Journal: Nucleic Acids Research

Article Title: A three-state model for the regulation of telomerase by TERRA and hnRNPA1

doi: 10.1093/nar/gkt695

Figure Lengend Snippet: Endogenous TERRA is bound to hnRNPA1. ( A ) Immunoprecipitation of hnRNPA1-myc in 293T nuclear extract. TERRA and centromeric RNA were detected by dot blot hybridization. Immunoprecipitation efficiency was determined by western blotting. Twenty-five percent of the RNA extracted from the input (I) and all RNA from the immunoprecipitates were spotted on the membrane and quantified. Bars represent the means ± SD of three independent experiments. ( B ) RNA-ChIP of TERRA associated with endogenous hnRNPA1 in 293T cells untreated (−) or transfected with siRNA against GFP or hnRNPA1. RNA was detected by dot blot hybridization. One percent of the input RNA or supernatant and half of the RNA from the immunoprecipitates was loaded. The same samples were treated with RNase A before loading (+ RNase). Bars represent the means ± SD of three independent experiments. ( C ) RNA-ChIP of TERRA associated with hnRNPA1-ZZ in HT1080 cells. RNA was detected as in (B). One percent of the input RNA or supernatant and half of the RNA from the immunoprecipitates was loaded. *** P = 0.0002. Bars represent the means ± SD of six independent experiments.

Article Snippet: For bacterial expression of hnRNPA1, pGEX-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pcDNA6-hnRNPA1 using BamH1-A1_F and A1-EcorRI_R primers ( Supplementary Table S1 ) and subcloned into the BamHI and EcoRI sites of pGEX-6P-1 vector (GE Healthcare). pcDNA6-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pCMV6-XL5-hnRNPA1 (Origene, clone NM_002136) using HindIII-A1_F and EcoRI-A1_R primers. cDNAs were subcloned into retroviral-based pCL vectors for expression of ZZ or hnRNPA1-ZZ proteins in HT1080 cells.

Techniques: Immunoprecipitation, Dot Blot, Hybridization, Western Blot, Transfection

Journal: Nucleic Acids Research

Article Title: A three-state model for the regulation of telomerase by TERRA and hnRNPA1

doi: 10.1093/nar/gkt695

Figure Lengend Snippet: Purification and characterization of recombinant GST-hnRNPA1. ( A ) GST and GST-hnRNPA1 fractions used for in vitro assays after the second purification step on the Mono Q sepharose column. The left SDS polyacrylamide gel was stained with Coomassie brilliant blue. Lane 1: GST, lane 2: GST-hnRNPA1, lanes 3 and 4: 200 and 400 ng of BSA. On the right are the corresponding western blots. Western blots were probed with α-GST antibody and α-hnRNPA1 antibody as indicated. ( B ) Recombinant GST-hnRNPA1 but not GST binds (UUAGGG) 3 . Electromobility shift assay and supershift of [ 32 P] 5′ end-labeled (UUAGGG) 3 (2 nM) with purified GST or GST-hnRNPA1 (180 nM) with antibody (Ab) or without (−)200 ng α-hnRNPA1 antibody. Lane 1: (UUAGGG) 3 alone. Lane 2: (UUAGGG) 3 incubated with GST. Lane 3: (UUAGGG) 3 incubated with GST and α-hnRNPA1 Ab. Lane 4: (UUAGGG) 3 incubated with GST-hnRNPA1. Lane 5: (UUAGGG) 3 incubated with GST-hnRNPA1 and α-hnRNPA1 Ab. GST-hnRNPA1- (UUAGGG) 3 and Ab-GST-hnRNPA1- (UUAGGG) 3 complexes (supershift) are indicated. ( C ) GST-hnRNPA1 interacts with TERRA RNA and telomeric DNA oligonucleotides but not with the TS primer. EMSA analysis of [ 32 P] 5′ end-labeled (UUAGGG) 3 , (TTAGGG) 3 or TS (2 nM) with purified GST-hnRNPA1 (35, 105 or 315 nM) or 315 nM GST. ( D ) GST-hnRNPA1 has similar affinity for TERRA and telomeric DNA oligonucleotides. EMSA analysis of [ 32 P] 5′ end-labeled (UUAGGG) 3 or (TTAGGG) 3 (2 nM) with purified GST-hnRNPA1 (12.5, 25, 37.5, 62.5, 75, 87.5, 112.5 or 137.5 nM). Kds are given and were determined from four independent experiments. ( E ) EMSA competition experiments to assess specificity of hnRNPA1 for TERRA and telomeric DNA oligonucleotides. On the left gel, GST-hnRNPA1 (60 nM) was incubated with [ 32 P] 5′ end-labeled (TTAGGG) 3 (2 nM) mixed with H 2 O or 17.5, 35, 175 or 350 nM of non-labeled dA 18 ; 175 or 350 nM TS; 17.5, 35, 175 or 350 nM of (TTAGGG) 3 ; 17.5, 35, 175 or 350 nM of rA 18 ; 17.5, 35, 175 or 350 nM of (UUAGGG) 3 . On the right gel, GST-hnRNPA1 (60 nM) was incubated with [ 32 P] 5′ end-labeled (UUAGGG) 3 (2 nM) mixed with H 2 O or 17.5, 35, 175 or 350 nM of rA 18 ; 17.5, 35, 175 or 350 nM of (UUAGGG) 3 ; 17.5, 35, 175 or 350 nM of non-labeled dA 18 ; 175 or 350 nM TS; 17.5, 35, 175 or 350 nM of (TTAGGG) 3 . For both gels, in lane 1, the radiolabeled oligonucleotide was incubated with the protein buffer only. The vertical line between lanes indicates juxtaposition of separate parts of the same gel.

Article Snippet: For bacterial expression of hnRNPA1, pGEX-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pcDNA6-hnRNPA1 using BamH1-A1_F and A1-EcorRI_R primers ( Supplementary Table S1 ) and subcloned into the BamHI and EcoRI sites of pGEX-6P-1 vector (GE Healthcare). pcDNA6-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pCMV6-XL5-hnRNPA1 (Origene, clone NM_002136) using HindIII-A1_F and EcoRI-A1_R primers. cDNAs were subcloned into retroviral-based pCL vectors for expression of ZZ or hnRNPA1-ZZ proteins in HT1080 cells.

Techniques: Purification, Recombinant, In Vitro, Staining, Western Blot, Electro Mobility Shift Assay, Labeling, Incubation

Journal: Nucleic Acids Research

Article Title: A three-state model for the regulation of telomerase by TERRA and hnRNPA1

doi: 10.1093/nar/gkt695

Figure Lengend Snippet: Purification of overexpressed telomerase and inhibiting effects of hnRNPA1 on telomerase activity by primer binding. ( A ) The enriched telomerase fraction used for direct telomerase assays does not contain detectable endogenous hnRNPA1 protein. Load, flow through and eluate from TEV cleavage were analyzed by western blotting using the indicated antibodies. ( B ) hnRNPA1 inhibits telomerase activity by binding to the primer. Direct telomerase assay with telomerase substrates B-(TTAGGG) 3 (left) and B-TS (right). The 3′ end biotinylated 10-mer was [ 32 P] 5′ end-labeled and used as a loading control (added during the purification step of the elongated products on streptavidin beads). The numbers on the left of the gels indicate the number of nucleotides added to the substrates. For both gels, lane 1: DTA performed in presence of the protein buffer, lanes 2, 3 and 4: DTA performed in presence of 17.5, 50 and 150 nM GST-hnRNPA1, lane 5: DTA performed with 150 nM GST.

Article Snippet: For bacterial expression of hnRNPA1, pGEX-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pcDNA6-hnRNPA1 using BamH1-A1_F and A1-EcorRI_R primers ( Supplementary Table S1 ) and subcloned into the BamHI and EcoRI sites of pGEX-6P-1 vector (GE Healthcare). pcDNA6-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pCMV6-XL5-hnRNPA1 (Origene, clone NM_002136) using HindIII-A1_F and EcoRI-A1_R primers. cDNAs were subcloned into retroviral-based pCL vectors for expression of ZZ or hnRNPA1-ZZ proteins in HT1080 cells.

Techniques: Purification, Activity Assay, Binding Assay, Western Blot, Telomerase Assay, Labeling

Journal: Nucleic Acids Research

Article Title: A three-state model for the regulation of telomerase by TERRA and hnRNPA1

doi: 10.1093/nar/gkt695

Figure Lengend Snippet: Alleviation of telomerase inhibition by TERRA with hnRNPA1. ( A ) Rescue of telomerase activity by GST-hnRNPA1 when using the TS primer. Lanes 1–4: DTA performed in the presence of protein buffer and water, 175 nM rA 18 , 17.5 nM (UUAGGG) 3 or 35 nM (UUAGGG) 3 . Lanes 5–7: DTA performed in presence of 17.5 nM GST-hnRNPA1 and 175 nM rA 18 , 17.5 nM (UUAGGG) 3 or 35 nM (UUAGGG) 3 . Lanes 8–10: DTA performed in the presence of 50 nM GST-hnRNPA1 and 175 nM rA 18 , 17.5 nM (UUAGGG) 3 or 35 nM (UUAGGG) 3 . Lanes 11–14: DTA performed in presence of 150 nM GST-hnRNPA1 and 175 nM rA 18 , 17.5 nM (UUAGGG) 3 , 35 nM (UUAGGG) 3 or 175 nM (UUAGGG) 3 . Lanes 15–18: DTA performed in presence of 150 nM GST and 175 nM rA 18 , 17.5 nM (UUAGGG) 3 , 35 nM (UUAGGG) 3 or 175 nM (UUAGGG) 3 . Lane 19: DTA performed in the presence of 25 mM EDTA. 3′ end biotinylated 10-mer was [ 32 P] 5′ end-labeled and used as a recovery and loading control. The numbers on the left of the gels indicate the number of nucleotides added to the substrates. ( B ) Quantification of (A) from two independent experiments. ( C ) and ( D ) same as (A) and (B), but the assays were performed with the (TTAGGG) 3 primer.

Article Snippet: For bacterial expression of hnRNPA1, pGEX-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pcDNA6-hnRNPA1 using BamH1-A1_F and A1-EcorRI_R primers ( Supplementary Table S1 ) and subcloned into the BamHI and EcoRI sites of pGEX-6P-1 vector (GE Healthcare). pcDNA6-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pCMV6-XL5-hnRNPA1 (Origene, clone NM_002136) using HindIII-A1_F and EcoRI-A1_R primers. cDNAs were subcloned into retroviral-based pCL vectors for expression of ZZ or hnRNPA1-ZZ proteins in HT1080 cells.

Techniques: Inhibition, Activity Assay, Labeling

Journal: Nucleic Acids Research

Article Title: A three-state model for the regulation of telomerase by TERRA and hnRNPA1

doi: 10.1093/nar/gkt695

Figure Lengend Snippet: ( A ) hnRNPA1 levels do not vary throughout the cell cycle. Left panel: hnRNPA1 protein level in synchronized HeLa cells after release from a double thymidine block. AS: asynchronous cells, 0 h: before release. Cells were collected every 2 h (2, 4, 6, 8, 10 and 12 h). Protein content was analyzed by western blotting using the indicated antibodies. CyclinE and CyclinB1 were used as markers for cell synchronization. Right panel: hnRNPA1 levels were quantified and normalized to tubulin levels and to the 0 h sample. Error bars correspond to standard deviation of two independent experiments. ( B ) Model: telomerase is switched on or off depending on the local concentrations of telomerase, hnRNPA1, TERRA and free 3′ telomeric single-stranded overhang. (1) TERRA < hnRNPA1: only a subset of hnRNPA1 is bound by TERRA and the remainder is bound to the 3′ telomeric single-stranded overhang, inhibiting the access for telomerase. (2) TERRA = hnRNPA1: TERRA and hnRNPA1 form an inert RNP complex. Telomerase can act on the telomeric single-stranded 3′ overhang. (3) TERRA > hnRNPA1: hnRNPA1-free TERRA will bind telomerase and inhibit its activity.

Article Snippet: For bacterial expression of hnRNPA1, pGEX-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pcDNA6-hnRNPA1 using BamH1-A1_F and A1-EcorRI_R primers ( Supplementary Table S1 ) and subcloned into the BamHI and EcoRI sites of pGEX-6P-1 vector (GE Healthcare). pcDNA6-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pCMV6-XL5-hnRNPA1 (Origene, clone NM_002136) using HindIII-A1_F and EcoRI-A1_R primers. cDNAs were subcloned into retroviral-based pCL vectors for expression of ZZ or hnRNPA1-ZZ proteins in HT1080 cells.

Techniques: Blocking Assay, Western Blot, Standard Deviation, Activity Assay

Journal: PLoS ONE

Article Title: Hsp72 (HSPA1A) Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment

doi: 10.1371/journal.pone.0149409

Figure Lengend Snippet: A. Beta-TC-6 cells were transfected and cell viability was assessed by MTS assay. Data represent percentage of live cells ± SD (filled bars) when transfected with vector pCMV6-XL5 (control) or human Hsp72::EGFP cDNA clone or m-proIAPP cDNA clone or h-proIAPP cDNA clone or both h-proIAPP and human Hsp72::EGFP vectors. Data represents the sum of three independently performed experiments. **p<0.01 versus respective controls, n = 3. B. At the end of the transfection period, phase contrast and fluorescence images were obtained. Data is a representative experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm. C. Beta-TC-6 cells were transfected with empty vector pCMV6-XL5 (control), or m-proIAPP cDNA clone or h-proIAPP cDNA clone or with both h-proIAPP and human Hsp72::EGFP vectors. After transfection, samples were collected and examined for the expression of h-IAPP by Western blot analysis. Data is a representative experiment from at least three independently performed experiments with similar results. D. Beta-TC-6 cells were transfected with empty vector pCMV6-XL5 (control), or human Hsp72::EGFP cDNA clone, or with both h-proIAPP and human Hsp72::EGFP vectors. After transfection, samples were collected and examined for the expression of Hsp72 by Western blot analysis. Data is a representative experiment from at least three independently performed experiments with similar results.

Article Snippet: When 70–80% confluency was reached, cells were transfected with h-proIAPP cDNA clone (vector pCMV6-XL5, Origene, Rockville, MD) or m-proIAPP cDNA clone (vector pCMV6-Kan/NEO, Origene) or h-Hsp72 cDNA clone (vector pEGFP, Addgene, Cambridge, MA) or both h-proIAPP and h-Hsp72 vectors.

Techniques: Transfection, MTS Assay, Plasmid Preparation, Fluorescence, Expressing, 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