Journal: bioRxiv

Article Title: Heparan sulfate promotes autoactivation of pro-Cathepsin K by destabilizing the propeptide-catalytic domain interaction

doi: 10.64898/2026.01.18.700217

Figure Lengend Snippet: Mouse femur section was co-stained with anti-CtsK (green), HS20 (purple) and anti-LAMP2 (red) and visualized with anti-rabbit Alexa488, anti-human Alexa594, and anti-rat Alexa647 secondary antibodies, respectively. Area shown is the cortical bone. The approximate boundary of the bone is shown with white dashed line. A blood vessel within the bone is indicated with yellow dashed line. Red blood cells in the vessel shown strong green autofluorescence. A closeup view of the large osteoclast is shown in the right bottom panel. Another osteoclast (likely only the tip of the osteoclast) is indicated with a yellow arrow in the merged image. Nuclei of several osteocytes are indicated with white arrow head. Images representative of three separate experiments.

Article Snippet: Following deparaffinization and citric acid-based antigen retrieval (pH7), sectioned were blocked and incubated overnight at 4 °C with the following primary antibodies: 0.2 μg/ml rabbit anti-mCtsK polyclonal antibody (described previously) , 1ug/ml human anti-HS mAb (HS20, from Bio X cell) , and 1ug/ml Rat anti-mouse Lamp2 (GL2A7, Developmental Studies Hybridoma Bank).

Techniques: Staining

Journal: bioRxiv

Article Title: Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

doi: 10.1101/2025.11.03.686439

Figure Lengend Snippet: Fluorescence imaging showing fraction of CTR1 (green) localized with respective endocytic vesicles (red) while DAPI (blue) marks the nucleus at copper (50 µM) and CDDP (25µM & 50µM). The Scale bar represents 10 microns. (A) Immunofluorescence image showing fraction of Ctr1 (green) with early endosomal marker EEA1 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-EEA1. (B) Immunofluorescence image showing fraction of Ctr1 (green) with retromer controlled Recycling endosomal vesicle VPS35 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-VPS35. (C) Immunofluorescence image showing fraction of Ctr1 (green) with late endosomal marker Rab7 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab7. (D) Immunofluorescence image showing fraction of Ctr1 (green) with retromer late endosomal marker Rab9 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab9. (E) Immunofluorescence image showing fraction of Ctr1 (green) with lysosomal resident protein Lamp2 (Red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Lamp2. (F) Quantitative analysis showing fraction of CTR1 residing in different endocytic vesicles upon 1 hr. treatment of copper (100 µM) and CDDP (100 µM) respectively graphically represented using a Box and Whisker plot with jitter points (n>50). The box represents the 25 to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5 × interquartile range (IQR) from the first and third quartile. ∗ p < 0.05, ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: The primary antibodies utilized in immunofluorescence studies are as follows: Rabbit anti-FLAG M2 ( :600,CST #14793), Rabbit Ctr1(1:800,# ab129067 Abcam), Mouse Na/K ATPase ( :600,#MA3-929 Invitrogen), Mouse VPS35 ( :500,#sc-374372, Santa Cruz Biotechnology), Mouse Rab7 ( :50,#sc-376362, Santa Cruz Biotechnology), Mouse Lamp2 ( :150, #H4B4 DSHB), Mouse Rab9 ( :100, #MA3-06 Invitrogen), Mouse EEA1(1:100 #610457, 1:400; BD Biosciences).The Secondary antibodies utilized in immunofluorescence studies are as follows: Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 ( :2000, #A-21206 Invitrogen), Donkey anti-Mouse Alexa 555 (#A-32773, 1:2000; Invitrogen).

Techniques: Fluorescence, Imaging, Immunofluorescence, Marker, Whisker Assay

Journal: bioRxiv

Article Title: Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

doi: 10.1101/2025.11.03.686439

Figure Lengend Snippet: (A) Immunofluorescence image showing fraction of Ctr1 (green) with early endosomal marker EEA1 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-EEA1. (B) Immunofluorescence image showing fraction of Ctr1 (green) with retromer controlled Recycling endosomal vesicle VPS35 (red) in HEK293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-VPS35. (C) Immunofluorescence image showing fraction of Ctr1 (green) with late endosomal marker Rab7 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab7. (D) Immunofluorescence image showing fraction of Ctr1 (green) with retromer late endosomal marker Rab9 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab9. (E) Immunofluorescence image showing fraction of Ctr1 (green) with lysosomal resident protein Lamp2 (Red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Lamp2. (F) Quantitative analysis showing fraction of CTR1 residing in different endocytic vesicles upon 1 hr. treatment of copper (100 µM) and CDDP (100 µM) respectively graphically represented using a Box and Whisker plot with jitter points (n>50). The box represents the 25 to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5 × interquartile range (IQR) from the first and third quartile. ∗ p < 0.05, ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: The primary antibodies utilized in immunofluorescence studies are as follows: Rabbit anti-FLAG M2 ( :600,CST #14793), Rabbit Ctr1(1:800,# ab129067 Abcam), Mouse Na/K ATPase ( :600,#MA3-929 Invitrogen), Mouse VPS35 ( :500,#sc-374372, Santa Cruz Biotechnology), Mouse Rab7 ( :50,#sc-376362, Santa Cruz Biotechnology), Mouse Lamp2 ( :150, #H4B4 DSHB), Mouse Rab9 ( :100, #MA3-06 Invitrogen), Mouse EEA1(1:100 #610457, 1:400; BD Biosciences).The Secondary antibodies utilized in immunofluorescence studies are as follows: Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 ( :2000, #A-21206 Invitrogen), Donkey anti-Mouse Alexa 555 (#A-32773, 1:2000; Invitrogen).

Techniques: Immunofluorescence, Marker, Whisker Assay

Journal: bioRxiv

Article Title: Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

doi: 10.1101/2025.11.03.686439

Figure Lengend Snippet: (A) Magic red, a substrate of Cathepsin B which signifies lysosomal activity showed via immunofluorescence images in Hek293T cell line. Red dots indicate cathepsin positive lysosomes in Copper and CDDP treated condition. (B) Cathepsin B activity measured using Corrected total cell fluorescence (CTCF) analysis and plotted (C) Size of cathepsin positive lysosomes analyzed and plotted. (D) Lysosensor Green, dye measuring change in lysosomal P.H used to measure acidic characteristics of Lysosome via Flow cytometry(n=3). (E) Expression of Lamp2, a lysosomal membrane resident glycoprotein associate with lysosomal number and function showed by Western blot. (F) Abundance of Lamp2 in Hek293T cells analyzed using Non-Parametric T test and plotted. (n=3) (G) Number of acidic lysosomal compartments measured using Lysotracker via Flow cytometry analyzed and plotted . (n=3)

Article Snippet: The primary antibodies utilized in immunofluorescence studies are as follows: Rabbit anti-FLAG M2 ( :600,CST #14793), Rabbit Ctr1(1:800,# ab129067 Abcam), Mouse Na/K ATPase ( :600,#MA3-929 Invitrogen), Mouse VPS35 ( :500,#sc-374372, Santa Cruz Biotechnology), Mouse Rab7 ( :50,#sc-376362, Santa Cruz Biotechnology), Mouse Lamp2 ( :150, #H4B4 DSHB), Mouse Rab9 ( :100, #MA3-06 Invitrogen), Mouse EEA1(1:100 #610457, 1:400; BD Biosciences).The Secondary antibodies utilized in immunofluorescence studies are as follows: Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 ( :2000, #A-21206 Invitrogen), Donkey anti-Mouse Alexa 555 (#A-32773, 1:2000; Invitrogen).

Techniques: Activity Assay, Immunofluorescence, Fluorescence, Flow Cytometry, Expressing, Membrane, Western Blot

Journal: Frontiers in Immunology

Article Title: Implication of LAMP proteins and autophagy markers in colorectal cancer aggressiveness

doi: 10.3389/fimmu.2025.1662830

Figure Lengend Snippet: Strong LAMP and autophagy markers expression in tumor buds within the front of tumor invasion in CRC. Tissue sections. Normal colon distal to the CRC area a- e: (A) Glandular parenchyma 0; stroma 2+ in the upper 1/3 of the mucosal chorion. Immunohistochemistry (IHC), anti-LAMP1 , x 200 (original magnification) ; (B) Glands 1 +. IHC, anti- LAMP2 , x200 (original magnification) ; (C) Expression 1 +. IHC, anti- LAMP2A , x200 (original magnification) ; (D) Left side of the microphotograph, tumor parenchyma 1+, stroma 3+ IHC, anti- LC3B , x50 (original magnification) ; (E) Expression 2 +. IHC, anti- BECLIN1, x200 (original magnification) ; Tumor parenchyma in well-differentiated adenocarcinoma (G1) f-j: (F) Tumor parenchyma predominantly apical cytoplasmic signal 2+; (IHC), anti-LAMP1 , x 400 (original magnification) ; (G) Tumor parenchyma 2+, stroma 2 +. IHC, anti- LAMP2 , x100 (original magnification) ; (H) In the tumor cells of tumor parenchyma 2 +. IHC, anti- LAMP2A , x200 (original magnification) ; (I) Right side of the microphotograph, tumor parenchyma 2+, stroma 3+ IHC, anti- LC3B , x200 (original magnification) ; (J) In the tumor cells of tumor parenchyma 2+ with expression enhanced (accentuated) around the lumen of the tumor glands. IHC, anti- BECLIN1, x200 (original magnification) Tumor parenchyma in poorly differentiated adenocarcinoma (G3) (K–O) : (K) Tumor parenchyma with marked diffuse cytoplasmic expression 3 +. (IHC), anti-LAMP1 , x 200 (original magnification) ; (l) Tumor parenchyma (top) and tumor front (bottom) with clearly visible expression gradient, with more pronounced expression; expression also in the stroma around the tumor front zone in the form of vacuoles and diffusely. IHC, anti- LAMP2 , x100 (original magnification) ; (M) In the tumor cells of the tumor parenchyma 2 +. IHC, anti- LAMP2A , x200 (original magnification) ; (N) Tumor parenchyma and stroma 3+ & internal positive normal colon with a nerve fiber 3+ (arrow). IHC, anti- LC3B , x400 (original magnification) ; (O) 3+ expression in tumor cells from the tumor parenchyma, with expression enhanced (accentuated) around the nucleus but diffuse in nature. IHC, anti- BECLIN1, x200 (original magnification) . Tumor front and budding p-u: (P) Tumor front (middle) with marked diffuse cytoplasmic 3+ expression and surrounding stroma with 3 +. (IHC), anti-LAMP1 , x 200 (original magnification) ; (Q) Tumor front (Budd 3) with more pronounced expression in tumor cells. IHC, anti- LAMP2 , x200 (original magnification) ; (R) Pronounced expression 3+ in tumor embolus (arrow). IHC, anti- LAMP2 , x400 (original magnification) ; (S) 3+ expression in the tumor cells of the tumor front. IHC, anti- LAMP2A , x400 (original magnification) ; (T) Tumor front 3 +. IHC, anti- LC3B , x200 (original magnification) ; (U) In the tumor front area of the above case (O) , the expression decreases; expression 1+ in the tumor front area. IHC, anti- BECLIN1, x200 (original magnification) ; LAMP1, LAMP2, LAMP2A, LC3B and BECLIN1 expression levels in tumor parenchyma, stroma and front are assessed on adenocarcinomatous cells (cytokeratin 20+/cytokeratin 7-/CDX2+ markers).

Article Snippet: All samples were incubated with primary anti-human LAMP1 and LAMP2 mouse monoclonal antibodies (clones H4A3 and H4B4, DSHB, Yowa University) at working dilution of 1:100; anti-LAMP2A (Abcam Cat. No Ab125068 ) at a dilution of 1:100; anti- Beclinazako1 (GeneTex Cat. No GTX31722) at a dilution of 1:300; anti- LC3B (GeneTex Cat. No GTX82986) at a dilution of 1:400 at 4°C, overnight.

Techniques: Expressing, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Implication of LAMP proteins and autophagy markers in colorectal cancer aggressiveness

doi: 10.3389/fimmu.2025.1662830

Figure Lengend Snippet: LAMP1, LAMP2, LAMP2A, BECLIN1 and LC3B tissue expression in CRC and healthy individuals. (A) Difference in the intensity of LAMP1, LAMP2, LAMP2A, BECLIN1 and LC3B expression in tumor parenchyma, stroma and front in CRC and in normal colon. Statistical significative differences of the protein expression levels in the tumor parenchyma and in the tumor stroma relative to the tumor front and negative controls, respectively. Expression levels were categorized into four groups (category 0- no expression, 1- low expression, 2- moderate expression, 3- high expression) in the tissue regions analyzed: (TP- tumor parenchyma; TS- tumor stroma; TF- tumor front; TNC- normal colon tissue in the CRC area; NC CRC- normal colon distal to the CRC area; NC- normal colon from nontumorous patients). Fisher-Freeman-Halton test. Differences are statistically significant with a Benjamini-Hochberg corrected p value ( * p< 0,05; ** p< 0,01; *** p< 0,001; not significant (NS), p> 0.05). For exact uncorrected and corrected p-values, see Supplementary Table S1 , a); (B) Kendall`s tau correlation matrix between LAMP1, LAMP2, LAMP2A, BECLIN1 and LC3B tissue expression, budding and other clinical variables. G, histological grade; pT, tumor stage; pN, invasion in lymph nodes; L, invasion in lymphatics; V, invasion in blood vessels; MSS/MSI, microsatellite stability/microsatellite instability; TP, tumor parenchyma; TS, tumor stroma; TF, tumor front. * Correlation is statistically significant with a Benjamini-Hochberg corrected p value <0.05. For exact uncorrected and corrected p-values, see Supplementary Table S1b ); The colors span from dark blue to dark red, where dark blue denotes a r value of - 1, and dark red indicates a r value of 1.

Article Snippet: All samples were incubated with primary anti-human LAMP1 and LAMP2 mouse monoclonal antibodies (clones H4A3 and H4B4, DSHB, Yowa University) at working dilution of 1:100; anti-LAMP2A (Abcam Cat. No Ab125068 ) at a dilution of 1:100; anti- Beclinazako1 (GeneTex Cat. No GTX31722) at a dilution of 1:300; anti- LC3B (GeneTex Cat. No GTX82986) at a dilution of 1:400 at 4°C, overnight.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Implication of LAMP proteins and autophagy markers in colorectal cancer aggressiveness

doi: 10.3389/fimmu.2025.1662830

Figure Lengend Snippet: Protein and gene expression levels of LAMPs and autophagy markers in blood samples. (A-D) Difference in the secretory plasma circulating form of LAMP1, LAMP2, and BECLIN1 in CRC and in healthy patients. Statistical significative differences of the protein plasma levels in the CRC blood samples relative to the healthy controls, respectively. Statistically significative differences were calculated by t-test with Welch`s correction, ****p<0,0001; not significant (ns), p> 0.05. (E–H) LAMP1, LAMP2, BECLIN1 and LC3B overexpression in CRC. qPCR analysis of LAMP1, LAMP2, BECLIN1 and LC3B mRNA levels in WBC in CRC and healthy donors. Data are shown as mean ± SD. Statistically significative differences were calculated by t-test. p value (*p< 0,05; **p<0,001; ***p< 0,0001; ****p<0,0001). (I) Тwo subcategories, with lower levels of BECLIN1 (within the red circle) and higher- above. BECLIN1 low plasma concentrations are associated with CRC patients displaying elevated transcript levels in WBCs (the figure on the right- t-test with Welch`s correction, box plot analysis). (J) Kendall`s tau rank correlation matrix between LAMP1, LAMP2, BECLIN1 and LC3B plasma protein and gene expression levels in WBC and other clinical variables. pT, tumor stage; pN, invasion in lymph nodes; * Correlation is statistically significant with a Benjamini-Hochberg corrected p value < 0.05. For exact uncorrected and corrected p-values, see Supplementary Table S1c ); The colors span from dark blue to dark red, where dark blue denotes a r value of - 1, and dark red indicates a r value of 1.

Article Snippet: All samples were incubated with primary anti-human LAMP1 and LAMP2 mouse monoclonal antibodies (clones H4A3 and H4B4, DSHB, Yowa University) at working dilution of 1:100; anti-LAMP2A (Abcam Cat. No Ab125068 ) at a dilution of 1:100; anti- Beclinazako1 (GeneTex Cat. No GTX31722) at a dilution of 1:300; anti- LC3B (GeneTex Cat. No GTX82986) at a dilution of 1:400 at 4°C, overnight.

Techniques: Gene Expression, Clinical Proteomics, Over Expression

Journal: Frontiers in Immunology

Article Title: Implication of LAMP proteins and autophagy markers in colorectal cancer aggressiveness

doi: 10.3389/fimmu.2025.1662830

Figure Lengend Snippet: LAMP and autophagy signatures as potential markers of invasiveness in CRC and their prognostic significance. (A) LAMP1, LAMP2, BECLIN1 and LC3B tissue expression in CRC and healthy individuals. Box plot analysis of LAMP1, LAMP2, BECLIN1 and LC3B mRNA levels in tissues in CRC TCGA COAD tumor and TCGA COAD normal and GTEx normal data, using the GEPIA 2 data platform-Copyright © 2018 Zhang’s Lab. For the gene expression profile were considered: Differential Method ANOVA; ILog2FCI Cutoff 1, q-value Cutoff: 0.0. (B) CRC stage distribution (I-IV) of LAMP1, LAMP2, BECLIN1 and LC3B in the TCGA data COAD tumor, using the GEPIA 2 data platform - Copyright © 2018 Zhang’s Lab. (C) Correlation analysis of LAMP1 and LAMP2 gene signatures in the TCGA data COAD tumor (left, r=0.34, p=4.9e-09) and in the TCGA Tumor/Normal and the GTEx data for the colon-sigmoid/transverse sections (right, r=0.62, p=0), using the GEPIA 2 data platform - Copyright © 2018 Zhang’s Lab. Pearson correlation coefficient was calculated using the expression data sets derived from TCGA COAD tumor (n=275), TCGA COAD normal (n=41), as well as GTEx colon sigmoid and colon transverse (n=308). (D) Cancer-specific survival analysis in the context of LAMP1, LAMP2, BECLIN1 and LC3B expression in patients with CRC (TCGA). Kaplan-Meier analysis was performed, comparing TCGA-COAD patients with high 75% Cutoff and patients with low 25% Cutoff expression values within 140 months, using the GEPIA 2 data platform - Copyright © 2018 Zhang’s Lab, where MSI-H (n=52), MSI-L (n=52) and MSS (n=184).

Article Snippet: All samples were incubated with primary anti-human LAMP1 and LAMP2 mouse monoclonal antibodies (clones H4A3 and H4B4, DSHB, Yowa University) at working dilution of 1:100; anti-LAMP2A (Abcam Cat. No Ab125068 ) at a dilution of 1:100; anti- Beclinazako1 (GeneTex Cat. No GTX31722) at a dilution of 1:300; anti- LC3B (GeneTex Cat. No GTX82986) at a dilution of 1:400 at 4°C, overnight.

Techniques: Expressing, Gene Expression, Derivative Assay