monoclonal mouse anti lamp2  (Developmental Studies Hybridoma Bank)

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

Journal: bioRxiv

Article Title: Artificial intelligence-augmented drug discovery identifies gefitinib as a potential treatment for ALS

doi: 10.1101/2025.03.06.641147

Figure Lengend Snippet: a) Healthy HEK293 cells were treated for 6h with 0.1% (v/v) DMSO vehicle control, 10 µM gefitinib, 500 nM rapamycin, with and without 100 nM bafilomycin A1 (Baf) co-treatment. The induction of autophagic flux, as indicated by LC3-II levels and LC3-II/LC3-I ratio, was subsequently measured via western blotting. b) Densitometry quantification of LC3-II – LC3-I ratio. Data normalised to DMSO vehicle = 1. Data = mean ± SD; n = 5. Paired t-test (Baf A1 vs Baf A1 + Gef), *P < 0.05. c) Densitometry quantification of LC3-II expression. Data normalised to DMSO vehicle = 100. Data = mean ± SD; n = 5. Paired t-test (Baf A1 vs Baf A1 + Gef), *P < 0.05. d) Healthy control iAstrocytes were treated for 16h with 0.1% (v/v) DMSO vehicle control (0.1% v/v), 10 µM gefitinib, 250 nM Torin 1, with and without 100 nM bafilomycin A1 co-treatment. The induction of autophagic flux, as indicated by LC3-II levels and LC3-II/LC3-I ratio, was subsequently measured via western blotting. e) Densitometry quantification of LC3-II expression. Data normalised to DMSO vehicle = 100. Data = mean ± SD; n=6 (Con_3 n=2, Con_4 n=2, Con_5 n=2). Paired t-test (Baf A1 vs Baf A1 + Gef), *P < 0.05. f) Densitometry quantification of LC3-II – LC3-I ratio. Data normalised to DMSO vehicle = 1. Data = mean ± SD; n=6 (Con_3 n=2, Con_4 n=2, Con_5 n=2). Paired t-test (Baf A1 vs Baf A1 + Gef), ns. g) Expression of LC3 in control, C9, and sALS iAstrocytes after treatment with DMSO 0.1% v/v or gefitinib 10 µM for 48h. h) Densitometry quantification of LC3-II expression. Data normalised to DMSO vehicle-treated Con_1 iAstrocytes = 100. Data = mean ± SD; n = 3 Two-way ANOVA with Šidák’s multiple comparison’s test, *P < 0.05; ***P < 0.001; ****P<0.0001. i) Densitometry quantification of LC3-II – LC3-I ratio. Data normalised to DMSO vehicle-treated Con_1 iAstrocytes = 1. Data = mean ± SD; n = 3-4. Two-way ANOVA with Šidák’s multiple comparison’s test, *P < 0.05. j) Example image of untreated C9_1 iAstrocyte line showing TDP-43 C-terminal cytoplasmic aggregate enclosed within a vimentin cage (white arrow). Scale = 20 μm. k) Representative images of C9_1 iAstrocytes immunostained for LC3B and LAMP2, following a 48h treatment with 0.1% (v/v) DMSO vehicle control or 10 µM gefitinib. Scale: 20 μm. l) Representative images of C9_1 iAstrocytes immunostained for TDP-43 C-terminal and LAMP2, following a 48h treatment with 0.1% (v/v) DMSO vehicle control or 10 µM gefitinib. Scale: 20 μm. m) Manders coefficient of TDP-43 co-localisation with LAMP2 was determined using JACoP plugin for Fiji for C9_1 iAstrocytes. Data = mean ± SD; n=3. Paired t-test, **P<0.005. n) Manders coefficient of TDP-43 co-localisation with LAMP2 was determined using JACoP plugin for Fiji for C9_1 iAstrocytes. Data = mean ± SD; n=3. Paired t-test, *P < 0.05.

Article Snippet: Cells were incubated overnight at 4°C with chicken polyclonal anti-vimentin (1:1,000, Merck Millipore, catalogue number: AB5733), rabbit polyclonal anti-TDP-43 C-terminal (1:300, Proteintech, catalogue number: 12892-1-AP), rabbit polyclonal anti-LC3B (1:1,000, Novus Biologics, catalogue number: 2220), or mouse monoclonal anti-LAMP2 (1:200, Santa Cruz, clone H4B4, catalogue number: sc-18822).

Techniques: Control, Western Blot, Expressing