ib4 biotinylated mouse rat rabbit goat griffonia simplicifolia vector  (Vector Laboratories)

Journal: Nature cancer

Article Title: Skeletal muscle endothelial dysfunction through the activin A–PGC1α axis drives progression of cancer cachexia

doi: 10.1038/s43018-025-00975-6

Figure Lengend Snippet: a-e . Characteristics of cachexia in control (3 and 5 month, referred to as 3 m and 5 m) and KPC (3 m and 5 m) mice. a . Body weight (n = 9 for control 3 m, n = 8 for KPC 3 m, n = 10 for control 5 m, n = 11 for KPC 5 m). Each dot represents one mouse. b-c . Representative flow plots of CD31 + CD45- muscle ECs ( b ) and quantification of muscle ECs by FACS analysis ( c , n = 3). Each dot represents one mouse. d . The vasculature of the lung and spleen from age-matched control and KPC-3-month mice was evaluated by IB4 IF analysis. e . Four limbs grip strength of mice (n = 10 for control 3 m, n = 8 for KPC 3 m, n = 13 for control 5 m and KPC 5 m). Each dot represents one mouse. f . Gastrocnemius (GC) mass (n = 6 for control 3 m, n = 9 for KPC 3 m, n = 10 for control 5 m and KPC 5 m). Each dot represents one mouse. g . Tibialis anterior (TA) mass (n = 7 for control 3 m, n = 8 for KPC 3 m, n = 10 for control 5 m, n = 9 for KPC 5 m). Each dot represents one mouse. h . Expression of cachexia markers, MuRF1 (n = 12 for control 5 m, n = 14 for KPC 3 m, n = 4 for KPC 5 m) and Atrogin1 (n = 8 for control 5 m, n = 12 for KPC 3 m, n = 4 for KPC 5 m) in TA muscles by RT-qPCR. Each dot represents one mouse. i-l . Control-5month and KPC-5month mice were assessed for changes in muscle fiber types and fat mass. i . Representative images for type 2a and type 1 muscle fiber in GC muscles. j-k . Quantification of muscle fiber types in GC ( j ) (n = 4) and TA ( k ) muscles (n = 3). Each dot represents one mouse. l . The weight (g) of adipose tissues was normalized using tibial length (mm) (n = 8 for sWAT and vWAT, n = 8 for control BAT, n = 7 for KPC 5 m BAT). Each dot represents one mouse. sWAT : subcutaneous white adipose tissue, vWAT ; visceral white adipose tissue, BAT ; brown adipose tissue. Data are presented as mean ± SEM. Statistical analysis was conducted using unpaired, two-tailed t-test ( c, j, k, l ) or one-way ANOVA with Tukey’s multiple comparison test ( a, e-h ).

Article Snippet: After washing with 1× PBS, the slides were permeabilized with 0.2% Triton X-100 for 10 min at room temperature and followed by blocking with blocking buffer (1× PBS, 2% BSA, 0.05% Tween-20 and 5% goat serum) for 1 h at room temperature and then stained with EC-specific CD31 antibody (ab28364; 1:100 dilution) or biotinylated IB4 (Vector Labs, B-1205-.5; 1:100 dilution) overnight at 4 °C, followed by staining with goat anti-rabbit Alexa Fluor 594 secondary antibody (A-11012, Invitrogen; 1:500 dilution) or FITC–streptavidin (Invitrogen, 11–4317-87; 1:500 dilution) for 1 h at room temperature.

Techniques: Control, Expressing, Muscles, Quantitative RT-PCR, Two Tailed Test, Comparison

Journal: Nature cancer

Article Title: Skeletal muscle endothelial dysfunction through the activin A–PGC1α axis drives progression of cancer cachexia

doi: 10.1038/s43018-025-00975-6

Figure Lengend Snippet: a – d , Control mice were intravenously injected with AAV-activin A low dose and AAV-activin A high dose and evaluated after 3 weeks. a , Representative 3D images for the surface volume of total CD31 + (red) or functional IB4 + (green) vessels in the GC muscles. b , Muscle vascular density (percentage of CD31 + or IB4 + vessel volume in 3D ROI volume; n = 3). Each dot represents one mouse. c , d , Muscle ECs isolated from mice overexpressing AAV-activin A low dose and AAV-activin A high dose were evaluated for the expression of apoptosis-related genes ( c ) or EndMT marker genes ( d ) by RT–qPCR ( n = 5). Each dot represents one mouse. e – h , Control 5-month-old mice and KPC mice (3 and 5 months old). e , Cross-sectioned GC muscles were used for an in situ TUNEL assay for apoptotic cells and costained with endothelial TF EGR1 for ECs. Top, the selected areas (white square boxes) were enlarged to show the colocalization of TUNEL and EGR1. White arrows indicate the double-positive cells for TUNEL (red) and EGR1 (green) signal. f , Number of double-positive cells of TUNEL and EGR1 in e ( n = 5 for control 5-month-old and KPC 3-month-old mice and n = 4 for KPC 5-month-old mice). Each dot represents one mouse. g , GC muscles were costained with IB4 for ECs and with anti-α-SMA antibody for mesenchymal cells. White arrows indicate the colocalization of IB4 (red) and α-SMA (green) signals. h , Quantification of IB4 and anti-α-SMA double-positive cells in g ( n = 3). Each dot represents one mouse. i , Muscle ECs isolated from control 5-month-old and KPC 5-month-old mice were evaluated for the expression of EndMT marker genes by RT–qPCR ( n = 5 for CD31 , n = 6 for Cdh5 , n = 4 for VEGFR2 , n = 5 for Twist and n = 7 for Snail ). Each dot represents one mouse. The nuclei were visualized with DAPI. Each gene level was normalized by PPIA levels and is presented as the FC. Data are presented as the mean ± s.e.m. Statistical analysis was conducted using an unpaired two-tailed t -test ( b – d and i ) or a one-way ANOVA with Tukey’s multiple-comparison test ( f and h ).

Article Snippet: After washing with 1× PBS, the slides were permeabilized with 0.2% Triton X-100 for 10 min at room temperature and followed by blocking with blocking buffer (1× PBS, 2% BSA, 0.05% Tween-20 and 5% goat serum) for 1 h at room temperature and then stained with EC-specific CD31 antibody (ab28364; 1:100 dilution) or biotinylated IB4 (Vector Labs, B-1205-.5; 1:100 dilution) overnight at 4 °C, followed by staining with goat anti-rabbit Alexa Fluor 594 secondary antibody (A-11012, Invitrogen; 1:500 dilution) or FITC–streptavidin (Invitrogen, 11–4317-87; 1:500 dilution) for 1 h at room temperature.

Techniques: Control, Injection, Functional Assay, Muscles, Isolation, Expressing, Marker, Quantitative RT-PCR, In Situ, TUNEL Assay, Two Tailed Test, Comparison

Journal: Nature cancer

Article Title: Skeletal muscle endothelial dysfunction through the activin A–PGC1α axis drives progression of cancer cachexia

doi: 10.1038/s43018-025-00975-6

Figure Lengend Snippet: a-d . C57BL/6 WT mice were intravenously injected with AAV-Activin-A in a dose-dependent manner, and Activin-A levels were measured every week for 3 weeks. a . Activin-A levels in blood plasma following AAV-Activin-A overexpression (n = 4 for PBS 1w and 3w, n = 5 for PBS 2w, n = 6 for AAV-Activin-A low-OE 1w, 2w, 3w, n = 6 for AAV-Activin-A high-OE 1w, 2w, 3w). Each dot represents one mouse. b . Four limbs’ grip strength (n = 3 for PBS, n = 6 for AAV-Activin-A low-OE , n = 6 for AAV-Activin-A high-OE ). Each dot represents one mouse. c . Body weight (n = 3 for PBS, n = 6 for AAV-Activin-A low-OE , n = 9 for AAV-Activin-A high-OE ). Each dot represents one mouse. d . GC and TA muscle mass (n = 3 for PBS, n = 6 for AAV-Activin-A low-OE , n = 9 for AAV-Activin-A high-OE ). Each dot represents one mouse. e-g . The HLMVECs were treated with Activin-A (25 ng/mL) for 24 h. e . HUVEC viability with and without Activin-A exposure (n = 6). Each dot represents one biological replicate. f-g . the apoptotic cells were evaluated with Annexin V-FITC + PI+ cells using FACS analysis. Representative flow plots ( f ) and quantification of apoptotic muscle ECs ( g ) (n = 3 for vehicle, n = 6 for Activin-A). Each dot represents one biological replicate. h . The expression of EndMT marker genes related genes by RT-qPCR (n = 6 for Cdh5 , n = 8 for VEGFR2 , n = 4 for Snail , n = 6 for Vimentin ). Each dot represents one biological replicate. i . HLMVECs were treated with Activin-A (25 ng/mL) for the indicated time course (0, 5, 15, 30, 60, or 90 min) and followed by Western blotting. j . Quantification of the phospho-Smad3 levels in i (n = 3). Each dot represents one biological replicate. The statistical analysis was performed using a t-test with comparisons vs the 0 min time point. k . In situ TUNEL assay for apoptotic ECs in GC muscles of control (n = 4) and melanoma-bearing mice (n = 4). Double-positive cells of TUNEL and ERG1 in total ECs (ERG1 + ) were considered apoptotic ECs. Each dot represents one mouse. l . The expression of apoptosis genes in isolated muscle ECs from control (n = 3) and LLC1 cachexia (n = 3) mice by RT-qPCR. Each dot represents one mouse. m . Quantification of EndMT cells (double-positive cells of IB4 and α-SMA in total IB4+ ECs) in GC muscles of control and melanoma 3w mice (n = 3 for control, n = 5 for melanoma 3w). Each dot represents one mouse. n . The expression of EndMT marker genes in isolated muscle ECs from control and melanoma cachexia mice by RT-qPCR (n = 3 for CD31 ,n = 4 for Cdh5 , VEGFR2 , Snail , and Vimentin ). Each dot represents one mouse. o . The expression of EndMT marker genes in isolated muscle ECs from control and LLC1 cachexia (n = 3) mice by RT-qPCR. Each dot represents one mouse. Data are presented as mean ± SEM. Each gene level was normalized by 18 s or PPIA levels and presented as a fold change. Statistical analysis was conducted using either unpaired, two-tailed t-test ( e , g , h , j , l , m , n , o ) or one-way ANOVA with Tukey’s multiple comparison test ( a-d , k ).

Article Snippet: After washing with 1× PBS, the slides were permeabilized with 0.2% Triton X-100 for 10 min at room temperature and followed by blocking with blocking buffer (1× PBS, 2% BSA, 0.05% Tween-20 and 5% goat serum) for 1 h at room temperature and then stained with EC-specific CD31 antibody (ab28364; 1:100 dilution) or biotinylated IB4 (Vector Labs, B-1205-.5; 1:100 dilution) overnight at 4 °C, followed by staining with goat anti-rabbit Alexa Fluor 594 secondary antibody (A-11012, Invitrogen; 1:500 dilution) or FITC–streptavidin (Invitrogen, 11–4317-87; 1:500 dilution) for 1 h at room temperature.

Techniques: Injection, Clinical Proteomics, Over Expression, Expressing, Marker, Quantitative RT-PCR, Western Blot, In Situ, TUNEL Assay, Muscles, Control, Isolation, Two Tailed Test, Comparison

Journal: Nature cancer

Article Title: Skeletal muscle endothelial dysfunction through the activin A–PGC1α axis drives progression of cancer cachexia

doi: 10.1038/s43018-025-00975-6

Figure Lengend Snippet: a-b . C57BL/6 WT mice were intravenously injected with AAV-Activin-A low dose and AAV-Activin-A high dose. After 3weeks, the mice were injected intravenously with IB4-A594 and fixable FITC-dextran before harvesting gastrocnemius muscles. a . Representative images of FITC-dextran+ (green) area. b . Intensity of FITC-dextran signal in a (n = 3). Each dot represents one mouse. c-e . The mice were intraperitoneally injected with pimonidazole (100 mg/kg) 1 h before harvesting muscle and the extent of hypoxia in gastrocnemius muscle was determined by the Hypoxyprobe Plus Kit. c . Muscle hypoxia in LLC1-cachexia mice. d . Quantification of surface volume of FITC-pimonidazole in c (n = 3). Each dot represents one mouse. e . Quantification of fluorescence intensity of FITC-pimonidazole in muscles of melanoma cachexia mice (n = 5). Each dot represents one mouse. f . H&E-stained abdominal muscles from control subjects and cancer patients. The yellow boxes were enlarged to show clear vessel structure and the infiltrated immune cells in near blood vessels. R1 and R2 in the upper panel indicate different regions from the same patient muscle. g . Gating strategy for flow cytometry analysis at – . . The proportion (%) of immune cell type relative to total immune cells from control and melanoma 3w mice in the scRNA-Seq data. i . The PGC1α mRNA levels in isolated muscle ECs (n = 4 for control 5 m and n = 6 for KPC 5 m, n = 8 for control and n = 12 for melanoma 3w, n = 3 for control and LLC1 3w). Each dot represents one mouse. j . Expression of PGC1α in control and cancer patient muscle ECs by IF analysis (n = 5). The yellow boxes are enlarged to show the colocalization of CD31 and PGC1α. k-l . HLMVECs were treated with Activin-A (25 ng/mL) for indicated times. k . Immunoblotting to assess phospho-FOXO1/3/4 levels. l . Quantification of p-FOXO1/3/4 immunoblots shown in k (n = 3). Each dot represents one biological replicate. The statistical analysis was performed using a t-test with comparisons versus the 0 min time point. m . HLMVECs were transfected with specific siRNA for control, FOXO-1, −3, or −4. The protein levels of PGC1α, FOXO-1, −3, or −4 were determined by immunoblotting. n . Quantification of PGC1α levels in m (n = 3). Each dot represents one biological replicate. Data are presented as mean ± SEM. Statistical analysis was conducted using an unpaired, two-tailed t-test ( b , d, e, i, l ) or one-way ANOVA with Dunnett’s multiple comparison test ( n ).

Article Snippet: After washing with 1× PBS, the slides were permeabilized with 0.2% Triton X-100 for 10 min at room temperature and followed by blocking with blocking buffer (1× PBS, 2% BSA, 0.05% Tween-20 and 5% goat serum) for 1 h at room temperature and then stained with EC-specific CD31 antibody (ab28364; 1:100 dilution) or biotinylated IB4 (Vector Labs, B-1205-.5; 1:100 dilution) overnight at 4 °C, followed by staining with goat anti-rabbit Alexa Fluor 594 secondary antibody (A-11012, Invitrogen; 1:500 dilution) or FITC–streptavidin (Invitrogen, 11–4317-87; 1:500 dilution) for 1 h at room temperature.

Techniques: Injection, Muscles, Fluorescence, Staining, Control, Flow Cytometry, Isolation, Expressing, Western Blot, Transfection, Two Tailed Test, Comparison

Journal: Nature cancer

Article Title: Skeletal muscle endothelial dysfunction through the activin A–PGC1α axis drives progression of cancer cachexia

doi: 10.1038/s43018-025-00975-6

Figure Lengend Snippet: a-d . Effect of Activin-A neutralizing antibody. a . Timelines for Activin-A neutralizing antibody treatment. b . Expression of EC marker and anti-apoptotic genes in isolated muscle ECs ( CD31 ; n = 8 for PBS, n = 12 for Melanoma + IgG, n = 8 for Melanoma + α-Activin-A ab, Bcl2; n = 8 for PBS, n = 12 for Melanoma + IgG, n = 8 for Melanoma + α-Activin-A ab, Mcl1; n = 8 for PBS, n = 12 for Melanoma + IgG, n = 8 for Melanoma + α-Activin-A ab). Each dot represents one mouse. c . The tumor growth (n = 4 for PBS, n = 5 for Melanoma + IgG and Melanoma + α-Activin-A ab). Each dot represents one mouse. d . The body weight was presented without (Δ) tumor weight (n = 8 for PBS, n = 4 for Melanoma + IgG, n = 5 for Melanoma + α-Activin-A ab). Each dot represents one mouse. e . Timelines for intramuscular injection of lenti-EC-PGC1α-GFP virus. f-i . Efficiency of local vascular overexpression of EC-PGC1α in muscles. f . All mice were perfused with IB4-A594 before harvesting the muscles, and the endothelial-specific expression of lenti-PGC1α-EGFP virus in muscle was determined by IF analysis for IB4. g . Percentage of GFP+ or IB4+ cells per field in f (n = 4). Each dot represents one mouse. h . Percentage of GFP+ cells in IB4+ cells in f (n = 4). Each dot represents one mouse. i . Expression of PGC1α in isolated muscle ECs (n = 7). j-o . Effect of local vascular overexpression of EC-PGC1α in melanoma bearing mice. j . H&E-stained GC muscles (n = 3). The ‘n’ represents the number of mice. k . Quantification of muscle fiber type 2a Fiber (n = 3). Each dot represents one mouse. l . Body weight was presented without (Δ) tumor weight (n = 4 for control, n = 3 for EC-PGC1α im-OE alone, n = 6 for melanoma alone, n = 7 for melanoma with EC-PGC1α im-OE ). Each dot represents one mouse. m . Melanoma growth (n = 4 for control, n = 3 for EC-PGC1α im-OE alone, n = 6 for melanoma alone, n = 6 for melanoma with EC-PGC1α im-OE ). Each dot represents one mouse. n . Melanoma weight (n = 6). Each dot represents one mouse. o . Adipose tissue weight (n = 4 for control, n = 3 for EC-PGC1α im-OE alone, n = 4 for melanoma alone, n = 4 for melanoma with EC-PGC1α im-OE ). Each dot represents one mouse. p-v . Effect of local overexpression of EC-PGC1α in CT26 bearing mice. p . 3D tissue images with IB4+ (green) functional vessels. q . Quantification of functional muscle vascular density (n = 3). Each dot represents one mouse. r . Muscle weight (n = 4 for control, n = 4 for EC-PGC1α im-OE alone, n = 5 for CT26 alone, n = 5 for CT26 with EC-PGC1α im-OE ). Each dot represents one mouse. s . Grip strength (n = 4 for control, n = 4 for EC-PGC1α im-OE alone, n = 5 for CT26 alone, n = 5 for CT26 with EC-PGC1α im-OE ). Each dot represents one mouse. t . Body weight without (Δ) tumor weight (n = 4 for control, n = 4 for EC-PGC1α im-OE alone, n = 6 for CT26 alone, n = 6 for CT26 with EC-PGC1α im-OE ). Each dot represents one mouse. u . CT26 tumor weight (n = 4 for control, n = 4 for EC-PGC1α im-OE alone, n = 6 for CT26 alone, n = 6 for CT26 with EC-PGC1α im-OE ). Each dot represents one mouse. v . Adipose tissue weight (n = 4 for control, n = 4 for EC-PGC1α im-OE alone, n = 7 for CT26 alone for sWAT and vWAT, n = 6 for CT26 alone for BAT, n = 6 for CT26 with EC-PGC1α im-OE ). Each dot represents one mouse. Data are presented as mean ± SEM. Statistical analysis was conducted using unpaired, two-tailed t-test ( i, n ) or one-way ANOVA with Tukey’s multiple comparison test ( b-d, k, l, m , o, q-v ).

Article Snippet: After washing with 1× PBS, the slides were permeabilized with 0.2% Triton X-100 for 10 min at room temperature and followed by blocking with blocking buffer (1× PBS, 2% BSA, 0.05% Tween-20 and 5% goat serum) for 1 h at room temperature and then stained with EC-specific CD31 antibody (ab28364; 1:100 dilution) or biotinylated IB4 (Vector Labs, B-1205-.5; 1:100 dilution) overnight at 4 °C, followed by staining with goat anti-rabbit Alexa Fluor 594 secondary antibody (A-11012, Invitrogen; 1:500 dilution) or FITC–streptavidin (Invitrogen, 11–4317-87; 1:500 dilution) for 1 h at room temperature.

Techniques: Expressing, Marker, Isolation, Injection, Virus, Over Expression, Muscles, Staining, Control, Functional Assay, Two Tailed Test, Comparison

Journal: Nature cancer

Article Title: Skeletal muscle endothelial dysfunction through the activin A–PGC1α axis drives progression of cancer cachexia

doi: 10.1038/s43018-025-00975-6

Figure Lengend Snippet: a – e , Mice were intravenously injected with anti-activin A neutralizing antibody or IgG1 isotype every 4 days, 1 week after melanoma implantation. a , Muscle vasculature by IF staining with CD31 antibody. Nuclei were stained with DAPI. b . Muscle vasculature density ( n = 3 for PBS and n = 5 for melanoma + IgG and melanoma + anti-activin A antibody). Each dot represents one mouse. c , Mouse grip strength ( n = 8 for PBS, n = 4 for melanoma + IgG and n = 5 for melanoma + anti-activin A antibody). Each dot represents one mouse. d , Muscle mass for GC ( n = 6 for PBS and n = 5 for melanoma + IgG and melanoma + anti-activin A antibody) and TA ( n = 8 for PBS and n = 5 for melanoma + IgG and melanoma + anti-activin A antibody). Each dot represents one mouse. e , Cachectic marker MuRF1 mRNA in TA muscle ( n = 6 for PBS and n = 5 for melanoma + IgG and melanoma + anti-activin A antibody). Each dot represents one mouse. f – l , After melanoma implantation, the mice were intramuscularly injected with lentiviral control and lenti-EC PGC1α–GFP. f – h , Mice were retro-orbitally injected with IB4–A594 before isolating muscles. f , Representative 3D vasculature after overlaying with CD31 + (red) and IB4 + (pseudocolored green) vessels in GC muscles. g , Quantification of CD31 + (red) muscle vasculature density in f ( n = 3). Each dot represents one mouse. h , Manders’ colocalization coefficients for CD31 and IB4 in f ( n = 3). Each dot represents one mouse. i , Mouse grip strength ( n = 8 for control, n = 3 for EC PGC1α im-OE alone, n = 8 for melanoma alone and n = 7 for melanoma with EC PGC1α im-OE ). Each dot represents one mouse. j , Muscle mass for GC ( n = 9 for control, n = 4 for EC PGC1α im-OE alone, n = 10 for melanoma alone and n = 10 for melanoma with EC PGC1α im-OE ) and TA ( n = 8 for control, n = 4 for EC PGC1α im-OE alone, n = 9 for melanoma alone and n = 9 for melanoma with EC PGC1α im-OE ). Each dot represents one mouse. k , mRNA levels of MuRF1 ( n = 3 for control and n = 5 for melanoma with or without EC PGC1α im-OE ) and Atrogin1 ( n = 6 for control, n = 5 for melanoma without EC PGC1α im-OE and n = 4 for melanoma with EC PGC1α im-OE ) by RT–qPCR. Each dot represents one mouse. l , Inflammatory genes in GC muscles by RT–qPCR ( TNF , n = 4 for control and n = 8 for melanoma with or without EC PGC1α im-OE ; IL1β , n = 8 for control, n = 12 for melanoma without EC PGC1α im-OE and n = 10 for melanoma with EC PGC1α im-OE ). Each dot represents one mouse. m , Graphical summary. All mice were examined 3 weeks after melanoma implantation. Data are presented as the mean ± s.e.m. Each gene level was normalized by PPIA levels and is presented as the FC. Statistical analysis was conducted using a one-way ANOVA with Tukey’s multiple-comparison tests ( b – e and g – l ).

Article Snippet: After washing with 1× PBS, the slides were permeabilized with 0.2% Triton X-100 for 10 min at room temperature and followed by blocking with blocking buffer (1× PBS, 2% BSA, 0.05% Tween-20 and 5% goat serum) for 1 h at room temperature and then stained with EC-specific CD31 antibody (ab28364; 1:100 dilution) or biotinylated IB4 (Vector Labs, B-1205-.5; 1:100 dilution) overnight at 4 °C, followed by staining with goat anti-rabbit Alexa Fluor 594 secondary antibody (A-11012, Invitrogen; 1:500 dilution) or FITC–streptavidin (Invitrogen, 11–4317-87; 1:500 dilution) for 1 h at room temperature.

Techniques: Injection, Staining, Marker, Control, Muscles, Quantitative RT-PCR, Comparison

Journal: Nature cancer

Article Title: Skeletal muscle endothelial dysfunction through the activin A–PGC1α axis drives progression of cancer cachexia

doi: 10.1038/s43018-025-00975-6

Figure Lengend Snippet: a . Timelines for systemic injection of EC-PGC1α lentivirus. The ‘ sys-OE’ indicates the systematical overexpression. b-i . After tumor implantation, the mice were intravenously injected with lenti-control and lenti-PGC1α-EGFP virus and evaluated 3 weeks later. b . IB4 positive (+) functional vessels in gastrocnemius muscle of control and melanoma 3w mice (n = 4). c . Expression levels of PGC1α in isolated muscle ECs (n = 6). Each dot represents one mouse. d-i . melanoma-bearing mice (n = 4) and CT26-bearing mice (n = 3 for CT26 alone, n = 5 for CT26 with EC-PGC1α sys-OE ). d . Mouse grip strength. e . Tumor growth. f . Body weight without (Δ) tumor weight. g . GC muscle weight. h . TA muscle weight. i . Adipose tissues’ weight. Data are presented as mean ± SEM. Statistical analysis was conducted using an unpaired, two-tailed t-test ( c-i ).

Article Snippet: After washing with 1× PBS, the slides were permeabilized with 0.2% Triton X-100 for 10 min at room temperature and followed by blocking with blocking buffer (1× PBS, 2% BSA, 0.05% Tween-20 and 5% goat serum) for 1 h at room temperature and then stained with EC-specific CD31 antibody (ab28364; 1:100 dilution) or biotinylated IB4 (Vector Labs, B-1205-.5; 1:100 dilution) overnight at 4 °C, followed by staining with goat anti-rabbit Alexa Fluor 594 secondary antibody (A-11012, Invitrogen; 1:500 dilution) or FITC–streptavidin (Invitrogen, 11–4317-87; 1:500 dilution) for 1 h at room temperature.

Techniques: Injection, Over Expression, Tumor Implantation, Control, Virus, Functional Assay, Expressing, Isolation, Two Tailed Test

Journal: Scientific reports

Article Title: Protein disulfide isomerase integrates toll-like receptor 4 and P2X7 receptor signaling pathways during lipopolysaccharide-induced neuroinflammation.

doi: 10.1038/s41598-025-92780-5

Figure Lengend Snippet: Fig. 1. Effects of P2X7R deletion on PDI expression and reactive gliosis in response to LPS. P2X7R deletion does not alter PDI expression level in the hippocampus under physiological condition. P2X7R ablation attenuates PDI upregulation and microglial (not astroglial) activation following LPS treatment. (A) Effects of P2X7R deletion on PDI expression in response to LPS. Representative Western blot images for PDI (left panels) and quantification of the effects of P2X7R deletion on PDI density (right panel) following LPS treatment (#,*p < 0.05 vs. control and P2X7R+/+mice, respectively; Kruskal–Wallis test, n = 7, respectively). (B) Representative photos for Iba-1 positive microglia in the hippocampus following LPS treatment. (C, D) Representative immunofluorescent images for PDI expression in microglia (C) and astrocytes. (E) Quantification of the effects of P2X7R deletion on IB4 (a microglial marker), GFAP (an astroglial marker) and PDI intensities under post-LPS condition (*p < 0.05 vs. P2X7R+/+mice, respectively; Mann–Whitney test, n = 7, respectively).

Article Snippet: Fluorescent intensity was performed by two Antigen Host Manufacturer (Catalog number) Dilution GFAP Mouse Millipore (#MAB3402) 1:2000 (IH) IB4 - Vector (#B-1205) 1:200 (histochemistry) Iba-1 Rabbit Biocare Medical (#CP290) 1:500 (IH) iNOS Rabbit Novus Biologicals (#NB300-605) 1:500 (WB) N-cadherin Rabbit Abcam (ab18203) 1:4000 (WB) NF-κB p65 Rabbit Abcam (ab16502) 1:1000 (WB) NF-κB p65 S276 Rabbit Abcam (ab106129) 1:100 (IH)1:1000 (WB) P2X7R Rabbit Alomone labs (#APR-008) 1:500 (WB) PDI Rabbit Proteintech (#11245-1-AP)Cell signaling (#2446)

Techniques: Expressing, Activation Assay, Western Blot, Control, Marker, MANN-WHITNEY

Journal: Scientific reports

Article Title: Protein disulfide isomerase integrates toll-like receptor 4 and P2X7 receptor signaling pathways during lipopolysaccharide-induced neuroinflammation.

doi: 10.1038/s41598-025-92780-5

Figure Lengend Snippet: Fig. 6. The effects of NO on microglial activation and p65 S276 phosphorylation under physiological condition. SNAP (a NO donor) leads to microglial activation and p65 phosphorylation under physiological condition, which are attenuated by P2X7R deletion. (A) Representative photos for p65 S276 phosphorylation in microglia following SNAP treatment. (B–C) Quantification of the effects of SNAP on IB4 (a microglial marker, B) and p65 S276 intensities (C; #,*p < 0.05 vs. vehicle and P2X7R+/+mice, respectively; Kruskal–Wallis test, n = 7, respectively).

Article Snippet: Fluorescent intensity was performed by two Antigen Host Manufacturer (Catalog number) Dilution GFAP Mouse Millipore (#MAB3402) 1:2000 (IH) IB4 - Vector (#B-1205) 1:200 (histochemistry) Iba-1 Rabbit Biocare Medical (#CP290) 1:500 (IH) iNOS Rabbit Novus Biologicals (#NB300-605) 1:500 (WB) N-cadherin Rabbit Abcam (ab18203) 1:4000 (WB) NF-κB p65 Rabbit Abcam (ab16502) 1:1000 (WB) NF-κB p65 S276 Rabbit Abcam (ab106129) 1:100 (IH)1:1000 (WB) P2X7R Rabbit Alomone labs (#APR-008) 1:500 (WB) PDI Rabbit Proteintech (#11245-1-AP)Cell signaling (#2446)

Techniques: Activation Assay, Phospho-proteomics, Marker