nM BafA1 for the final 4 hr. All cells were stained with 50 nM MitoTracker DR 30 min prior to fixing. Fixed cells were stained with either LAMP1 (upper panel), p62 (middle panel), or LC3 (lower panel) antibody and imaged by confocal microscopy. Arrowheads point to SYTL5/RAB27A-positive structures. Scale bars: 10 μm, 2 μm (insets). ( C ) SYTL5/RAB27A dKO U2OS cells rescued with SYTL5-EGFP and mScarlet-RAB27A were treated with 1 μM MRT68921 for 2 hr (upper panel) or 5 μM Vps34-IN1 for 2 hr (lower panel). All cells were stained with 50 nM MitoTracker DR 30 min prior to live imaging confocal microscopy. Scale bars: 10 μm, 2 μm (insets). Figure 4—figure supplement 1—source data 1. Original image files for . Figure 4—figure supplement 1—source data 2. Original image files for . Figure 4—figure supplement 1—source data 3. Original image files for .

Article Snippet: Antibody , Mouse monoclonal RAB27A , Santa Cruz Biotechnology , #sc-74586 , 1:1000.

Techniques: Staining, Confocal Microscopy, Imaging

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) U2OS cells were transfected with 20 nM control siRNA, SYTL5 siRNA #1 or RAB27A siRNA #1 for 48 hr followed by 24 hr incubation with 1 mM DFP or 1 mM DMOG. Cell lysates were analysed by western blot for BNIP3L. Actin was used as a loading control. Error bars represent the mean with standard deviation between replicates (n=3). ( B ) U2OS cells were transfected with 20 nM control siRNA or SYTL5 siRNA oligos for 72 hr. Transfection efficiency was analysed by qPCR where the relative mRNA expression of SYTL5 was normalised to the expression levels of TBP and siControl. Error bars represent the standard deviation between replicates (n=3). Significance was determined by one-way ANOVA followed by Dunnett multiple comparison test, ***=p < 0.001. ( C ) U2OS cells expressing the mitochondrial matrix reporter NIPSNAP1 aa1-53 -EGFP-mCherry (referred to as IMLS cells) were transfected with 20 nM control siRNA or SYTL5 siRNA oligos for 48 hr before 24 hr incubation with 1 mM DFP in the absence or presence of 100 nM BafA1 for the last 2 hr. After fixation, cell nuclei were stained with Hoechst and widefield images were obtained using a high-content imaging microscope. The area of red-only puncta per cell (>1000 cells analysed, representing mitochondria delivered to lysosomes as the EGFP signal is quenched in the acidic lysosome) was quantified using a Cell Profiler pipeline. The obtained results were normalised to the control siRNA, and error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Bonferroni multiple comparison test. ( D ) U2OS IMLS cells expressing PARKIN were transfected with 20 nM control siRNA or SYTL5 siRNA oligos for 48 hr before a 16 hr incubation with 20 μM CCCP to induce mitophagy, with treatment with 100 nM BafA1 or not for the last 2 hr. The area of red-only puncta per cell (>1000 cells analysed) was quantified using Cell Profiler from widefield images obtained using a high-content imaging microscope. The obtained results were normalised to the control siRNA, and error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. ( E ) U2OS IMLS cells were transfected with 20 nM control siRNA or RAB27A siRNA oligos for 48 hr before 24 hr incubation with 1 mM DFP in the absence or presence of 100 nM BafA1 for the last 2 hr. After fixation, cell nuclei were stained with Hoechst and widefield images were obtained using a high-content imaging microscope. The area of red-only puncta per cell (>1000 cells analysed) was quantified using a Cell Profiler pipeline. The obtained results were normalised to the siControl, and error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Bonferroni multiple comparison test. ( F ) WT (control) or SYTL5 KO U2OS cells were starved in EBSS for 4 hr or incubated with complete medium in the presence or absence of 100 nM BafA1 the last 2 hr. Cell lysates were harvested and analysed by western blot for LC3B and actin proteins. LC3B-II band intensity was quantified relative to actin and normalised to control. Error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by one-way ANOVA followed by Bonferroni multiple comparison test. Figure 4—figure supplement 2—source data 1. Original uncropped blots for . Figure 4—figure supplement 2—source data 2. Uncropped blots with the relevant bands clearly labelled for . Figure 4—figure supplement 2—source data 3. Values plotted in .

Article Snippet: Antibody , Mouse monoclonal RAB27A , Santa Cruz Biotechnology , #sc-74586 , 1:1000.

Techniques: Transfection, Control, Incubation, Western Blot, Standard Deviation, Expressing, Comparison, Staining, Imaging, Microscopy

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) Mitochondrial oxygen consumption rate was analysed in U2OS control cells, SYTL5 KO, RAB27A KO, and dKO cells using the Seahorse XF analyser. OCR was measured after sequential addition of oligomycin, CCCP and a combination of rotenone and antimycin-A into the assay medium. Error bars represent the mean with standard deviation between replicates (n=3). ( B ) The four basal OCR measurements per well (before oligomycin addition) were averaged to obtain the basal OCR value and the non-mitochondrial respiration was subtracted to determine the basal respiration associated with each condition. The ATP production was calculated by subtracting the proton leak from the maximal respiratory capacity. Error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Bonferroni multiple comparison test, *=p < 0.05, **=p < 0.01. ( C ) The extracellular acidification rate (ECAR) was analysed in U2OS control cells, SYTL5 KO, RAB27A KO, and dKO cells using the Seahorse XF analyser. ECAR was measured upon addition of glucose, oligomycin, and 2-DG using control and SYTL5-depleted cells. Error bars represent the mean with standard deviation between replicates (n=3). ( D ) The glycolysis rate of each condition was calculated by subtracting the ECAR value after 2-DG treatment from the ECAR after addition of glucose (Glycolysis = ECAR after addition of glucose − ECAR after 2-DG treatment). Error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by one-way ANOVA followed by Tukey multiple comparison test, *=p < 0.05. ( E ) Cell lysates from U2OS control, RAB27A KO, SYTL5 KO, and dKO cells were analysed by western blot for the indicated electron transport chain complex-II, -III, -V, and -V proteins. GAPDH was used as a loading control. ( F ) Quantification of data in E. Band intensities were quantified relative to GAPDH and normalised to control. Error bars represent the mean with standard deviation between replicates (n=4). Significance was determined by two-way ANOVA followed by Bonferroni multiple comparison test, **=p < 0.01. ( G ) U2OS cells were transfected with 20 nM control siRNA, SYTL5 siRNA #1, or RAB27A siRNA #1 for 48 hr followed by 24 hr incubation with 1 mM DFP or 1 mM DMOG. Cell lysates were analysed by western blot for COXIV. Actin was used as a loading control. Error bars represent the mean with standard deviation between replicates (n=3). Figure 5—source data 1. Original uncropped blots for . Figure 5—source data 2. Uncropped blots with the relevant bands clearly labelled for . Figure 5—source data 3. Values plotted in .

Article Snippet: Antibody , Mouse monoclonal RAB27A , Santa Cruz Biotechnology , #sc-74586 , 1:1000.

Techniques: Control, Standard Deviation, Comparison, Western Blot, Transfection, Incubation

Journal: eLife

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.7554/eLife.105541

Figure Lengend Snippet: ( A ) SYTL5 expression levels in normal/healthy tissues, using data from the GTEx project ( v8 ). Shown are the 10 tissue sites for which SYTL5 is most highly expressed (i.e. based on median expression levels). TPM = transcripts per million. Median is represented by a red solid line and quartiles by red dashed lines. ( B ) Comparison of SYTL5 gene expression levels in normal adrenal gland samples (GTEx, n=128) and adrenocortical carcinoma samples (The Cancer Genome Atlas (TCGA) ACC cohort, n=77). Median expression is represented by a red solid line and quartiles by red dashed lines. Statistical analysis was performed using the unpaired t-test with Welch’s correction. TPM = transcripts per million. ( C ) Kaplan-Meier plot for ACC patient survival related to SYTL5 expression levels. The events related to high SYTL5 expression (expression level above median) are indicated in red, and the dotted lines represent 95% confidence interval. Events related to low SYTL5 expression (expression level below median) are indicated in blue, and the dotted lines represent 95% confidence interval. Statistical analysis was performed using the Logrank (Mantel-Cox) test. ( D ) Comparison of RAB27A gene expression levels in normal adrenal gland samples (GTEx, n=128) and adrenocortical carcinoma samples (The Cancer Genome Atlas (TCGA) ACC cohort, n=77). Median expression is represented by a red solid line and quartiles by red dashed lines. Statistical analysis was performed using the unpaired t-test with Welch’s correction. ( E ) Kaplan-Meier plot for ACC patient survival related to RAB27A expression levels. The events related to high RAB27A expression (expression level above median) are indicated in red, and the dotted lines represent 95% confidence interval. Events related to low RAB27A expression (expression level below median) are indicated in blue, and the dotted lines represent 95% confidence interval. Statistical analysis was performed using the Logrank (Mantel-Cox) test. Figure 6—source data 1. Values plotted in .

Article Snippet: Antibody , Mouse monoclonal RAB27A , Santa Cruz Biotechnology , #sc-74586 , 1:1000.

Techniques: Expressing, Comparison, Gene Expression

Journal: Cancer Biology & Therapy

Article Title: Challenges and caveats in manipulating extracellular vesicle secretion from pancreatic cancer cells

doi: 10.1080/15384047.2025.2569946

Figure Lengend Snippet: Rab27a and Rab35 KO do not impact KPC tumor growth. (a) Schematic of the involvement of Rab27a and Rab35 in EV secretion. (b) Immunoblot validation of Rab27a and Rab35 KO probed for Rab27a, Rab35, and α-tubulin. (c) Particle concentration of EVs derived from WT, Rab27a KO, and Rab35 KO cells. (d) Growth of WT, Rab27a KO, and Rab35 KO cells. (e) Tumor volume, weight, and immunofluorescence staining for Ki-67 in KPC WT, Rab27a KO, and Rab35 KO 14-d orthotopic pancreatic tumors implanted into C57BL6 mice. p -values were calculated using an ordinary one-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Cell lines were developed to express the tetracycline-inducible shRab27a for mouse Rab27a (pLKO-Tet-On-shRab27a, Addgene #120930).

Techniques: Western Blot, Biomarker Discovery, Concentration Assay, Derivative Assay, Immunofluorescence, Staining

Journal: Cancer Biology & Therapy

Article Title: Challenges and caveats in manipulating extracellular vesicle secretion from pancreatic cancer cells

doi: 10.1080/15384047.2025.2569946

Figure Lengend Snippet: Pooled shRab27a does not KD Rab27a. (a) Schematic of EV production through the action of Rab27a. (b) DNA validation of the full Tet-On shRab27a lentiviral construct following transduction. (c) Growth of KPC cells treated with increasing concentrations of dox. (d) Relative Rab27a expression normalized to Gapdh in KPC PalmGRET tCD19 shRab27a cells with siRab27a or dox treatment (+ = 0.6 µg/mL, ++ = 3 µg/mL. (e) Immunoblotting of shRab27a cells after 48 and 72 h of siRNA or 3 µg/mL dox treatment. (f) Relative Rab27a expression normalized to Gapdh in 5 clones with and without 0.3 µg/mL dox treatment. (g) Immunoblotting of shRab27a clone 4 with and without 3 µg/mL dox at 48 and 72 h probed for Rab27a and α-tubulin. (h) Nanoparticle tracking analysis of EVs isolated from shRab27a clone 4 with and without 3 µg/mL dox. p -values were calculated using an unpaired two-tailed Student's t -test. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Cell lines were developed to express the tetracycline-inducible shRab27a for mouse Rab27a (pLKO-Tet-On-shRab27a, Addgene #120930).

Techniques: Biomarker Discovery, Construct, Transduction, Expressing, Western Blot, Clone Assay, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Schematic overview of experimental strategy for monitoring the 3D nuclear position of the MyoD1 locus in living myoblasts by inserting a 64x LacO array downstream of MyoD1 coupled with expression of GFP-LacR. b Representative 3D reconstructed z stack of images showing intranuclear 3D position of the MyoD1 locus in proliferating myoblasts (green dots). Scale bars are indicated on images. See also Supplementary Movie . c Immunofluorescence microscopy of proliferating and 4-days differentiated MyoD1 and Pax7 reporter myoblasts. MyoD1 and Pax7 loci are indicated by yellow arrowheads. Lamin A/C staining (red) marks nuclear border. Scale bars, 10 μm. d Schematic drawing depicting the analysis of the radial distance of gene loci to the nuclear periphery and normalization based on nuclear height. e Density plots of the normalized radial distance of loci to the periphery. Left: Distribution of MyoD1 in proliferating (red) and differentiating (day 4, green) C2C12 myoblasts. n prolif = 106, n diff = 120, p = 1 × 10 −4 . Right: Distribution of MyoD1 and Pax7 loci in proliferating myoblasts. n MyoD1 = 208, n Pax7 = 79, p = 6.3 × 10 −4 . p values for loci were calculated using two-sample, two-sided KS test and corrected for multiple testing using Hochberg method. Numeric values displayed on violin plots represent median values. f Normalized radial position distribution of data shown in ( e ) displayed in violin plots. H3K9me2-marked region (gray-shaded horizontal bar) represents peripheral heterochromatin layer as measured in ( g ). **** p = 1 × 10 − 4 , *** p = 6.3 × 10 −4 p values were calculated using two-sample, two-tailed KS test and corrected for multiple testing using Hochberg method. g H3K9me2 immunofluorescence staining of MyoD1 C2C12 reporter myoblasts. Width of H3K9me2-marked layer is measured in rendered images (middle panel, yellow dots and lines illustrate measurement method for heterochromatin layer), bar graph depicts normalized mean thickness ± SEM of heterochromatin layer measured at 100 different locations. Scale bars, 10 μm. Images in ( c ) were processed with Fiji and in ( b ) and ( g ) with Imaris. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Expressing, Immunofluorescence, Microscopy, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Immunoblot analysis of total cell extracts of proliferating wildtype and knockout C2C12 clones using antibodies to the indicated antigens. Normalized radial distance of MyoD1 to the nuclear border in wildtype C2C12 myoblasts versus cells with ( b ) single knockouts, and ( c ) double or triple knockouts of indicated proteins. H3K9me2-marked heterochromatin layer is shown as gray-shaded bar. Dotted line indicates that experiments were performed separately using slightly different laser settings. n Wt = 208, n emerin KO = 333, n LAP2β KO = 140; n Wt = 141, n LA/C KO = 403, n LBR KO = 283; n Wt = 722, n LA/C&LBR KO = 453, n LA/C, LBR&Emd KO = 300. * p Emd KO = 0.02, remaining p values are non-significant ( p > 0.05; two-sample, two-tailed KS test, Hochberg correction for multiple testing). Numeric values displayed on violin plots represent median values. LA/C lamin A/C, Emd emerin. d Localization of emerin assessed by immunofluorescence microscopy in control and lamin A/C-LBR double knockout myoblasts. Scale bars, 10 μm. Images processed with Fiji. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Western Blot, Knock-Out, Clone Assay, Two Tailed Test, Immunofluorescence, Microscopy, Control, Double Knockout

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Immunostaining and 3D reconstruction of H3K9me3-marked heterochromatin in wildtype and lamin A/C-LBR double knockout C2C12 cells. Scale bars, 10 μm. b Graphs show quantification of mean area per intranuclear H3K9me3-positive spot (left), and number of intranuclear foci per cell (right). Bar graphs represent mean ± SD. n Wt = 40, n KO = 31, n KO2 = 31 (single data points are displayed). Left graph: H(2) = 11.72, ** p KO = 0.0096, ** p KO2 = 0.0058; right graph: H(2) = 52.07, **** p KO = 9.8 × 10 -8 , **** p KO2 = 6.6 × 10 −11 (Kruskal-Wallis test). c Nucleoplasmic over peripheral H3K9me3 signal ratio determined using line intensity profiles across the nucleus in wildtype and lamin A/C-LBR double knockout cells. Line graph on the right shows a representative H3K9me3 fluorescence intensity plot measured along the dashed lines in the images shown in ( a ). Bar graph displays mean values ± SD ( n Wt = 4, n KO = 5). t (7) = 8.31, **** p = 7.1 × 10 −5 (two-tailed t-test). d Lamin B1 immunostaining of indicated cell lines. Scale bars, 10 μm. e Lamin B1 ChIP-qPCR analyses demonstrating loss of lamin B1 binding to LADs at the nuclear periphery of lamin A/C-LBR double knockout cells. Data represent mean values ± SEM of three biological replicates. Horizontal dashed line indicates the signal obtained using unspecific IgG antibodies in control ChIP. The genomic positions of tested LADs are indicated in the table. f Representative confocal images of fluorescence in-situ hybridization (FISH) signal using a GFP-labeled BAC probe for a LAD region (green dots). Dashed line indicates nuclear border. Scale bars, 10 μm. g Radial distance of LAD region to the nuclear periphery upon depletion of lamin A/C and LBR as detected by FISH. n Wt = 909, n KO = 666, **** p = 7.6 × 10 -4 (two-sample, two-tailed KS test). Images in ( a ) processed with Imaris, and in ( d ) and ( f ) with Fiji. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Immunostaining, Double Knockout, Fluorescence, Two Tailed Test, ChIP-qPCR, Binding Assay, Control, In Situ Hybridization, Labeling

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Schematic overview of the strategy for generating multiple protein knockout cell lines. MyoD1 reporter C2C12 myoblasts constitutively expressing Cas9 were transfected with three synthetic sgRNAs targeting the gene of interest. Depletion of various candidate proteins was performed consecutively. b Detection of normalized radial distance of MyoD1 to periphery in proliferating myoblasts following depletion of indicated protein(s). Violin plots were generated from at least 450 data points. n Wt = 722, n 4KO = 454, n 3KO = 898, n 2KO = 685, n NET39 KO = 500; n Tmem38a KO = 571, n WFS1 KO = 517. *** p 4KO = 2 × 10 −4 , **** p 3KO = 3.2 × 10 −6 , **** p 2KO = 7.9 × 10 −5 , p NET39 KO = 0.6; *** p Tmem38a KO = 2 × 10 −4 , **** p WFS1 KO = 1 × 10 −6 (two-sample two-tailed KS test, Hochberg correction for multiple testing). Numeric values displayed on the violin plot represent median values. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Knock-Out, Expressing, Transfection, Generated, Two Tailed Test