Journal: Cancer Research Communications

Article Title: Defining the Ovarian Cancer Precancerous Landscape through Modeling Fallopian Tube Epithelium Reprogramming Driven by Extracellular Vesicles

doi: 10.1158/2767-9764.CRC-25-0064

Figure Lengend Snippet: Validation of 1-day and 14-day treatment transcriptomics data using IHC. A, Left, CCL2 transcript expression in OVCAR3-, FT240-, and PBS-treated secretory cells after 1 day. Right, IHC staining for upregulated CCL2 in FTE from three individual patients. B, Left, FLNA transcript expression in OVCAR3-, FT240-, and PBS-treated secretory cells after 1 day. Right, IHC staining for downregulated FLNA in FTE from three individual patients. C, Left, VCAM1 transcript expression in OVCAR3-, FT240-, and PBS-treated secretory cells after 1 day. Right, IHC staining for upregulated VCAM1 in FTE from three individual patients. D, Left, TPI1 transcript expression in OVCAR3-, FT240-, PBS-treated secretory cells after 1 day. Right, IHC staining for downregulated protein TPI1 in FTE from three individual patients. E, Left, TXNIP transcript expression in OVCAR3-, FT240-, and PBS-treated secretory cells after 14 days. Right, IHC staining for downregulated TXNIP in FTE from three individual patients. F, Left, VCAM1 transcript expression in OVCAR3-, FT240-, and PBS-treated ciliated cells after 14 days. Right, IHC staining for upregulated VCAM1 in FTE from three individual patients. In all boxplots, each point represents normalized transcript abundance measured in a single segment. Scale bar, 20 μm; arrows indicate epithelium.

Article Snippet: Primary antibodies against CCL2 (Novus, cat. #NBP1-07035SS, RRID: AB_1625611), VCAM1 (Thermo Fisher Scientific, cat. #MA5-31965, RRID: AB_2809259), FLNA (Proteintech, cat. #67133-1-Ig, RRID: AB_2882432), TPI1 (Proteintech, cat. #10713-1-AP, RRID: AB_2207716), and TXNIP (Thermo Fisher Scientific, cat. #40-3700, RRID: AB_2533462) were incubated overnight at 4°C, followed by detection using a horseradish peroxidase–linked secondary antibody and DAB substrate (the full procedure is provided in the Supplementary Methods).

Techniques: Biomarker Discovery, Expressing, Immunohistochemistry

Journal: Nature Communications

Article Title: The CCL2-CCR2 axis drives neuromuscular denervation in amyotrophic lateral sclerosis

doi: 10.1038/s41467-025-62351-3

Figure Lengend Snippet: A Proteome profiler analysis of 111 inflammatory proteins in gastrocnemius (GC), tibialis anterior (TA) and extensor digitorum longus (EDL) muscles and spinal cord (SC) samples from late symptomatic (postnatal day 19) hTDP-43 Tg/Tg mice. Fold change in protein levels is presented relative to wild-type (WT) littermate controls. In the GC muscle, CCL2, CCL3, CCL4, CCL5 and CCL6 chemokines showed the greatest increase in protein levels (magnified and highlighted in the top right panel). Exact fold changes are available in the Source Data file. B –E Quantitative analysis of CCL2 ( B ), CCL3 ( C ), CCL4 ( D ) and CCL5 ( E ) proteins in the GC and TA muscles and SC using Legendplex immunoassay. Data is presented as mean ± sem. The Legendplex multiplex immunoassay and statistical analysis was performed with n = 4 mice in the GC, TA and SC of the WT group and TA and SC of the Tg/Tg group. The GC of the Tg/Tg group was quantified in n = 3 mice. Data analysis: In each case ( B – E ), unpaired, one-tailed t-test was performed for direct comparison of WT vs Tg/Tg groups; ( B) gastrocnemius : p = 0.0350; ( C) g astrocnemius : p = 0.0174; ( C) tibialis anterior: p = 0.0318; ( C) spinal cord: p = 0.0083; ( D) gastrocnemius: p = 0.0209; ( E) gastrocnemius: p = 0.0031; ( E) tibialis anterior: p = 0.0456. * = p < 0.05; ** = p < 0.01.

Article Snippet: To assess the effects of local immunomodulatory treatment on NMJ denervation and immune cell infiltration, hTDP-43 Tg/Tg mice received two intramuscular injections (at P7 and P14) of either CCL2 neutralising antibody [Cat# AF-479-NA, RRID: AB_354500, R&D Systems, Minneapolis, MN, USA; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in phosphate buffer saline (PBS)] or isotypic normal goat IgG (Cat# AB-108-C, RRID:AB_354267, R&D Systems; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in PBS) in the severely affected GC muscle.

Techniques: Muscles, Multiplex Assay, One-tailed Test, Comparison

Journal: Nature Communications

Article Title: The CCL2-CCR2 axis drives neuromuscular denervation in amyotrophic lateral sclerosis

doi: 10.1038/s41467-025-62351-3

Figure Lengend Snippet: A–C CCL2 staining (green) in the GC muscle of wild-type (WT; A ) and late symptomatic postnatal day 19 (P19) hTDP-43 Tg/Tg (Tg/Tg; B, C ) mice. Arrows indicate CCL2 + cells in the close vicinity of neuromuscular junctions (NMJs), visualised with α-bungarotoxin (BTX; magenta). C High magnification image of CCL2 + cells around a single NMJ in the GC muscle of late stage hTDP-43 Tg/Tg mice. Innervation is shown through NEFH/SV2a (gray) staining. Asterisks label individual nuclei of CCL2 + cells. 3D-reconstructed version of this image is available as Supplementary movie . D–F Quantitative analysis of the ratio of NMJs with perisynaptic CCL2-expressing cells in presymptomatic ( D ), early symptomatic ( E ) and late symptomatic ( F ) mice. Data is presented as mean ± sem. Scale bar represents 50 µm on ( A, B ) and 10 µm on ( C ). Data analysis: ( D ) Presymptomatic stage (postnatal day 7): n = 4 (WT), n = 3 (Tg/Tg), unpaired, two-tailed t-test, p = 0.0466; ( E ) Early symptomatic stage (postnatal day 15): n = 3 (WT, Tg/Tg), unpaired, two-tailed t-test, p = 0.0088; ( F ) Late symptomatic stage: n = 4 (WT, Tg/Tg), unpaired, two-tailed t-test, p = 0.0011. * = p < 0.05; ** = p < 0.01.

Article Snippet: To assess the effects of local immunomodulatory treatment on NMJ denervation and immune cell infiltration, hTDP-43 Tg/Tg mice received two intramuscular injections (at P7 and P14) of either CCL2 neutralising antibody [Cat# AF-479-NA, RRID: AB_354500, R&D Systems, Minneapolis, MN, USA; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in phosphate buffer saline (PBS)] or isotypic normal goat IgG (Cat# AB-108-C, RRID:AB_354267, R&D Systems; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in PBS) in the severely affected GC muscle.

Techniques: Staining, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: The CCL2-CCR2 axis drives neuromuscular denervation in amyotrophic lateral sclerosis

doi: 10.1038/s41467-025-62351-3

Figure Lengend Snippet: A Schematic of experimental design. B–D Representative images of NMJ denervation in the gastrocnemius (GC) muscle of untreated ( B ), IgG-treated ( C ) and CCL2-neutralising antibody-treated ( D ) late symptomatic postnatal day 19 (P19) hTDP-43 Tg/Tg mice. Red arrows indicate fully innervated NMJs, as revealed by the presynaptic NEFH/SV2a staining. Scale bar represents 40 µm. E–G Representative images of the CD45 + leukocyte infiltration in the innervation zone of the GC muscle in untreated ( E ), IgG-treated ( F ) and CCL2-neutralising antibody-treated ( G ) late symptomatic hTDP-43 Tg/Tg mice. Scale bar represents 40 µm. H Qualitative analysis of NMJ innervation, representing the ratio of fully innervated NMJs in the GC muscle. Analysis was performed with n = 7 (untreated) and n = 4 (IgG, CCL2) mice/group and one-way ANOVA with Fisher’s LSD post-hoc, no treatment vs. IgG: p < 0.0001, no treatment vs. CCL2: p < 0.0001. On average 30-40 NMJs were analysed per muscle. I–K Quantitative morphometric analysis of the NMJs revealed improvement in axonal diameter ( I ), nerve terminal area ( J ) and overlap ( K ) variables. The exact p values and statistical analyses are detailed in Table . L, M Quantitative analysis of CD45 + ( L ) and CCR2 + ( M ) cell count in the late symptomatic GC muscle of hTDP-43 Tg/Tg mice. Analysis was performed on n = 8 (untreated) and n = 4 (IgG, CCL2) mice/group. Data analysis: ( L) CD45 analysis: Kruskal-Wallis test and uncorrected Dunn’s post-hoc [Shapiro-Wilk normality test, ALS untreated group: p = 0.0091, ALS IgG group: p = 0.7802, ALS CCL2 group p = 0.3820], no treatment vs. IgG: p = 0.0114, no treatment vs. CCL2: p = 0.0053; ( M) CCR2 analysis: one-way ANOVA with Fisher’s LSD post-hoc, no treatment vs. IgG: p < 0.0001, no treatment vs. CCL2: p = 0.0002. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Data is presented as mean ± sem.

Article Snippet: To assess the effects of local immunomodulatory treatment on NMJ denervation and immune cell infiltration, hTDP-43 Tg/Tg mice received two intramuscular injections (at P7 and P14) of either CCL2 neutralising antibody [Cat# AF-479-NA, RRID: AB_354500, R&D Systems, Minneapolis, MN, USA; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in phosphate buffer saline (PBS)] or isotypic normal goat IgG (Cat# AB-108-C, RRID:AB_354267, R&D Systems; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in PBS) in the severely affected GC muscle.

Techniques: Staining, Cell Counting