lamp2 mouse monoclonal antibodies  (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

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a-b) Lysosomal accumulations of mTOR are lost in concanamycin A (ConA)-treated cells (100 nM, 6 h). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (a). Quantification of mTOR/LAMP2 colocalization in (b). n = 50 individual cells from 5 independent fields per condition. (c) ConA treatment (100 nM) preferentially diminishes phosphorylation of the lysosomal substrate TFEB but not of the cytoplasmic substrates S6K and 4E-BP1 under basal culture conditions. ConA (or DMSO as control, Ctrl) was added directly in the media for 6 hours before lysis. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before lysis. For AA starvation (–AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. ConA (or DMSO) was also included in the treatment media. The composition of all media is described in the Methods (see ‘Cell culture treatments’). (d-f) As in (a-c) but for treatments with chloroquine (CQ; 50 μM, 6 h). Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. For all panels, representative data from one out of three independent replicate experiments are shown. Data in graphs shown as mean ± SEM. **** p < 0.0001. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Phospho-proteomics, Control, Lysis, Cell Culture

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of the pharmacological inhibition of lysosomal function by BafA1 targeting the v-ATPase. b , c , Basal lysosomal proteolysis in HEK293FT cells shown by accumulation of LC3B upon BafA1 treatment (100 nM, 6 h before fixation) ( b ) and quantification of LC3B signal ( c ). n Ctrl = 49 and n BafA1 = 50 individual cells from five independent fields per condition. d , A schematic representation of the treatment strategy followed in this study, assessing mTORC1 activity under basal (unchallenged cells), starvation or acute re-activation (AA add-back) conditions. AA levels are shown by a black line, and mTORC1 activity by a red line (see also ). e , f , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, treated as indicated, using confocal microscopy (magnified insets shown on the right; scale bars, 25 μm and for insets, 5 μm) ( e ) and quantification of colocalization ( f ). n = 50 individual cells from five independent fields per condition. g , Immunoblots with lysates from HEK293FT WT cells treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, and BafA1 as shown, probed with the indicated antibodies. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. For e – g , BafA1 (100 nM) (or DMSO as control, Ctrl) was added directly in the media for 6 h before fixation ( e and f ) or lysis ( g ). For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation or lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation or lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. BafA1 (or DMSO) was also included in the treatment media. The composition of all media is described in . Data in graphs shown as mean ± s.e.m. ** P < 0.01, **** P < 0.0001. Source numerical data and unprocessed blots are available in . See also Extended Data Figs. and .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Inhibition, Activity Assay, Activation Assay, Marker, Confocal Microscopy, Western Blot, Control, Lysis

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of the pharmacological inhibition of lysosomal proteases by PepA and E64 blocking local AA production. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells ( b ) and its quantification ( c ), treated as indicated, using confocal microscopy. PepA (50 μM) and E64 (25 μM) (or DMSO as control, Ctrl) were added directly in the media for 16 h before fixation (magnified insets shown to the right; scale bars, 25 μm and for insets, 5 μm). n = 56 individual cells from three independent fields per condition. Data shown as mean ± s.e.m. **** P < 0.0001. d , Immunoblots with lysates from HEK293FT WT cells, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, and protease inhibitors (PepA + E64) as shown, probed with the indicated antibodies. PepA (50 μM) and E64 (25 μM) were added directly in the media for 16 h before lysis. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation or lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. PepA + E64 (or DMSO) were also included in the treatment media. The composition of all media is described in . Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Source numerical data and unprocessed blots are available in .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Inhibition, Blocking Assay, Marker, Confocal Microscopy, Control, Western Blot, Lysis

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of lysosomal enzyme sorting at the Golgi and delivery to lysosomes that depends on the GNPTAB enzyme. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells using confocal microscopy ( b ) and its quantification ( c ). Cells were transiently transfected with siRNAs targeting GNPTAB or a control RNAi duplex (siCtrl) and treated as indicated. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. The composition of all media is described in . n = 44–50 individual cells from five independent fields per condition (see also ). d , Immunoblots with lysates from HEK293FT WT cells transiently transfected with siRNAs targeting GNPTAB or a control RNAi duplex (siCtrl) and treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions as described in b , probed with the indicated antibodies. e , f , Functional characterization of GNPTAB KO HEK293FT cells. g , h , Lysosomal accumulations of mTOR are lost in GNPTAB KOs ( g ) and quantification of mTOR/LAMP2 colocalization ( h ). n = 50 individual cells from five independent fields per condition. For microscopy, magnified insets are shown to the right. Scale bars, 25 μm and for insets, 5 μm. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. ** P < 0.01, **** P < 0.0001. Source numerical data and unprocessed blots are available in .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Transfection, Control, Western Blot, Functional Assay, Microscopy

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , b , mTOR/LAMP2 colocalization ( a ) and its quantification ( b ). mTOR delocalizes away from lysosomes already after 2 h of BafA1 treatment. Time course of BafA1 treatment (100 nM, 2–8 h) to block lysosomal function in HEK293FT cells. Magnified insets shown to the right ( a ). Scale bars, 25 μm and insets, 5 μm. n = 49–50 individual cells from five independent fields per condition (see also ). c – e , Dephosphorylation kinetics of lysosomal (TFEB) and cytoplasmic (S6K and 4E-BP1) substrates of mTORC1 upon BafA1 treatment (100 nM, 1–8 h) in HEK293FT cells showing a rapid drop in TFEB phosphorylation, whereas that of S6K/4E-BP1 remains largely unaffected even at much later timepoints ( c ). Quantification of TFEB phosphorylation in ( d ) and S6K phosphorylation in ( e ). f , g , The rapamycin time course (20 nM, 1–30 min) in control (WT) and RagA/B KO cells, assessing S6K dephosphorylation kinetics ( f ) and the quantification of S6K phosphorylation ( g ). The rate of S6K dephosphorylation is similar between Rag-proficient and Rag-deficient cells. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., non-significant. Source numerical data and unprocessed blots are available in .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Blocking Assay, De-Phosphorylation Assay, Phospho-proteomics, Control

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model for the genetic removal of the Rag GTPases. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT and RagA/B KO cells ( b ) and its quantification ( c ), when treated as indicated, using confocal microscopy. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in . Magnified insets shown to the right in b . Scale bars, 25 μm and for insets, 5 μm. n = 55–60 individual cells from three or four independent fields per condition (see also ). d , Lyso-IP experiments with WT and RagA/B KO HEK293FT cells stably expressing HA-tagged TMEM192 (or FLAG-TMEM192 as negative control). Intact lysosomes were immunopurified by anti-HA IPs under native conditions, and the presence of the indicated proteins in lysosomal and non-lysosomal fractions as well as in whole-cell lysates was analysed by immunoblotting. e , Immuno-EM analysis of mTOR localization. Control (WT) or RagA/B KO MEFs, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, treated as described in b , were stained with antibodies against endogenous mTOR (10 nm gold particles) and LAMP2 (5 nm gold particles) ( e ). Magnified insets shown on the right side; the area used for magnification is marked with a white square. Scale bars, 500 nm and for insets, 80 nm. LY, LAMP2-positive lysosomes. f , g , Quantification of mTOR localization at lysosomes or the cytoplasm in WT ( f ) or RagA/B KO MEFs ( g ), treated and analysed by immuno-EM as in e . Samples incubated with secondary antibodies only (no primary ab) were used as negative controls for background staining. Values represent number of gold particles per μm 2 . n WT = 58–60 ( f ), n KO = 60 ( g ) randomly selected areas (1 μm 2 each) from three independent grids per condition. Data shown as mean ± s.e.m. * P < 0.05, **** P < 0.0001. n.s., non-significant. Source numerical data and unprocessed blots are available in . See also Extended Data Fig. .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Stable Transfection, Expressing, Negative Control, Western Blot, Control, Staining, Incubation

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a-b) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT or RagC/D KO cells, treated as indicated in the figure, using confocal microscopy. For basal (+AA) conditions, culture media were replaced with +AA treatment media 90 min before fixation. For AA starvation (–AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in the Methods (see ‘Cell culture treatments’). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (a). Quantification of colocalization in (b). n WT(+AA) = 50, n CDKO(+AA) = 50, n WT(–AA) = 48, n CDKO(–AA) = 49, n WT(–/+AA) = 49, n CDKO(–/+AA) = 50 individual cells from 5 independent fields per condition. Representative data from one out of three independent experiments are shown. (c) Immunoblots with lysates from HEK293FT WT and RagC/D KO cells, treated with media containing or lacking AAs, in basal (+AA), starvation (–AA) or add-back (–/+AA) conditions, probed with the indicated antibodies. Treatments were performed as in (a). n = 3 independent experiments. (d-e) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in WT or RagA/B KO MEF cells, treated as indicated in the figure, using confocal microscopy. Treatments were performed as in (a). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (d). Quantification of colocalization in (e). n WT(+AA) = 51, n ABKO(+AA) = 50, n WT(–AA) = 49, n ABKO(–AA) = 50, n WT(–/+AA) = 49, n ABKO(–/+AA) = 49 individual cells from 3 independent fields per condition. Representative data from one out of two independent experiments are shown. (f) As in (c), but with WT and RagA/B KO MEFs. n = 3 independent experiments. (g) As in (c), but with WT and RagA/B KO SW-620 cells. n = 3 independent experiments. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form; S: SUMOylated form. Data in graphs shown as mean ± SEM. *** p < 0.001, **** p < 0.0001, ns: non-significant. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Cell Culture, Western Blot

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , Immunoblots with lysates from HEK293FT WT and RagA/B KO cells, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, probed with the indicated antibodies. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in . b , In vitro kinase assays with mTORC1 immunopurified from WT or RagA/B KO HEK293FT cells and recombinant 4E-BP1 protein used as substrate, with 4E-BP1 phosphorylation detected by immunoblotting. No ATP samples (−ATP) used as negative controls. c , Lyso-IP experiments in WT and RagA/B KO HEK293FT cells stably expressing HA-tagged TMEM192 (or FLAG-TMEM192 as negative control). Intact lysosomes immunopurified by anti-HA IPs under native conditions, and the presence of the indicated proteins in the lysosomal and non-lysosomal fractions, as well as in whole-cell lysates, analysed by immunoblotting. Note the absence of S6K from lysosomal fractions and the presence of phospho-TFEB in the lysosomal fractions only of control cells. n = 2 independent experiments. d , e , Phosphorylation of multiple mTORC1 substrates is largely unaffected by BafA1 treatment (100 nM, 6 h) ( d ) or loss of Rag GTPases ( e ). In e , Torin1 (250 nM, 1 h) was used as a control for mTOR inhibition. f , g , GRASP55 phosphorylation by mTORC1 is retained in RagA/B KO ( f ) or BafA1-treated cells (100 nM, 6 h) ( g ), similarly to that of S6K. In g , starvation was performed as in a . Torin1 (250 nM, 1 h) was used as a control for mTOR inhibition. h , RagC is an additional lysosomal mTORC1 substrate that requires properly functioning lysosomes for its phosphorylation, similarly to TFEB/TFE3. AA starvation or blockage of lysosomal function with BafA1 (100 nM, 6 h) decrease RagC phosphorylation (shown as elevated RagC signal with #5466). Treatments performed as in a . i , j , Lysosomal localization of RagC is unaffected by BafA1 treatment (100 nM, 6 h) ( i ). Quantification of RagC/LAMP2 colocalization in ( j ). Scale bars, 25 μm and for insets, 5 μm. n = 50 individual cells from five independent fields per condition. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. n.s., non-significant. Source numerical data and unprocessed blots are available in . See also Extended Data Figs. – .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Western Blot, Lysis, In Vitro, Recombinant, Phospho-proteomics, Stable Transfection, Expressing, Negative Control, Control, Inhibition

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a) Schematic model of cytoplasmic AA sensing and signaling upstream of the Rags. See text for details. (b-c) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, using confocal microscopy. Cells were transiently transfected with siRNAs targeting Mios or a control RNAi duplex (siCtrl) and treated as indicated. For basal (+AA) conditions, culture media were replaced with +AA treatment media 90 min before fixation. For AA starvation (–AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in the Methods (see ‘Cell culture treatments’). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (b). Quantification of colocalization in (c). n siCtrl(+AA) = 49, n siMios(+AA) = 46, n siCtrl(–AA) = 49, n siMios(–AA) = 47, n siCtrl(–/+AA) = 50, n siMios(–/+AA) = 46 individual cells from 5 independent fields per condition. Representative data from one out of two independent experiments are shown. (d) Immunoblots with lysates from HEK293FT WT cells, transiently transfected with siRNAs targeting Mios or a control RNAi duplex (siCtrl), and treated with media containing or lacking AAs, in basal (+AA), starvation (–AA) or add-back (–/+AA; 10 or 30 min) conditions, probed with the indicated antibodies. Treatments were performed as in (b). n = 3 independent experiments. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. Data in (c) shown as mean ± SEM. * p < 0.05, *** p < 0.001, ns: non-significant. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Transfection, Control, Cell Culture, Western Blot

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a) Expression analysis of LAMTOR1 by qPCR confirms successful knockdown in HEK293FT cells. n = 2 independent experiments. (b) Schematic model of lysosomal tethering of the Rag dimer by the LAMTOR complex. (c-d) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, using confocal microscopy. Cells were transiently transfected with siRNAs targeting LAMTOR1 or a control RNAi duplex (siCtrl). Magnified insets shown to the right. Scale bars = 10 μm (for insets, 5 μm) (c). Quantification of colocalization in (d). n siCtrl = 50, n siLAMTOR1 = 48 individual cells from 3 independent fields per condition. Representative data from one out of two independent experiments are shown as mean ± SEM. **** p < 0.001. (e) Immunoblots with lysates from HEK293FT WT cells, transiently transfected with siRNAs targeting LAMTOR1 or a control RNAi duplex (siCtrl), cultured under basal conditions, and probed with the indicated antibodies. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. n = 3 independent experiments. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Expressing, Knockdown, Marker, Confocal Microscopy, Transfection, Control, Western Blot, Cell Culture