Journal: Neuropathology and Applied Neurobiology

Article Title: Collablots: Quantification of Collagen VI Levels and Its Structural Disorganisation in Cell Cultures From Patients With Collagen VI‐Related Dystrophies

doi: 10.1111/nan.70020

Figure Lengend Snippet: (A) Illustration of collablot technique workflow. The first part of the workflow consists of cell culture and collagen secretion stimulation. The second is the collablot, where cells are fixed and incubated with a primary antibody, followed by its secondary antibody and then collagen or disorganisation is quantified. (B) Set up of the plates. Plates are divided into permeabilised and non‐permeabilised. Wells in red indicate staining with DRAQ5, and wells in green indicate staining with collagen VI antibody or CHP.

Article Snippet: Samples were then incubated with DNA stain DRAQ5 (Thermo Fisher Scientific, Waltham, MA, US) diluted 1:1000 in PBS for 1 h, after which the plates were scanned using an Odyssey M imaging system (Odyssey M: LI‐COR Biosciences, Lincoln, NE, US).

Techniques: Cell Culture, Incubation, Staining

Journal: Neuropathology and Applied Neurobiology

Article Title: Collablots: Quantification of Collagen VI Levels and Its Structural Disorganisation in Cell Cultures From Patients With Collagen VI‐Related Dystrophies

doi: 10.1111/nan.70020

Figure Lengend Snippet: (A) Immunocytochemical analysis of the three different primary antibodies for collagen VI (ab6588, MAB1944 and MAB3303; 1:2500) was carried out using the CTRL‐1 line, permeabilised and non‐permeabilised. A single replicate with two repeats. (B) Collablot analysis with two different primary antibodies for collagen VI (MAB1944 and MAB3303) titrations (1:1000, 1:2000, 1:4000, 1:10000 and 1:25000) carried out using CTRL‐1 line, permeabilised (total collagen) and non‐permeabilised (extracellular collagen). ColVI signal refers to the DRAQ5‐adjusted ColVI signal. A single replicate with two repeats. Data was analysed using the non‐parametric Mann–Whitney test.

Article Snippet: Samples were then incubated with DNA stain DRAQ5 (Thermo Fisher Scientific, Waltham, MA, US) diluted 1:1000 in PBS for 1 h, after which the plates were scanned using an Odyssey M imaging system (Odyssey M: LI‐COR Biosciences, Lincoln, NE, US).

Techniques: MANN-WHITNEY

Journal: Neuropathology and Applied Neurobiology

Article Title: Collablots: Quantification of Collagen VI Levels and Its Structural Disorganisation in Cell Cultures From Patients With Collagen VI‐Related Dystrophies

doi: 10.1111/nan.70020

Figure Lengend Snippet: (A) Mechanism of action of the Collagen‐Hybridising Peptide (CHP). (B) Immunocytochemical analysis of the colocalisation of the CHP (1:5) and the collagen VI (1:2500) was carried out using a non‐permeabilised CTRL‐1 line. A single replicate with two repeats. (C) Collablot analysis of CHP titrations (1:5, 1:10 and 1:20) carried out using CTRL‐1 line, permeabilised and non‐permeabilised. CHP signal refers to the DRAQ5‐adjusted CHP signal. A single replicate with three repeats. Data was analysed using a non‐parametric Mann–Whitney test.

Article Snippet: Samples were then incubated with DNA stain DRAQ5 (Thermo Fisher Scientific, Waltham, MA, US) diluted 1:1000 in PBS for 1 h, after which the plates were scanned using an Odyssey M imaging system (Odyssey M: LI‐COR Biosciences, Lincoln, NE, US).

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Mitoxantrone inhibits and downregulates ER α through binding at the DBD-LBD interface

doi: 10.1101/2025.01.07.631371

Figure Lengend Snippet: ( A,B ) MTO (1 μM) treatment reduces ER protein levels over time in MCF7 ( A ) and T47D ( B ) cells expressing wildtype or Y537S/D538G knock-in mutant ER, quantified relative to β-actin. ( C ) In MCF7 cells expressing exogenous HA-tagged Y537S ER mutants, MTO decreases both endogenous and exogenous ER levels, with HA-tagged mutant quantified relative to β-actin. ( D ) Immunofluorescence microscopy reveals dose-dependent reduction of ER protein in MCF7 cells treated with MTO (0.1, 0.5, and 1 μM, 48 hours). ( E ) In-cell western assay shows ER levels in MCF7 cells after 18-hour treatment with E2, MTO, or fulvestrant, normalized to DNA content (DRAQ5) and expressed as percentage of DMSO control. ( F ) Time-course immunofluorescence in T47D cells shows nuclear ER reduction after 2-hour MTO treatment. ( G ) Subcellular fractionation reveals early (2-hour) ER accumulation in cytoplasm with concurrent nuclear depletion. Data represent mean ±SEM (n=4). One-way ANOVA with Dunnett’s test: * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: ER protein expression was quantified using a LI-COR Odyssey imaging system and normalized to DRAQ5 DNA stain (Thermo Scientific, # 62251, 1:10,000).

Techniques: Expressing, Knock-In, Mutagenesis, Immunofluorescence, Microscopy, In-Cell ELISA, Control, Fractionation