Journal: Autophagy

Article Title: Autophagy flux in CA1 neurons of Alzheimer hippocampus: Increased induction overburdens failing lysosomes to propel neuritic dystrophy

doi: 10.1080/15548627.2016.1239003

Figure Lengend Snippet: Primers used for qPCR analyses.

Article Snippet: , LAMP2 , lysosomal associated membrane protein 2 , Hs00174474_m1.

Techniques: Binding Assay, Membrane

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of Histone Deacetylases Prevents Cardiac Remodeling After Myocardial Infarction by Restoring Autophagosome Processing in Cardiac Fibroblasts.

doi: 10.1159/000493672

Figure Lengend Snippet: Fig. 2. TSA inhibits MI-induced impairment of autophagosome clearance in vivo. A, Immunoblots and (B) quantitative analysis of LC3, P62, beclin 1, LAMP2, Bcl2 and Bax protein expression in mice myocardium from the different groups (n=3). C, Representative immunofluorescence and quantitative analysis of P62 and the cardiac fibroblast marker vimentin (n=3). *P<0.05 vs the indicated group. MI, myocardial infarction; TSA, trichostatin A.

Article Snippet: Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against LC3 (Sigma-Aldrich, L8918), p62 (Cell Signaling Technology, #5114), GAPDH (Cell Signaling Technology, #5174), LAMP2 (Cell Signaling Technology, #49067), Bcl2 (Cell Signaling Technology, #2872), and Bax (Cell Signaling Technology, #14796).

Techniques: In Vivo, Western Blot, Expressing, Immunofluorescence, Marker

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of Histone Deacetylases Prevents Cardiac Remodeling After Myocardial Infarction by Restoring Autophagosome Processing in Cardiac Fibroblasts.

doi: 10.1159/000493672

Figure Lengend Snippet: Fig. 5. Restoration of autophagic flux is responsible for the protective effects of TSA against hypoxia-induced cell death. A, Immunoblotting analysis of LAMP2 protein levels. B, Mitochondrial membrane potential (B) and cell death (C) after hypoxia with or without CQ and adenovirus encoding short hairpin RNA of LAMP2a. *P<0.05 vs the indicated group (n=3). TMRE, tetramethylrhodamine ethyl ester; TSA, trichostatin A; CQ, chloroquine.

Article Snippet: Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against LC3 (Sigma-Aldrich, L8918), p62 (Cell Signaling Technology, #5114), GAPDH (Cell Signaling Technology, #5174), LAMP2 (Cell Signaling Technology, #49067), Bcl2 (Cell Signaling Technology, #2872), and Bax (Cell Signaling Technology, #14796).

Techniques: Western Blot, Membrane, shRNA

Journal: Molecular Psychiatry

Article Title: Mitochondrial hypermetabolism precedes impaired autophagy and synaptic disorganization in App knock-in Alzheimer mouse models

doi: 10.1038/s41380-023-02289-4

Figure Lengend Snippet: A Chord plot of significantly (FDR < 0.1) DEGs related to autophagy. The color of the circle edge boxes indicate up- (red) or down- (blue) regulation. B The relative mRNA expression of selected genes was normalized to Tubb3 ( n = 3; size effect for Trim30a 6-month-old App NL-G-F = 4.004 ± 1.676, for Rubcnl 6-month-old App NL-G-F = 0.9521 ± 0.2321, for Lamp2 6-month-old App NL-G-F = 0.4017 ± 0.1530, for Rab7b 6-month-old App NL-G-F = 2.354 ± 0.6304, 12-month-old App NL-G-F = 4.910 ± 0.2842, 12-month-old vs 2-month-old App NL-G-F = 4.678 ± 0.2835). Statistical significance was analyzed using Kruskal-Wallis tests followed by Dunn’s multiple comparison test. * p < 0.05. * vs age matched WT mice, # vs 2-month-old genotype-matched mice. C Co-immunofluorescence staining of Aβ, LC3 and synaptophysin with 12 month-old WT and App NL-G-F mice ( n = 4). Red: Aβ, Green: LC3, Yellow: Synaptophysin, Blue: nucleus. Scale bar: 20 µm. D , E Signal intensity of Aβ (Red), LC3 (Green), Synaptophysin (Yellow) and nucleus (Blue) under the white line of WT and App NL-G-F mouse brain staining gated in ( C ). F – I Phospho-p62 (S403), total p62, LC3-I and LC3-II protein levels in hippocampal crude synaptosomal fraction (P2) or soluble fraction (S2) from 12-month-old App NL-G-F hippocampus were visualized by Western blotting ( n = 4; size effect for p-p62 in P2 = 0.8713 ± 0.2675 and p-p62 in S2 = 1.387 ± 0.4519, for p62 in P2 = 0.3021 ± 0.08530, for LC3-II in P2 = 1.350 ± 0.2286). Protein levels were normalized to β3-tubulin. Statistical significance was analyzed using unpaired t -test. * p < 0.05, ** p < 0.01.

Article Snippet: The TaqMan Fast Advanced Master Mix (Thermo Fisher Sci., Cat. no: 4444557) was used to perform the qPCR using the following TaqMan mouse gene expression assays (FAM) (Thermo Fisher Sci., Cat. no: 4331182): Mm04225236_g1 for Atp8 ; Mm00432648_m1 for Cox8b ; Mm00444593_m1 for Ndufa1 ; Mm01352366_m1 for Sdha ; Mm01615741_gH for Uqcrb ; Mm01183349_m1 for Clec7a ; Mm00441259_g1 for Ccl3 ; Mm04209424_g1 for Trem2 ; Mm00437893_g1 for C4b ; Mm01312230_m1 for Chrna4 ; Mm00495267_m1 for Lamp2 ; Mm00523599_g1 for Rab7b ; Mm01310727 for Rubcnl ; Mm01274264_m1 for Trim30a ; Mn00450314_m1 for Vamp8 .

Techniques: Expressing, Comparison, Immunofluorescence, Staining, Western Blot

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: American Journal of Physiology - Cell Physiology

Article Title: Intestinal epithelial tight junction barrier regulation by autophagy-related protein ATG6/beclin 1

doi: 10.1152/ajpcell.00246.2018

Figure Lengend Snippet: Tat-beclin 1 increases the rate of occludin endocytosis. A: following surface biotinylation assay, as detailed in experimental procedures, and SDS-PAGE, immunoblots were probed with anti-occludin antibody. Caco-2 cells treated with Tat-beclin 1 peptide showed an increased amount of biotinylated occludin. B: %endocytosed occludin compared with total occludin contents at indicated time points, from 3 independent experiments. *P < 0.01 vs. Tat-scrambled peptide, unpaired Student’s t-test; n = 4, representation of 3 independent experiments. C: Tat-beclin 1 induces increased lysosomal targeting of occludin. In Tat-beclin 1-treated cells, occludin showed increased colocalization with lysosomal marker lysosome-associated membrane protein-2 (LAMP2) compared with Tat-scrambled peptide (Tat-scr)-treated cells. Occludin, green; LAMP2, red. Only merged signals (yellow) are retained in the colocalization images. Scale bars = 5 μm. Representation of 20 areas examined in 3 independent experiments.

Article Snippet: Primary antibodies used in this study included occludin (33-1500; Invitrogen; and LS-C-140253-100; LifeSpan BioSciences), zona occludens protein-1 (ZO-1, 617300; Invitrogen), claudin-1 (51-9000; Invitrogen), claudin-2 (51-6100; Invitrogen), claudin-3 (34-1700; Thermo Fisher Scientific), claudin-4 (PA-5-16875; Thermo Fisher Scientific), caveolin-1 (3238; Cell Signaling Technology), early endosome antigen 1 (EEA1, E-7659; Sigma-Aldrich), Ras-related protein Rab-5 (Rab5, R-4654; Sigma-Aldrich), beclin 1 (ab-114071 and ab-207612; Abcam), microtubule-associated protein 1A/1B-light chain 3B (LC3B, L-7543; Sigma-Aldrich), ATG16 (PA5-35207; Thermo Fisher Scientific), lysosome-associated membrane protein-2 (LAMP2, NBP-2-22217; Novus Biologicals), and β-actin (MA-5-15739; Thermo Fisher Scientific).

Techniques: Surface Biotinylation Assay, SDS Page, Western Blot, Marker, Membrane

Journal: Autophagy

Article Title: Zika virus NS2A protein induces the degradation of KPNA2 (karyopherin subunit alpha 2) via chaperone-mediated autophagy

doi: 10.1080/15548627.2020.1823122

Figure Lengend Snippet: List of primers used in this study

Article Snippet: The primary mouse monoclonal antibodies against KPNA1 (Santa Cruz Biotechnology, sc-101,292), KPNA2 (Santa Cruz Biotechnology, sc-55,537), hemagglutinin (HA) tag (ThermoFisher Scientific, 26,183), GFP (Biolegend, 75,818–584), HSPA8/HSC70 (Santa Cruz Biotechnology, sc-7298), ubiquitin (Santa Cruz Biotechnology, sc-8017), GAPDH (Santa Cruz Biotechnology, sc-365,062), and TUBB1/β-tubulin (Sigma, T7816), and rabbit polyclonal antibodies against ZIKV NS4B (GeneTex, GTX133311), ZIKV E (GeneTex, GTX133314), NS2B (GeneTex, GTX133318), NS5 (GeneTex, GTX133329) and LAMP2A (Boster, M01573) were used in this study.

Techniques: