Biology subjects

Pepperkok, R.

Publications and source records attributed to Pepperkok, R..

17 recordsLinked to original sources

bioRxiv2026

An Advanced Mobile Laboratory to enable field-based microbial ecology and cell biology across scales

Microbial biodiversity is central to ecosystem function, yet mechanistic insights into the cell biology of environmental organisms remain limited. The underlying challenges are twofold: most microbes remain uncultivable, and a persistent gap exists between field sampling and laboratory analyses. Here, we introduce the Advanced Mobile Laboratory (AML), a field-deployable platform that integrates confocal microscopy, image-enabled cell sorting, and cryo-preparation for expansion and electron microscopy. This setup enables immediate, standardized processing and analysis of environmental communities directly at the sampling site. We demonstrate its capability using marine eukaryotic plankton, showing how the AML enables multiscale investigations, from live imaging of natural communities to enabling ultrastructural and single-cell omics analyses, while minimizing sample degradation and enabling on-site experimentation. By bringing high-end sample preparation and analytical capacity into the field, the AML enables studying life in its natural context to mechanistically understand lifes diversity in the environment.

cell biology↗

bioRxiv2025

iss-nf: A Nextflow-based end-to-end in situ sequencing decoding workflow

In situ sequencing (ISS) offers a powerful approach for spatially resolved gene expression profiling within tissue samples, but the complexity of analyzing the resulting data has limited its broader use. Here, we present iss-nf, a Nextflow-based, end-to-end workflow designed to streamline the decoding of large ISS datasets. The workflow automates critical steps, including image registration, fluorescent spot detection, transcript decoding, and quality control (QC), offering a scalable, reproducible, and user-friendly solution for ISS data analysis. We successfully applied iss-nf on multiple large datasets, including publicly available mouse brain and breast cancer tissue datasets, as well as an in-house non-small cell lung cancer (NSCLC) ISS dataset. The workflow is designed to be accessible for both experienced researchers, as well as newcomers to spatial transcriptomics, providing a robust tool for analyzing large-scale ISS data. Our results suggest that iss-nf is a valuable contribution to the growing field of spatial transcriptomics, enabling precise, modular, reproducible, and, by means of automated tiling and parallelization, scalable analysis of tissue-specific gene expression.

bioinformatics↗

bioRxiv2025

Smart Microscopy: Current Implementations and a Roadmap for Interoperability

Smart microscopy is transforming life sciences by automating experimental imaging workflows and enabling real-time adaptation based on feedback from images and other data streams. This shift increases throughput, improves reproducibility, and expands the functional capabilities of microscopes. However, the current landscape is highly fragmented. Academic researchers often develop custom solutions for specific scientific needs, while industry offerings are typically proprietary and tied to specific hardware. This diversity, while fostering innovation, also creates major challenges in interoperability, reproducibility, and standardization, which slows progress and adaption. This article presents a collaborative effort between academic and industry leaders to survey the current state of smart microscopy, highlight representative implementations, and identify common technical and organizational barriers. We propose a framework for greater interoperability based on shared standards, modular software design, and community-driven development. Our goal is to support collaboration across the field and lay the groundwork for a more connected, reusable, and accessible smart microscopy ecosystem. We conclude with a call to action for researchers, hardware developers, and institutions to join in building an open, interoperable foundation that will unlock the full potential of smart microscopy in life science research.

cell biology↗

bioRxiv2024

Dynamic multi-omics and mechanistic modeling approach uncovers novel mechanisms of kidney fibrosis progression

Kidney fibrosis, characterized by excessive extracellular matrix deposition, is a progressive disease that, despite affecting 10% of the population, lacks specific treatments and suitable biomarkers. This study presents a comprehensive, time-resolved multi-omics analysis of kidney fibrosis using an in vitro model system based on human kidney PDGFR{beta}+ mesenchymal cells aimed at unraveling disease mechanisms. Using transcriptomics, proteomics, phosphoproteomics, and secretomics we quantified over 14,000 biomolecules across seven time points following TGF-{beta} stimulation. This revealed distinct temporal patterns in the expression and activity of known and potential kidney fibrosis markers and modulators. Data integration resulted in time-resolved multi-omic network models which allowed us to propose mechanisms related to fibrosis progression through early transcriptional reprogramming. Using siRNA knockdowns and phenotypic assays, we validated predictions and regulatory mechanisms underlying kidney fibrosis. In particular, we show that several early-activated transcription factors, including FLI1 and E2F1, act as negative regulators of collagen deposition and propose underlying molecular mechanisms. This work advances our understanding of the pathogenesis of kidney fibrosis and provides a resource to be further leveraged by the community. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/618507v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@d2bfaborg.highwire.dtl.DTLVardef@257d34org.highwire.dtl.DTLVardef@13f06f3org.highwire.dtl.DTLVardef@e16e6c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

systems biology↗

bioRxiv2024

Exploring the Antifibrotic Potential of the heparan sulfate mimetic OTR4120: Insights from Preclinical Models

RationaleIdiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive deposition of extracellular matrix (ECM), resulting in lung function impairment. Heparan sulfate mimetics (HSm) have been suggested to have potential antifibrotic effects by regulating ECM. This study aims to investigate the impact of a specific HSm named OTR4120 on fibrotic processes in ex vivo, in vitro and in vivo models. MethodsHuman Precision Cut Lung Slices (hPCLS) treated with a fibrotic cocktail alone or with OTR4120 were evaluated using second-harmonic imaging microscopy (SHIM) to assess collagen deposition. Human embryonic fibroblast WI-38 cell line and primary fibroblasts obtained from human donors were differentiated into myofibroblasts (MYF) using TGF-{beta}1 and treated with OTR4120 or a control vehicle. Gene expression analysis for MYF markers was performed using quantitative PCR (qPCR). Protein expression of MYF markers was evaluated using immunofluorescence techniques. Bulk-RNA sequencing analysis on WI-38 cells cultured under different experimental conditions was conducted. Finally, the therapeutic effects of OTR4120 on a bleomycin-induced fibrosis mouse model were investigated. ResultsSHIM analysis on OTR4120-treated hPCLS showed a decrease in collagen deposition. OTR4120 treatment of primary fibroblasts and WI-38 cells exposed to TGF-{beta}1 significantly reduced the expression of MYF markers. Bulk-RNA sequencing analysis on OTR4120-treated WI-38 cells showed significant impacts on fibrosis-related processes. Therapeutic application of OTR4120 in vivo to bleomycin-induced fibrosis mice resulted in enhanced fibrosis resolution. ConclusionOTR4120 has potential therapeutic benefits as an antifibrotic agent in the context of lung fibrosis. Further investigations are necessary to understand the precise mechanism through which OTR4120 exerts its antifibrotic effects.

cell biology↗

bioRxiv2024

Origins of de novo chromosome rearrangements unveiled by coupled imaging and genomics

Chromosomal instability results in widespread structural and numerical chromosomal abnormalities (CAs) during cancer evolution1-3. While CAs have been linked to mitotic errors resulting in the emergence of nuclear atypias4-7, the underlying processes and basal rates of spontaneous CA formation in human cells remain under-explored. Here we introduce machine learning-assisted genomics-and-imaging convergence (MAGIC), an autonomously operated platform that integrates automated live-cell imaging of micronucleated cells, machine learning in real-time, and single-cell genomics to investigate de novo CA formation at scale. Applying MAGIC to near-diploid, non-transformed cell lines, we track CA events over successive cell cycles, highlighting the common role of dicentric chromosomes as an initiating event. We determine the baseline CA rate, which approximately doubles in TP53-deficient cells, and show that chromosome losses arise more rapidly than gains. The targeted induction of DNA double-strand breaks along chromosomes triggers distinct CA processes, revealing stable isochromosomes, amplification and coordinated segregation of isoacentric segments in multiples of two, and complex CA outcomes, depending on the break location. Our data contrast de novo CA spectra from somatic mutational landscapes after selection occurred. The large-scale experimentation enabled by MAGIC provides insights into de novo CA formation, paving the way to unravel fundamental determinants of chromosome instability.

genomics↗

bioRxiv2024

Reflection confocal microscopy for quantitative assessment of airway surface liquid dysregulation and pharmacological rescue in cystic fibrosis under near-physiological conditions

Proper regulation of airway surface liquid (ASL) is essential for effective mucociliary clearance (MCC) in healthy airways, and ASL depletion due to deficient cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion/fluid secretion plays an important role in the pathogenesis of mucociliary dysfunction and chronic muco-obstructive lung disease in patients with cystic fibrosis (CF). The current standard for quantitative measurements of ASL height is confocal fluorescence microscopy that has the disadvantage that it requires apical addition of volume for fluorescent staining, and hence perturbation of the ASL. Therefore, our aim was to develop a method that enables studies of ASL regulation under unperturbed conditions using reflected light by confocal microscopy of primary airway epithelial cultures grown at air-liquid interface (ALI). After apical volume addition to primary tracheal mouse cultures, confocal reflection microscopy yielded comparable ASL height as confocal fluorescence microscopy on cultures of wild-type mice, and was sensitive to detect ASL depletion on cultures of {beta}ENaC-Tg mice. Under unperturbed conditions, ASL determined by confocal reflection microscopy was significantly higher in wild-type and {beta}ENaC-Tg mice compared to values obtained by confocal fluorescence microscopy. Studies in normal and CF primary human airway epithelial cultures showed that confocal reflection microscopy was sensitive to detect effects of low temperature rescue and pharmacological modulation including improvement of CFTR function by VX-809 and VX-770 in cultures from CF patients with the F508del mutation. Our results support confocal reflection microscopy as a novel sensitive technique for quantitative studies of ASL regulation and response to therapeutic intervention under unperturbed near-physiological conditions in healthy and CF airways. NEW & NOTEWORTHYMeasurement of airway surface liquid (ASL) height by confocal fluorescence microscopy is an important tool to investigate ASL dysregulation and effects of therapeutic strategies aiming at restoring ASL volume to improve mucociliary clearance and lung function in patients with cystic fibrosis. However, confocal fluorescence microscopy has the disadvantage that it requires apical addition of volume for fluorescent staining of the ASL leading to perturbation of its height and composition. Here, we developed confocal reflection microscopy as a new method that enables quantitative assessment of ASL on highly-differentiated primary airway epithelial cultures under unperturbed near-physiological conditions by detection of refracted light.

cell biology↗

bioRxiv2023

Dextromethorphan inhibits collagen transport in the endoplasmic reticulum eliciting an anti-fibrotic response in ex-vivo and in vitro models of pulmonary fibrosis

Excessive deposition of fibrillar collagen in the interstitial extracellular matrix (ECM) of human lung tissue causes fibrosis, which can ultimately lead to organ failure. Despite our understanding of the molecular mechanisms underlying the disease, a cure for pulmonary fibrosis has not yet been found. In this study, we screened an FDA-approved drug library containing 712 drugs and found that Dextromethorphan (DXM), a cough expectorant, significantly reduces the amount of excess fibrillar collagen deposited in the ECM in in-vitro cultured primary human lung fibroblasts (NHLF) and ex-vivo cultured human precision-cut lung slice (hPCLS) models of lung fibrosis. Reduced extracellular fibrillar collagen levels in the ECM upon DXM treatment are due to a reversible trafficking inhibition of collagen type I (COL1) in the endoplasmic reticulum (ER) in TANGO1 and HSP47 positive structures. Mass spectrometric analysis shows that DXM causes hyper-hydroxylation of proline and lysine residues on Collagen (COL1, COL3, COL4, COL5, COL7, COL12) and Latent-transforming growth factor beta-binding protein (LTBP1 and LTBP2) peptides coinciding with their secretion block. In addition, thermal proteome profiling of cells treated with DXM shows increased thermal stability of prolyl-hydroxylases such as P3H2, P3H3, P3H4, P4HA1 and P4HA2, suggesting a change in activity. Transcriptome analysis of pro-fibrotic stimulated NHLFs and hPCLS upon DXM treatment showed activation of an anti-fibrotic program via regulation of pathways such as those involved in the MMP-ADAMTS axis, WNT, and fibroblast-to-myofibroblast differentiation. Taken together, the data obtained from both in-vitro and ex-vivo models of fibrogenesis show that Dextromethorphan has potent anti-fibrotic activity by efficient inhibition of COL1 membrane trafficking in the ER.

cell biology↗

bioRxiv2022

Phenotype-based single-cell transcriptomics reveal compensatory pathways involved in Golgi organization and associated transport.

The Golgi is a dynamic organelle with a unique morphology that has implications on its function. How the structural integrity of the Golgi is maintained despite its dynamic nature has been a long-standing question. Several siRNA-based screens have addressed this question and have identified a number of key players required for Golgi integrity. Interestingly, they also reported heterogeneity of phenotypic responses with regards to Golgi morphology. Although never systematically investigated, this variability has generally been attributed to poor transfection efficiency or cell cycle specific responses. Here we show that this heterogeneity is the result of differential response to the siRNA knockdown in different Golgi phenotypes, independent of transfection efficiency or cell cycle phases. To characterize the observed Golgi phenotype-specific responses at the molecular level we have developed an automated assay which enables microscopy-based phenotype classification followed by phenotype-specific single-cell transcriptome analysis. Application of this novel approach to the siRNA mediated knockdown of USO1, a key trafficking protein at the ER to Golgi boundary, surprisingly suggests a key involvement of the late endosomal/endocytic pathways in the regulation of Golgi organization. Our pipeline is the first of its kind developed to study Golgi organization, but can be applied to any biological problem that stands to gain from correlating morphology with single-cell readouts. Moreover, its automated and modular nature allows for uncomplicated scaling up, both in throughput and in complexity, helping the user achieve a systems level understanding of cellular processes.

systems biology↗

bioRxiv2022

The Golgi complex is a regulatory hub for homologous recombination-mediated DNA repair.

The Golgi complex is a key organelle of the secretory pathway, but also serves as a critical hub for maintaining cellular homeostasis, orchestrating various signalling pathways and essential cellular processes such as membrane trafficking and post-translational modifications. Despite its central role, the communication between the Golgi and the cell nucleus has remained largely unexplored. To bridge this gap, we have analysed and siRNA-validated localisation data of the Human Protein Atlas, finding an unexpected and significant level of interconnectivity between the proteomes of the Golgi complex and nucleus, including an intriguing involvement in DNA repair processes. Here, we uncovered a cluster of DNA repair proteins present in distinct sub-Golgi localisations as well as in the nuclear compartment. In response to genotoxic stress these proteins redistribute dynamically between the Golgi complex and the nucleus, with the specific type of DNA injury influencing this distribution pattern. To probe this Golgi-nucleus link, we examined the Homologous Recombination (HR) regulator RAD51C and find that DNA damage triggers ATM-dependent release of a Giantin-tethered Golgi pool and Importin-{beta}-dependent nuclear import, where repair-associated nuclear foci form. Downregulation of Giantin prematurely releases RAD51C, producing RAD51C nuclear foci lacking key HR markers, reducing ATM activation, elevating genome instability, and accelerating cell proliferation. These findings support a timing-based pathway in which the Golgi acts as a spatiotemporal coordination node for HR regulators and other DDR factors. Our study sheds light on the dynamic interplay between the Golgi and the nucleus in safeguarding genomic stability while maintaining the delicate balance of cellular homeostasis.

cell biology↗

bioRxiv2022

Quantitative imaging reveals PI3Kδ inhibition reduces rhinovirus-induced damage of small airway epithelia in ex vivo cultured human precision cut lung slices from COPD patients

Chronic obstructive pulmonary disease (COPD) is one of the major causes of disability and death worldwide and a significant risk factor for respiratory infections. Rhinoviral infections are the most common trigger of COPD exacerbations which lead to a worsening of disease symptoms, decline in lung function and increased mortality. The lack of suitable disease models to study the relevant cellular and molecular mechanism hinders the discovery of novel medicines that prevent disease progression in exacerbating COPD patients. We used quantitative multi-color imaging of COPD and control patient derived human precision-cut lung slices (hPCLS) to study the impact of rhinovirus infection on the structure and function of the small airway epithelium. Data analysis highlighted that COPD-derived hPCLS have a higher cellular density and basal cell hyperplasia, more unciliated airway surface areas with mucus overproduction, and shorter cilia length compared to control hPCLS. In response to rhinovirus 16 infection, COPD-derived hPCLS secreted higher amounts of pro-inflammatory cytokines and displayed decreased epithelial integrity and reduced airway ciliation. Finally, treatment with a selective PI3K{delta} inhibitor reduced secretion of rhinovirus-induced cytokines and ameliorated rhinovirus-induced damage to COPD small airway epithelia. Thus, these data demonstrate the potential of quantitative imaging to assess complex airway functions in a patient-derived lung tissue model system, and indicate that targeting PI3K{delta} might be a promising therapeutic opportunity to limit rhinovirus-induced airway damage in exacerbating COPD patients. SummaryPI3K{delta} inhibition reduces rhinovirus-mediated damage of small airway epithelia from chronic obstructive pulmonary disease (COPD) patients

pathology↗

bioRxiv2021

CLEMSite, a software for automated phenotypic screens using light microscopy and FIB-SEM

Correlative light and electron microscopy (CLEM) combines two imaging modalities, balancing out the limits of one technique with the other. In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume acquisition at the ultrastructural level. We present a toolset for adherent cultured cells that enables tracking and finding cell regions previously identified in light microscopy, in the FIB-SEM along with automatic acquisition of high-resolution volume datasets. We detect a grid pattern in both modalities (LM and EM), which identifies common reference points. The novel combination of these techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscopes field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5 {micro}m accuracy, while iterating between different targets and implementing unattended data acquisition. Our results demonstrate that executing high throughput volume acquisition in electron microscopy is possible.

cell biology↗

bioRxiv2021

A high throughput SEC23 functional interaction screen reveals a role for focal adhesion and extracellular matrix signalling in the regulation of COPII subunit SEC23A

Proteins that enter the secretory pathway are transported from their place of synthesis in the Endoplasmic Reticulum to the Golgi complex by COPII coated carriers. The components of the COPII transport machinery have been well characterized, but the network of proteins that regulate these components in response to extracellular cues have remained largely elusive. The discovery of such regulatory proteins is crucial for understanding how cells integrate extracellular and intracellular information to fine-tune membrane traffic in the secretory pathway. A key group of proteins that plays a central role in the regulation of membrane traffic is associated with the cytoskeleton. Using high throughput microscopy of the well-established VSVG transport from the ER to the plasma membrane, we comprehensively screened 378 cytoskeleton-associated proteins for their functional interaction with the COPII components SEC23A and SEC23B using a double knockdown approach. Ranking of the transport effectors identified 20 cytoskeleton-associated proteins as the strongest functional interactors of SEC23, most of them not previously associated with the secretory pathway. Knockdown of a subgroup of these interactors (FERMT2, MACF1, MAPK8IP2, NGEF, PIK3CA, ROCK1), associated with cell adhesion, not only induced changes in focal adhesions and the expression of adhesion-related genes but led to the specific downregulation of SEC23A, a SEC23 paralogue that has been implicated in the secretion of extracellular matrix (ECM) components. Furthermore, SEC23A downregulation could also be recapitulated by plating cells on ECM and was dependent on focal adhesions function. Altogether, our results identify a network of cytoskeleton-associated proteins connecting focal adhesions and ECM-related signalling with the gene expression of the COPII secretory machinery.

cell biology↗

bioRxiv2020

Pairwise genetic interactions modulate lipid plasma levels and cellular uptake

Genetic interactions (GIs), the joint impact of different genes or variants on a phenotype, are foundational to the genetic architecture of complex traits. However, identifying GIs through human genetics is challenging since it necessitates very large population sizes, while findings from model systems not always translate to humans. Here, we combined exome-sequencing and genotyping in the UK Biobank with combinatorial RNA-interference (coRNAi) screening to systematically test for pairwise GIs between 30 lipid GWAS genes. Gene-based protein-truncating variant (PTV) burden analyses from 240,970 exomes revealed additive GIs for APOB with PCSK9 and LPL, respectively. Both, genetics and coRNAi identified additive GIs for 12 additional gene pairs. Overlapping non-additive GIs were detected only for TOMM40 at the APOE locus with SORT1 and NCAN. Our study identifies distinct gene pairs that modulate both, plasma and cellular lipid levels via additive and non-additive effects and nominates drug target pairs for improved lipid-lowering combination therapies.

genetics↗

bioRxiv2020

Global distribution patterns of marine nitrogen-fixers by imaging and molecular methods

Biological nitrogen fixation plays a critical role in marine primary production, yet, our understanding of marine N2-fixers (diazotrophs) is hindered by limited observations. Here, we developed a quantitative image analysis pipeline in concert with mapping of molecular markers for mining >2,000,000 images and >1,300 metagenomes in surface, deep chlorophyll maximum and mesopelagic samples across 6 size fractions (<0.2-2000 m). Imaging and PCR-free molecular data were remarkably congruent. Sequences from diazotrophs were detected from the ultrasmall bacterioplankton (<0.2 m) to mesoplankton (180-2000 m), while images predicted symbiotic and colonial-forming diazotrophs (>20 {micro}m). Imaging and molecular data estimated that polyploidy can significantly impact gene abundances of symbiotic vs colonial-forming diazotrophs. In general our results support the canonical view that larger sized diazotrophs (>10 m) dominate the tropical belts, while sequences from unicellular cyanobacterial and non-cyanobacterial diazotrophs were globally distributed in surface and the mesopelagic. Co-occurring diazotrophic lineages of different lifestyles were frequently encountered, and several new high density regions of diazotrophs were identified in the global ocean. Overall, this work provides an update of marine diazotroph biogeographical diversity and contributes a new bio-imaging-informatic workflow.

microbiology↗

bioRxiv2020

BigDataProcessor2: A free and open-source Fiji plugin for inspection and processing of TB sized image data

Modern bioimaging and related areas such as sensor technology has seen tremendous development the last years allowing several contemporary imaging techniques, electron microscopy (EM) and light sheet microscopy in particular, to generate datasets frequently reaching the size of several terabytes (TB). As a consequence, even seemingly simple data operations such as cropping, chromatic- and drift-corrections and even visualisation, poses challenges when applied to thousands of time points or tiles. To address this we developed BigDataProcessor2 - a Fiji plugin facilitating processing workflows for TB sized image datasets. Availability and implementationBigDataProcessor2 is available as a Fiji plugin via the BigDataProcessor update site. The application is implemented in Java and the code is publicly available on GitHub (https://github.com/bigdataprocessor/bigdataprocessor2). Contactchristian.tischer@embl.de, nils.norlin@med.lu.se

bioinformatics↗

bioRxiv2019

Multiomic and quantitative label-free microscopy-based analysis of ex vivo culture and TGFbeta1 stimulation of human precision-cut lung slices

Fibrosis can affect any organ resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by expansion of connective tissue due to excessive deposition of extracellular matrix proteins (ECM), including the fibrillar forms of collagen. A significant hurdle for discovering cures for fibrosis is the lack of suitable models and techniques to quantify mature collagen deposition in tissues. Here we have extensively characterized an ex-vivo cultured human lung derived, precision-cut lung slices model (hPCLS) using live fluorescence light microscopy as well as mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint. Using an integrated approach of multiple readouts such as quantitative label-free Second Harmonic Generation (SHG) imaging to measure fibrillar collagen in the extracellular matrix and ELISA-based methods to measure soluble ECM biomarkers, we investigated TGFbeta1-mediated pro-fibrotic signalling in hPCLS. We demonstrate that hPCLS are viable and metabolically active with mesenchymal, epithelial, endothelial, and immune cells surviving for at least two weeks in ex vivo culture. Analysis of hPCLS-conditioned supernatants showed strong induction of ECM synthesis proteins P1NP and fibronectin upon TGFb stimulation. Importantly, this effect translated into an increased deposition of fibrillar collagen in ECM of cultured hPCLS as measured by a novel quantitative SHG-based imaging method only following addition of a metalloproteinase inhibitor (GM6001). Together the data show that an integrated approach of measuring soluble pro-fibrotic markers and quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing anti-fibrotic agents.

physiology↗