Journal: Cell

Article Title: Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells

doi: 10.1016/j.cell.2018.10.014

Figure Lengend Snippet:

Article Snippet: Anti-human CD107b APC (H4B4) , Miltenyi Biotec , Cat#130-103-960; RRID: AB_2654504.

Techniques: Purification, Control, Recombinant, Selection, Staining, Software

Journal: bioRxiv

Article Title: Chaperone-mediated Autophagy Deficiency Reprograms Cancer Metabolism Via TGFβ Signaling to drive Mesenchymal Tumor Growth

doi: 10.1101/2022.07.07.499098

Figure Lengend Snippet: (A) CRISPR/Cas9 genome editing and PCR genotyping of LAMP2A KO. (B) qRT-PCR analysis of LAMP1, LAMP2A, LAMP2B and LAMP2C expression in WT and KO cells from HT1080 and A549 cell lines (C) Western blot analysis of LAMP2A and LAMP1 in WT and KO (D) Confocal analysis of immunofluorescence with anti-LAMP2A (Green) and anti-LAMP1 (Red) antibodies in WT and KO cells from HT1080 and A549 cell lines. DAPI was used to counterstain cell nuclei. The scale bar (in white) is 20 µm. Insets at higher magnification as indicated. (E) Western blot analysis of LAMP2A, LAMP1 and LAMP2B in WT and KO xenograft tumors from HT1080 and A549 cell lines. Immunofluorescence staining for (F) LAMP2A or (G) ID1 in WT and KO HT1080 or A549 tumors. Scale bar is100 μm. Insets at higher magnification as indicated. Quantification of the ID1+ cell ratio per 20× field (bar graph). (H) Western blot analysis of ID1 in WT and KO xenograft tumors from HT1080 cell lines. Error bars, ±SD. ***p<0.001; two-tailed student’s t -test.

Article Snippet: Plasmid pCMV6-XL5-LAMP2A was purchased from Origene (Cat nr #: SC118738) and used for transfection with ViaFect from Promega according to the manufacturers recommended procedure.

Techniques: CRISPR, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Chaperone-mediated Autophagy Deficiency Reprograms Cancer Metabolism Via TGFβ Signaling to drive Mesenchymal Tumor Growth

doi: 10.1101/2022.07.07.499098

Figure Lengend Snippet: (A) proliferation (B) clonogenic growth (C) experimental overview of LAMP2KO implantation of 4 tumors (2 KO vs. 2 WT) per mice. (D) Immunohistochemical staining for LAMP2A and LAMP1 in WT and KO tumors from HT1080 and A549 cell lines (n=3 mice). The scale bar (in black) is 50 µm. Insets at higher magnification as indicated. (E) Representative images of the xenograft mice and tumors. (F and G) Tumor growth and weight (n=3 mice). (H) Representative image of EdU positive (EdU+, Green) and DAPI-(blue) labeled HT1080 WT and KO tumor slides (n=3). Scale bar is 100 μm (H, left). Quantification of the EdU+ cell ratio per 20× field (bar graph). Error bars, ±SD. ***p<0.001; ns= non-significant; two-tailed student’s t -test.

Article Snippet: Plasmid pCMV6-XL5-LAMP2A was purchased from Origene (Cat nr #: SC118738) and used for transfection with ViaFect from Promega according to the manufacturers recommended procedure.

Techniques: Immunohistochemical staining, Staining, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Chaperone-mediated Autophagy Deficiency Reprograms Cancer Metabolism Via TGFβ Signaling to drive Mesenchymal Tumor Growth

doi: 10.1101/2022.07.07.499098

Figure Lengend Snippet: (A) The transcript abundance of LAMP2A in three non-neoplastic human lung tissues compared to 8 primary human lung tumors. (B) LAMP2A expression in human metastatic and primary cancer tissues (n=5, for each patient). (C) Schematic picture of human NSCLC primary tumors and brain metastasis in cancer patient. (D) Comparative RNA expression analysis of human NSCLC primary tumors to match brain metastasis for EMT markers. (E) Tissue-Microarray analysis (TMA) of the LAMP2A and Vimentin protein expression level in 184 human multiorgan metastasis array. Two representative cores are shown in the figure to illustrate the inverse LAMP2A/Vimentin staining patterns. Scale bar 50 µm. (F) table revealing the number of patients with inverse expression of LAMP2A/Vimentin and its association with tumor grade. Error bars, ±SD. **p<0.01, ***p<0.001; ns= non-significant; two-tailed student’s t -test.

Article Snippet: Plasmid pCMV6-XL5-LAMP2A was purchased from Origene (Cat nr #: SC118738) and used for transfection with ViaFect from Promega according to the manufacturers recommended procedure.

Techniques: Expressing, RNA Expression, Microarray, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Chaperone-mediated Autophagy Deficiency Reprograms Cancer Metabolism Via TGFβ Signaling to drive Mesenchymal Tumor Growth

doi: 10.1101/2022.07.07.499098

Figure Lengend Snippet: (A) Western blot analysis of three anti-LAMP2A antibodies in A549 WT and KO cells. (B) The immunostaining with three anti-LAMP2A antibodies in A549 WT and KO cells (red). DAPI was used to counterstain cell nuclei (Blue). The scale bar (in white) is 5 µm. Insets at higher magnification as indicated. (C) Immunohistochemical staining for LAMP2A in A549 WT and KO tumors by three LAMP2A antibodies (n = 3-4 mice). Red arrows mark the area of staining in the mouse stroma (tumor-supporting mice tissues). The scale bar (in black) is 50 µm. Insets at higher magnification as indicated.

Article Snippet: Plasmid pCMV6-XL5-LAMP2A was purchased from Origene (Cat nr #: SC118738) and used for transfection with ViaFect from Promega according to the manufacturers recommended procedure.

Techniques: Western Blot, Immunostaining, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Chaperone-mediated Autophagy Deficiency Reprograms Cancer Metabolism Via TGFβ Signaling to drive Mesenchymal Tumor Growth

doi: 10.1101/2022.07.07.499098

Figure Lengend Snippet: (A) qPCR analysis of LAMP2 expression in the indicated set of cell lines. (B) The effect of TGFβ treatment on VIM, CDH1 and LAMP2A mRNA. The effect of TGFβ signaling inhibition (by Tranilast) or CMA activation on LAMP2A mRNA in OVPA8 cells. (C) The effect of CMA activation on TGFβR2 protein level in the ES2 cell line. (D) The effect of siRNA-mediated knockdown by two independent siRNAs targeting TGFβR2 on LAMP2A in FU-OV1 cells (left panel) and ES2 cells (right panel). (E) The effect of CMA activation, Tranilast, Tranilast +chloroquineon and CMA +chloroquineon treatment on TGFβR2. (F) The effect of LAMP2A knockdown on the TGFβR2 lysosomal enrichment as analyzed by mass spectrometry. (G) The effect of siRNA-mediated knockdown by two independent siRNAs targeting LAMP2A, on TGFβR2. (H) The effect of re-expression (RE) of LAMP2A in the LAMP2A KO HT1080 cells. Error bars, ±SD. **p<0.01, ***p<0.001; ns= non-significant; two-tailed student’s t -test.

Article Snippet: Plasmid pCMV6-XL5-LAMP2A was purchased from Origene (Cat nr #: SC118738) and used for transfection with ViaFect from Promega according to the manufacturers recommended procedure.

Techniques: Expressing, Inhibition, Activation Assay, Mass Spectrometry, Two Tailed Test

Journal: Autophagy

Article Title: Direct homophilic interaction of LAMP2A with the two-domain architecture revealed by site-directed photo-crosslinks and steric hindrances in mammalian cells

doi: 10.1080/15548627.2021.1911017

Figure Lengend Snippet: The N-domain truncation of mouse LAMP2A impairs the CMA activity. (A) Domain architectures and orientations of five LAMP family proteins. The core domain common to LAMPs 1–5 has four conserved cysteine residues that can form two disulfide bonds (pink). Both LAMP1 and LAMP2 have two core domains, whereas LAMP3 and LAMP4 have a mucin-like domain (gray) in addition to the core domain [ , ] and LAMP5 is composed of the single core domain . (B) Schematic representations of the full-length and N-domain truncated LAMP2A proteins used in this study. (C, D) rbc1cc1 −/- MEFs either with or without LAMP2 (generated by guide RNA #2) were transfected with GAPDH-HT. The full-length or truncated FLAG-LAMP2A (the N-terminally FLAG-tagged) was expressed together with GAPDH-HT in LAMP2-deficient rbc1cc1 −/- MEFs. The fluorescence images after labeling with TMR-HT ligand (red) for 18–20 h are shown. Nuclei were stained with Hoechst (blue). (D) Quantitative analysis of GAPDH-HT puncta. n = 50, 42, 51, 47. ***P < 0.001. Essentially the same results were obtained in three independent experiments, including one using guide RNA #1. (E) Quantitative analysis of GAPDH-HT puncta upon expression of the full-length or truncated LAMP2A, which is not tagged (left panel, n = 51, 59), or the C-terminally FLAG-tagged LAMP2A (right panel, n = 64, 53), in LAMP2-deficient rbc1cc1 −/- MEFs. (F) GAPDH-HT was transiently expressed in rbc1cc1 −/- MEFs (lane 2) or in the clones of LAMP2-deficient rbc1cc1 −/- MEFs stably expressing the full-length (FL) or truncated (TR) N-terminally FLAG-tagged LAMP2A (lanes 3, 4 and 5, 6, respectively). As a control, rbc1cc1 −/- MEFs were used without transfection of GAPDH-HT (lane 1). Twenty-four hours after the transfection of GPADH-HT, biotin-HT ligand was added to the medium and the cells were cultured for an additional 24 h (lanes 3–6). The proteins in the lysates and retrieved by NeutrAvidin beads (Pulldown) were analyzed by immunoblotting using an anti-HaloTag protein antibody, and reprobed with an anti-tubulin antibody. Closed and open triangles show GAPDH-HT (70 kDa) and likely the HT protein (30 kDa), respectively. Essentially the same results were obtained in an independent experiment. See also Fig. S2

Article Snippet: The mouse Lamp1 and Lamp2a cDNA clones in pCMV6-AC-GFP and the human LAMP2 , transcript variant A, cDNA clone in pCMV6 were obtained from ORIGENE (MG225631, MG222878, and RC221216, respectively).

Techniques: Activity Assay, Generated, Transfection, Fluorescence, Labeling, Staining, Expressing, Clone Assay, Stable Transfection, Cell Culture, Western Blot