Journal: Communications Biology

Article Title: PARP1 activation increases expression of modified tumor suppressors and pathways underlying development of aggressive hepatoblastoma

doi: 10.1038/s42003-018-0077-8

Figure Lengend Snippet: PARP1, Ku80, and Ku70 are dramatically elevated in patients with aggressive HBL and bind to the core sequence of ALCDs. a The core 18BPS was linked to biotin beads and was incubated with nuclear extracts isolated from HBL with low and high levels of TSPs. Coomassie staining identified four proteins (shown on the right) that specifically interacted with the 18BPS in nuclear extracts from aggressive HBL. b Large-scale isolation and mass spec analysis of the interacting proteins. The proteins which are specifically interacting with 18BPS are PARP1, Ku80, Ku70, and several additional proteins, some of them belong to nuclear matrix (Supplementary Fig. ). c , d Expression of PARP1, Ku80, and Ku70 is dramatically increased in patients with aggressive HBL. QRT-PCR ( c ) and western blotting ( d ) were performed with mRNAs and proteins isolated from HBL samples with low and high TSPs levels. SHP is a small heterodimer partner, which was detected by re-probe of the PARP1 membrane and which serves as a good control for protein loading. e PARP1, Ku80, and Ku70 form a complex in livers of patients with aggressive HBL. Ku70 and Ku80 were precipitated and PARP1 and Ku70 or Ku80 were determined in these IPs. IgG: heavy chains of IgG. f Fractionation of nuclear proteins from background (blue) and HBL (black) sections of aggressive HBLs by HPLC-based size exclusion chromatography (SEC). Red arrow shows elevation of optical density in the area of high MW protein–protein complexes. g Top: Examination of PARP1/Ku80/Ku70 complexes in SEC fractions. Western blotting shows the amount of proteins in each fraction. Bottom: PARP1-IP shows immunoprecipitation of PARP1 and analysis of Ku70 in these IPs. Images below show hypothetical compositions of PARP1 complexes. Error bars represent standard error of the mean ( c )

Article Snippet: Human: β-actin: Hs01060665_g1, NR1H4: Hs01026590_m1, PSMD10: Hs01100439_g1, CYP3A4: Hs00604506_m1, PCK1: Hs01572978_g1, POU5F1: Hs04260367_gH, EPCAM: Hs00901885_m1, THY-1: Hs00264235_s1, AFP: Hs00173490_m1, HNF4α: Hs00230853_m1, RB1: Hs01078066_m1, TP53: Hs01034249_m1, CELF1: Hs00198069_m1, CEBPα: Hs00269972_s1, ALB: Hs00910225_m1, CDKN1A: Hs00355782_m1, RUNDC1: Hs00405433_m1, HACE1: Hs00410879_m1, MYO18B: Hs00261714_m1, PGAP1: Hs01088726_m1, REG3A: Hs01055563_gH, PARP1; Hs00242302_m1, Ku70: Hs01922655_g1, and Ku80: Hs00897854_m1.

Techniques: Sequencing, Incubation, Isolation, Staining, Mass Spectrometry, Expressing, Quantitative RT-PCR, Western Blot, Membrane, Control, Fractionation, Size-exclusion Chromatography, Immunoprecipitation

Journal: Communications Biology

Article Title: PARP1 activation increases expression of modified tumor suppressors and pathways underlying development of aggressive hepatoblastoma

doi: 10.1038/s42003-018-0077-8

Figure Lengend Snippet: ALCDs are activated in aggressive HBL samples and in hepatoblastoma cancer cells by PARP1/Ku80/Ku70 complexes. a ChIP analysis of the ten representative ALCDs and two negative controls, which contain the inactive 250 bp domain (shown on the left and right) in background and HBL sections of the livers. M marker, In input, B beads, PP1 PARP1, H3K9-Ac histone H3 acetylated at K9, 3-me histone H3 trimethylated at K9. b Quantitative presentation of ChIP analysis. Amounts of PARP1/Ku80/Ku70 complexes were calculated as average of PARP1, Ku80, and Ku70 signals and then as percentage of this average signal to input. c ChIP analysis of ten representative ALCDs in HepG2 cells with and without treatment with 100 µM DPQ. d Quantitative presentations of ChIP analysis. Calculations were performed as described above. e Cell proliferation assay in HepG2, Huh6, and B6-2 cells treated with 100 µM DPQ for 72 h. Representative images highlighting changes in cell proliferation after DPQ treatment are shown in Supplementary Fig. . f Inhibition of PARP1 by DPQ reduces PARP1/Ku80/Ku70 complexes in HeLa and in HepG2 cells and reduces expression of TSPs and cell cycle proteins. Cells were treated with DPQ for 24 and 48 h and western blotting and Co-IPs were performed as described above. Error bars represent standard error of the mean ( e )

Article Snippet: Human: β-actin: Hs01060665_g1, NR1H4: Hs01026590_m1, PSMD10: Hs01100439_g1, CYP3A4: Hs00604506_m1, PCK1: Hs01572978_g1, POU5F1: Hs04260367_gH, EPCAM: Hs00901885_m1, THY-1: Hs00264235_s1, AFP: Hs00173490_m1, HNF4α: Hs00230853_m1, RB1: Hs01078066_m1, TP53: Hs01034249_m1, CELF1: Hs00198069_m1, CEBPα: Hs00269972_s1, ALB: Hs00910225_m1, CDKN1A: Hs00355782_m1, RUNDC1: Hs00405433_m1, HACE1: Hs00410879_m1, MYO18B: Hs00261714_m1, PGAP1: Hs01088726_m1, REG3A: Hs01055563_gH, PARP1; Hs00242302_m1, Ku70: Hs01922655_g1, and Ku80: Hs00897854_m1.

Techniques: Marker, Proliferation Assay, Inhibition, Expressing, Western Blot

Journal: Communications Biology

Article Title: PARP1 activation increases expression of modified tumor suppressors and pathways underlying development of aggressive hepatoblastoma

doi: 10.1038/s42003-018-0077-8

Figure Lengend Snippet: Inhibition of PARP1 by FDA-approved inhibitor DPQ and by si-PARP1 siRNA eliminates PARP1 complexes leading to silence of ALCDs and to inhibition of cell proliferation. a Western blotting of proteins isolated from Huh6 cells treated with 100 µM DPQ for 48 h. Ku80-IP: Ku80 was immunoprecipitated and PARP1 was examined in these IPs. Bar graphs below show levels of proteins as ratios to β-actin. b HepG2 and Huh6 cells were transfected with siRNA-targeting PARP1. Expression of proteins shown on the left and right was analyzed by western blotting. Ku80-IP: western shows the disruption of PARP1/Ku80/Ku70 complexes by inhibition of PARP1. c Cell proliferation assay of HepG2, Huh6, and B6-2 cells transfected with si-PARP1. The assay was performed 48 h after transfection of siRNA. d Representative images highlighting changes in cell proliferation with si-PARP1 transfection in B6-2 cells. e Scratch assay of proliferation of HepG2 cells untreated (con) and treated with DPQ. Percentage shows % of not-closed scratches (inhibition of proliferation) at 48 h after scratch. f Cell proliferation assay shows inhibition of proliferation of HepG2 cells by low concentrations of DPQ and olaparib (Ola). Right image shows Co-IP of PARP1/Ku80/70 complexes and western blotting of downstream targets of ALCDs. g ChIP assay of the ALCD regions of within C/EBPα, HACE1, p53, and β-catenin genes in control and olaparib-treated HepG2 cells. Experiment was performed as described in legend to Fig. . Bottom images show quantitation of ChIP for ALCDs in these genes. h A diagram showing hypothesis which is based on the results of these studies. Aggressive liver cancer is associated with elevation of PARP1, which forms complexes with Ku80 and Ku70 and subsequent chromatin remodeling around ALCDs that leads to a dramatic activation of multiple pathways of liver cancer. An important part of this hypothesis is that certain TSPs are also activated by PARP1-ALCDs axis, but they are posttranslationally modified and are converted into proteins with potential oncogenic activities. Oncogenic activities of posttranslationally modified C/EBPα are shown in our recent publication . Error bars represent standard error of the mean ( c , f )

Article Snippet: Human: β-actin: Hs01060665_g1, NR1H4: Hs01026590_m1, PSMD10: Hs01100439_g1, CYP3A4: Hs00604506_m1, PCK1: Hs01572978_g1, POU5F1: Hs04260367_gH, EPCAM: Hs00901885_m1, THY-1: Hs00264235_s1, AFP: Hs00173490_m1, HNF4α: Hs00230853_m1, RB1: Hs01078066_m1, TP53: Hs01034249_m1, CELF1: Hs00198069_m1, CEBPα: Hs00269972_s1, ALB: Hs00910225_m1, CDKN1A: Hs00355782_m1, RUNDC1: Hs00405433_m1, HACE1: Hs00410879_m1, MYO18B: Hs00261714_m1, PGAP1: Hs01088726_m1, REG3A: Hs01055563_gH, PARP1; Hs00242302_m1, Ku70: Hs01922655_g1, and Ku80: Hs00897854_m1.

Techniques: Inhibition, Western Blot, Isolation, Immunoprecipitation, Transfection, Expressing, Disruption, Proliferation Assay, Wound Healing Assay, Co-Immunoprecipitation Assay, Control, Quantitation Assay, Activation Assay, Modification

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Statins induce apoptosis through inhibition of Ras signaling pathways and enhancement of Bim and p27 expression in human hematopoietic tumor cells.

doi: 10.1177/1010428317734947

Figure Lengend Snippet: Figure 5. Induction of cell-cycle arrest at G1 phase and p27 expression by statins on HL-60 cells. (a) HL-60 cells were treated with fluvastatin or simvastatin for 24 h. The cell-cycle distribution changes were monitored by flow cytometry. Relative percentages of cells in each phase of the cell cycle as indicated. (b) HL-60 cells were treated with fluvastatin or simvastatin for 1, 3, 6, 12, or 24 h. Whole-cell lysates were generated and immunoblotted with antibodies against p53, p21, p27, and β-actin.

Article Snippet: Membranes were blocked with a solution containing 3% skim milk and then incubated overnight at 4°C with each of the following antibodies: anti-Ras (C-4) antibody, anti-Bim (H-191) antibody, anti-p53 (FL-393) antibody, anti-p21 (H-164) antibody, anti-p27 (F-8) antibody, anti-ubiquitin (FL-76) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-p44/42 mitogen-activated protein kinase (MAPK; ERK1/2) (Thr202/Tyr204) antibody, antiphospho-mTOR (Ser2448) antibody, anti-phospho-Bim (Ser69) antibody, anti-p44/42 MAPK (ERK1/2) antibody, anti-mTOR antibody (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin antibody (Sigma).

Techniques: Expressing, Flow Cytometry, Generated

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Statins induce apoptosis through inhibition of Ras signaling pathways and enhancement of Bim and p27 expression in human hematopoietic tumor cells.

doi: 10.1177/1010428317734947

Figure Lengend Snippet: Figure 6. U0126 and rapamycin induce cell death via Bim and p27 expression. (a) HL-60 cells were treated with 5 μM U0126, 10 μM rapamycin, 5 μM fluvastatin, and 10 μM simvastatin for 24 h. Whole-cell lysates were generated and immunoblotted with antibodies against phosphorylated ERK1/2 (phospho-ERK1/2), phosphorylated mTOR (phospho-mTOR), and mTOR. (b) HL-60 cells were treated with 5 μM U0126, 10 μM rapamycin, 5 μM fluvastatin, and 10 μM simvastatin for 72 h. Cell viability was measured by the trypan blue dye exclusion assay. The results are representative of five independent experiments. *p < 0.01 versus control (ANOVA with Dunnett’s test). (c and d) HL-60 cells were treated with U0126 and rapamycin for 24 h. (c) Whole-cell lysates were generated and immunoblotted with antibodies against phospho-Bim, BimEL, and β-actin. (d) Proteins immunoprecipitated with anti-ubiquitin antibody were immunoblotted with anti-Bim antibody. Ubiquitylated Bim was detected as upper shifted bands in anti-Bim blotting. (e) HL-60 cells were treated with U0126 and rapamycin for 24 h. The cell-cycle distribution changes were monitored by flow cytometry. Relative percentages of cells in each phase of the cell cycle as indicated. (f) HL-60 cells were treated with U0126 and rapamycin for 1, 3, 6, 12, or 24 h. Whole-cell lysates were generated and immunoblotted with antibodies against p53, p21, p27, and β-actin.

Article Snippet: Membranes were blocked with a solution containing 3% skim milk and then incubated overnight at 4°C with each of the following antibodies: anti-Ras (C-4) antibody, anti-Bim (H-191) antibody, anti-p53 (FL-393) antibody, anti-p21 (H-164) antibody, anti-p27 (F-8) antibody, anti-ubiquitin (FL-76) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-p44/42 mitogen-activated protein kinase (MAPK; ERK1/2) (Thr202/Tyr204) antibody, antiphospho-mTOR (Ser2448) antibody, anti-phospho-Bim (Ser69) antibody, anti-p44/42 MAPK (ERK1/2) antibody, anti-mTOR antibody (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin antibody (Sigma).

Techniques: Expressing, Generated, Exclusion Assay, Control, Immunoprecipitation, Ubiquitin Proteomics, Flow Cytometry

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Statins induce apoptosis through inhibition of Ras signaling pathways and enhancement of Bim and p27 expression in human hematopoietic tumor cells.

doi: 10.1177/1010428317734947

Figure Lengend Snippet: Figure 5. Induction of cell-cycle arrest at G1 phase and p27 expression by statins on HL-60 cells. (a) HL-60 cells were treated with fluvastatin or simvastatin for 24 h. The cell-cycle distribution changes were monitored by flow cytometry. Relative percentages of cells in each phase of the cell cycle as indicated. (b) HL-60 cells were treated with fluvastatin or simvastatin for 1, 3, 6, 12, or 24 h. Whole-cell lysates were generated and immunoblotted with antibodies against p53, p21, p27, and β-actin.

Article Snippet: Membranes were blocked with a solution containing 3% skim milk and then incubated overnight at 4°C with each of the following antibodies: anti-Ras (C-4) antibody, anti-Bim (H-191) antibody, anti-p53 (FL-393) antibody, anti-p21 (H-164) antibody, anti-p27 (F-8) antibody, anti-ubiquitin (FL-76) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-p44/42 mitogen-activated protein kinase (MAPK; ERK1/2) (Thr202/Tyr204) antibody, antiphospho-mTOR (Ser2448) antibody, anti-phospho-Bim (Ser69) antibody, anti-p44/42 MAPK (ERK1/2) antibody, anti-mTOR antibody (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin antibody (Sigma).

Techniques: Expressing, Flow Cytometry, Generated

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Statins induce apoptosis through inhibition of Ras signaling pathways and enhancement of Bim and p27 expression in human hematopoietic tumor cells.

doi: 10.1177/1010428317734947

Figure Lengend Snippet: Figure 6. U0126 and rapamycin induce cell death via Bim and p27 expression. (a) HL-60 cells were treated with 5 μM U0126, 10 μM rapamycin, 5 μM fluvastatin, and 10 μM simvastatin for 24 h. Whole-cell lysates were generated and immunoblotted with antibodies against phosphorylated ERK1/2 (phospho-ERK1/2), phosphorylated mTOR (phospho-mTOR), and mTOR. (b) HL-60 cells were treated with 5 μM U0126, 10 μM rapamycin, 5 μM fluvastatin, and 10 μM simvastatin for 72 h. Cell viability was measured by the trypan blue dye exclusion assay. The results are representative of five independent experiments. *p < 0.01 versus control (ANOVA with Dunnett’s test). (c and d) HL-60 cells were treated with U0126 and rapamycin for 24 h. (c) Whole-cell lysates were generated and immunoblotted with antibodies against phospho-Bim, BimEL, and β-actin. (d) Proteins immunoprecipitated with anti-ubiquitin antibody were immunoblotted with anti-Bim antibody. Ubiquitylated Bim was detected as upper shifted bands in anti-Bim blotting. (e) HL-60 cells were treated with U0126 and rapamycin for 24 h. The cell-cycle distribution changes were monitored by flow cytometry. Relative percentages of cells in each phase of the cell cycle as indicated. (f) HL-60 cells were treated with U0126 and rapamycin for 1, 3, 6, 12, or 24 h. Whole-cell lysates were generated and immunoblotted with antibodies against p53, p21, p27, and β-actin.

Article Snippet: Membranes were blocked with a solution containing 3% skim milk and then incubated overnight at 4°C with each of the following antibodies: anti-Ras (C-4) antibody, anti-Bim (H-191) antibody, anti-p53 (FL-393) antibody, anti-p21 (H-164) antibody, anti-p27 (F-8) antibody, anti-ubiquitin (FL-76) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-p44/42 mitogen-activated protein kinase (MAPK; ERK1/2) (Thr202/Tyr204) antibody, antiphospho-mTOR (Ser2448) antibody, anti-phospho-Bim (Ser69) antibody, anti-p44/42 MAPK (ERK1/2) antibody, anti-mTOR antibody (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin antibody (Sigma).

Techniques: Expressing, Generated, Exclusion Assay, Control, Immunoprecipitation, Ubiquitin Proteomics, Flow Cytometry

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Optimization of pGC-A expression in Sf9 cells. Western blot (anti-pGC-A) of total cell protein over different virus multiplicities of infection (MOI) and transfection time. MOI of 0 indicates no virus transfection. pGC-A (red arrow) is approximately 120 kDa.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Expressing, Western Blot, Infection, Transfection

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Determination of expressed full-length pGC-A functionality via whole-cell activity assay. The competitive ELISA assay measured the cGMP yield level in Sf9 cells expressing full-length pGC-A versus control Sf9 cells (n = 2). Both cell types were incubated with different concentrations of MANP ligand (0 to 10 7 pmol). Incubation with 0 pmol MANP served as a negative control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Activity Assay, Competitive ELISA, Expressing, Incubation, Negative Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purification of full-length pGC-A via affinity and size exclusion columns. ( A ) Western blot (anti-pGC-A) of samples from cell lysis to the affinity column. pGC-A (red arrow) is ~ 120 kDa. ( B ) Coomassie blue stain from cell lysis to the affinity column. ( C ) Coomassie blue stain of the eluted fraction from Superose 6 that was used for protein crystallization. M: marker; P: membrane pellet from ultracentrifugation after solubilization with n-dodecyl-β-D-maltoside (DDM) and cholesteryl hemisuccinate (CHS); FT: flowthrough from the affinity column; 50, 100, 500: imidazole (mM) at elution from the affinity column.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, Western Blot, Lysis, Affinity Column, Staining, Crystallization Assay, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Presence of pGC-A in crystals was confirmed via western blot. The anti-pGC-A antibody was used in the western blot. The pGC-A monomer is 120 kDa. Lane 1: combined mix: combined crystallization drops collected in the PCR tube. Lane 2: supernatant: the supernatant collected from the centrifuged combined mix. Lane 3: washed pellet: the crystal pellet was washed with the precipitant solution and collected again via centrifugation. Lane 4: washed supernatant: the supernatant from washed crystal pellet. Lane 5: crystallization drop: one crystallization hanging drop directly mixed with SDS sample buffer. Lane 6: crystallization sample: purified pGC-A before crystallization, serving as a positive control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Western Blot, Crystallization Assay, Centrifugation, Purification, Positive Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Indexed diffraction patterns from pGC-A microcrystals collected via serial crystallography at the Advanced Photon Source. Three indexable diffraction patterns with the highest resolution of 3 Å. ( A ) Blue/pink diffraction graphs show diffraction patterns analyzed using CrystFEL software. ( B ) The original diffraction patterns shown with white background. All diffraction dots were manually circled in red for better visualization.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Software

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Dynamic oligomeric states of pGC-A seen in replicate runs of Superose 6 size exclusion chromatography may be dependent on protein concentration. ( A ) The Superose 6 10/300GL column performance profile. Five standard proteins were used to generate relative molecule elution points based on different molecular sizes. Thyroglobulin (669 kDa) eluted at 14.19 mL, ferritin (440 kDa) eluted at 15.96 mL, aldolase (158 kDa) eluted at 17.58 mL, ovalbumin (44 kDa) eluted at 18.49 mL, and aprotinin (6.5 kDa) eluted at 21.60 mL. ( B-D ) Size exclusion chromatography of pGC-A. ( B ) The peak intensity at 17.3 mL corresponds to the pGC-A monomeric state (120 kDa). pGC-A monomer is the major peak determined by chromatography. Other ratios were faded out in the background and served as supplemental comparison. ( C ) pGC-A tetramer and monomer present similar ratios in the chromatographic separation. The peak intensity at 14.76 mL and 17.29 mL corresponds to pGC-A tetrameric (480 kDa) and monomeric states, respectively. ( D ) The pGC-A tetramer is the major peak. The peak intensity at 15.05 mL corresponds to the pGC-A tetrameric state.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Size-exclusion Chromatography, Protein Concentration, Chromatography

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stain of high resolution clear-native PAGE from pGC-A Superose 6 fractions. Superose 6 column eluted tetramer (480 kDa) and monomer (120 kDa) size peaks were concentrated and analyzed in a 4–16% native gel. M, marker.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Silver Staining, Clear Native PAGE, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stained clear-native PAGE of different oligomeric samples treated with or without dithiothreitol (DTT) overnight. Three peak samples, which represent the tetramer, dimer, and monomer of full-length pGC-A, were concentrated and split in half for treatment with or without DTT. S, concentrated sample only; S + DTT, concentrated sample incubated with 1 M DTT overnight.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Staining, Clear Native PAGE, Incubation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purified full-length pGC-A in vitro functional activity test. ( A ) Functional activity for two pGC-A oligomeric states with ATP incubation. The control group was GTP and ATP in sample buffer. For activity values in units of mg of purified pGC-A, the y-axis values of pmol/mL can be converted to nmol/mg protein by multiplying by 0.00873. ( B ) Functional activity for two pGC-A oligomeric states without ATP incubation. The control group was GTP only in the sample buffer. ( C ) Competitive cGMP ELISA standard fit in four parameters logistic (4PL) curve. The left Y-axis is the B/B0 (%) value and represents the percentage of bound cGMP. The right Y-axis represents the average net optical density (OD) reading at 405 nm. Both standard curves were generated with a 95% confidence interval. ( D ) The cGMP yield differences between pGC-A oligomer samples incubated with or without ATP were analyzed. All raw data points were analyzed via the ROUT method (Q = 1%) to remove significantly impossible outlier values before data analysis. One-way ANOVA was used to determine the statistical significance between samples and control in graphs ( A ) and ( B ). Two-way ANOVA was used to determine the statistical significance in graph ( D ). Each dot represented to the sample point and plotted as mean ± standard deviation (SD). * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001 and ns P ≥ 0.05, Not significant.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, In Vitro, Functional Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Generated, Standard Deviation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Proposed native state and three-step mechanism of full-length pGC-A. First, the full-length pGC-A forms a tetramer complex in the native state by non-covalent interactions (e.g., hydrogen bond and hydrophobic interactions). In each tetramer complex, there are two functional units, and each functional unit may represent a dimer. The narrowest part of the tetramer is the transmembrane domain. Second, the pGC-A signal transduction mechanism is not ATP-dependent. The current ATP-dependent two-step activation mechanism should instead be three-step. The first step is ligand (ANP) binding, which moderately activates the pGC-A; the second step is binding ATP, which partially boosts protein activity; the third step is the pGC-A phosphorylation, which fully activates the guanylyl cyclase.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Functional Assay, Transduction, Activation Assay, Binding Assay, Activity Assay

Journal: Genes & Development

Article Title: An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model

doi: 10.1101/gad.275891.115

Figure Lengend Snippet: Reduced DNA damage-mediated apoptosis in cells from the S47 mouse. ( A ) MEFs were generated from Hupki embryos containing either wild-type (WT) p53 or the S47 variant. MEFs were treated with 20 μM etoposide for 24 h, and protein lysates were analyzed by Western blot for the proteins indicated. The data depicted are representative of multiple experiments in multiple independent batches of MEFs. ( B ) Quantification of the relative intensity of cleaved lamin A blots from three independent experiments (as depicted in A ) in primary MEFs from the wild-type and S47 mice untreated or treated with 20 μM etoposide for 24 h. Error bars mark standard deviation. ( C ) Immunohistochemical analysis of the small intestine of wild-type (Wt) and S47 Hupki mice 4 h following exposure to 5 Gy of γ irradiation ( right panels) or untreated ( left panels) for total p53. Bars, 100 μm. ( D ) Quantification of the number of cells staining positively for p53 antisera in equal millimeters of crypts from the small intestines and colons of mice with wild-type p53 or S47. Error bars mark standard error. IR indicates 5 Gy of ionizing radiation. ( E ) Immunohistochemical analysis of the small intestine of wild-type and S47 Hupki mice 4 h following exposure to 5 Gy of γ irradiation ( right panels) or untreated ( left panels) for apoptotic cells (cleaved lamin A). Red arrows mark apoptotic cells. Bars, 100 μm. ( F ) Quantification of apoptosis in the wild-type and S47 small intestines ( left ) and colons ( right ) as cells positive for cleaved lamin A following 5 Gy of radiation. The data depicted are averaged from three fields from three independent experiments in which equal millimeters of crypts were analyzed and quantified. Error bars represent standard deviation. (*) P -value <0.05.

Article Snippet: Primary antibodies used for Western blotting included p53 (ab6) (Calbiochem, OP43), p53 Ser-46-P (Abcam, ab122898), p53 Ser-15-P (Cell Signaling, 9284), MDM2 (ab1 and ab2) (Calbiochem, OP46T and OP115), p21 (ab6) (Calbiochem, OP79), cleaved lamin A (Cell Signaling, 2035), cleaved caspase-3 (Cell Signaling, 9061), GAPDH (14C10) (Cell Signaling, 2118), GPX4 (Abcam, 125066), and GLS2 (Abcam, ab113509).

Techniques: Generated, Variant Assay, Western Blot, Standard Deviation, Immunohistochemical staining, Irradiation, Staining

Journal: Genes & Development

Article Title: An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model

doi: 10.1101/gad.275891.115

Figure Lengend Snippet: Marked impairment of cisplatin-mediated apoptosis in S47 cells and mice. ( A ) Primary MEFs from the wild-type (Wt) or S47 Hupki mouse were treated with 10 μM cisplatin (CDDP) for the time points indicated, and protein lysates were analyzed by Western blot analysis for the proteins indicated. The data depicted are representative of three independent experiments in a minimum of three independent batches of MEFs. ( B ) Primary MEFs from the wild-type and S47 mice as well as the p53 knockout mouse (p53 −/− ) were treated with 10 μM CDDP for 24 h, and protein lysates were analyzed by Western blot analysis for the proteins indicated. ( C ) Flow cytometric analysis of Annexin V-positive cells from primary wild-type and S47 MEFs treated with 10 μM CDDP for 24 h. The totals represent an average of three independent experiments normalized to untreated controls. Error bars represent standard deviations. ( D ) IC 50 analysis for cisplatin (CDDP) in primary wild-type (WT) and S47 MEFs treated with the indicated concentrations of cisplatin for 72 h and analyzed for viability by the Alamar blue assay. The depicted data represent an average of four independent experiments on independent batches of MEFs. Error bars represent standard deviation. ( E ) Human LCLs homozygous for wild-type p53 and the S47 variant were treated with 10 μM CDDP for 24 h, and protein lysates were analyzed by Western blot for the proteins indicated. ( F ) Flow cytometric analysis of Annexin V-positive cells from wild-type and S47 human LCLs treated with 10 μM CDDP for 24 h. The totals represent an average of three independent experiments normalized to untreated controls. Error bars represent standard deviations. ( G ) IC 50 analysis for cisplatin (CDDP) in wild-type and S47 LCLs treated with the indicated concentrations of cisplatin for 48 h and analyzed for viability by Alamar blue staining. The depicted data represent an average of three independent experiments. Error bars represent standard deviation. ( H ) IC 50 analysis for adriamycin in wild-type and S47 LCLs treated with the indicated concentrations of adriamycin for 48 h and analyzed for viability by Alamar blue staining. The depicted data represent an average of three independent experiments. Error bars represent standard deviation. ( I ) Cisplatin-mediated apoptosis, as assessed by cells positive for cleaved lamin A, in the kidneys of wild-type or S47 mice following injection with 20 mg/kg CDDP and analyzed after 48 h. Data are representative of n = 3 per mice group. Bar, 100 μm. ( J ) Clonogenic survival of shARF immortalized wild-type and S47 MEFs treated with the indicated concentrations of cisplatin (CDDP), plated at equal cell numbers 48 h later, and stained with crystal violet after 7 d. ( K ) Quantification of clonogenic survival of immortalized wild-type and S47 MEFs following cisplatin treatment. All values were normalized to the untreated control averaged from three independent experiments. Error bars represent standard deviation. (*) P -value < 0.05.

Article Snippet: Primary antibodies used for Western blotting included p53 (ab6) (Calbiochem, OP43), p53 Ser-46-P (Abcam, ab122898), p53 Ser-15-P (Cell Signaling, 9284), MDM2 (ab1 and ab2) (Calbiochem, OP46T and OP115), p21 (ab6) (Calbiochem, OP79), cleaved lamin A (Cell Signaling, 2035), cleaved caspase-3 (Cell Signaling, 9061), GAPDH (14C10) (Cell Signaling, 2118), GPX4 (Abcam, 125066), and GLS2 (Abcam, ab113509).

Techniques: Western Blot, Knock-Out, Alamar Blue Assay, Standard Deviation, Variant Assay, Staining, Injection, Control

Journal: Genes & Development

Article Title: An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model

doi: 10.1101/gad.275891.115

Figure Lengend Snippet: The S47 variant is impaired for transactivation of a subset of p53 target genes, including Gls2 , Noxa ( Pmaip1 ), and Sco2 . ( A ) qRT–PCR analysis of p53 target genes in primary wild-type (Wt) and S47 MEFs treated with 10 μM cisplatin (CDDP) for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P < 0.05. ( B ) qRT–PCR analysis of the p53 target genes indicated in independent batches of primary MEFs from wild-type and S47 mice treated with 10 μM CDDP for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P < 0.05. ( C ) qRT–PCR analysis of the p53 target genes indicated in human LCLs that are homozygous for wild-type p53 or S47 treated with 10 μM CDDP for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P ≤0.05. SCO2 was not expressed in LCL cells, so these data are not depicted. ( D ) Western analysis for the proteins indicated in wild-type and S47 MEFs pretreated with 10 μM p38MAPK inhibitor SB203580 for 2 h followed by 10 μM cisplatin (CDDP) for 24 h. GAPDH served as the loading control. ( E ) qRT–PCR analysis of the cells in D for the p53 target genes indicated, normalized to control (cyclophilin A). The depicted data represent the average of three independent experiments. Error bars represent standard deviation. (*) P <0.05.

Article Snippet: Primary antibodies used for Western blotting included p53 (ab6) (Calbiochem, OP43), p53 Ser-46-P (Abcam, ab122898), p53 Ser-15-P (Cell Signaling, 9284), MDM2 (ab1 and ab2) (Calbiochem, OP46T and OP115), p21 (ab6) (Calbiochem, OP79), cleaved lamin A (Cell Signaling, 2035), cleaved caspase-3 (Cell Signaling, 9061), GAPDH (14C10) (Cell Signaling, 2118), GPX4 (Abcam, 125066), and GLS2 (Abcam, ab113509).

Techniques: Variant Assay, Quantitative RT-PCR, Control, Standard Deviation, Western Blot

Journal: Genes & Development

Article Title: An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model

doi: 10.1101/gad.275891.115

Figure Lengend Snippet: Impaired DNA-binding ability of the S47 variant. ( A ) ChIP of primary wild-type (Wt) and S47 MEFs treated with 10 μM CDDP for 24 h analyzed using antisera to p53 (CM5) or IgG. The percentage binding normalized to input from qPCR analysis is shown. The data depicted are averaged from three independent experiments normalized to input. Error bars represent standard deviation. (*) P -value < 0.05. ( B ) ChIP analysis of p53 binding to the consensus elements from the genes indicated in human H1299 (p53-null) cells containing doxycycline-inducible wild-type or S47 forms of p53 in the absence and presence of 100 ng/mL doxycycline plus 10 μM cisplatin for 24 h. ChIP was performed using antisera to p53 (fl393G) or normal rabbit IgG. The data depicted are averaged from three independent experiments normalized to input. Error bars represent standard deviation. (*) P -value < 0.05. The IgG results are depicted for the CDKN1A/p21 p53-binding site but were comparable for all other sites analyzed. NOXA was not expressed in this cell line, so this gene was not analyzed.

Article Snippet: Primary antibodies used for Western blotting included p53 (ab6) (Calbiochem, OP43), p53 Ser-46-P (Abcam, ab122898), p53 Ser-15-P (Cell Signaling, 9284), MDM2 (ab1 and ab2) (Calbiochem, OP46T and OP115), p21 (ab6) (Calbiochem, OP79), cleaved lamin A (Cell Signaling, 2035), cleaved caspase-3 (Cell Signaling, 9061), GAPDH (14C10) (Cell Signaling, 2118), GPX4 (Abcam, 125066), and GLS2 (Abcam, ab113509).

Techniques: Binding Assay, Variant Assay, Standard Deviation

Journal: Genes & Development

Article Title: An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model

doi: 10.1101/gad.275891.115

Figure Lengend Snippet: Impaired ferroptosis in S47 cells. ( A ) Representative phase-contrast images of primary MEFs containing wild-type p53 (WT), heterozygous S47/wild type, or S47 treated with 4 μM ferroptosis inducer erastin or vehicle (DMSO) for 8 h (magnification 10×). Data represent the average of three independent studies. Bar, 20 μm. ( B ) Cell viability (Alamar blue) analysis of wild-type, S47/wild-type, or S47 primary MEFs treated with erastin for 72 h. The data represent the average of four independent experiments. Error bars represent standard error of the mean. ( C ) Western blot analysis for GLS2 in wild-type MEFs, wild-type MEFs infected with a lentiviral short hairpin for Gls2 (shGls2), and S47 MEFs untreated or treated with 4 µM erastin for 24 h. GAPDH served as the loading control. In the bottom panel, the percent viability using the Trypan blue exclusion assay is shown. Error bars represent standard deviation. ( D ) qRT–PCR analysis of slc7a11 normalized to cyclophilin A. The data are averaged from three independent biological replicates. Error bars represent standard deviation. ( E ) qRT–PCR analysis of Ptgs2 normalized to cyclophilin A. The data are averaged from three independent biological replicates. Error bars represent standard deviation. ( F ) Immunoblot analysis for GPX4 in wild-type and S47 MEFs following treatment with 10 µM CDDP for 24 h. ( G ) Cell viability analysis of wild-type and S47 human LCLs treated with RSL3 for 48 h. The data represent the average of three independent experiments. Error bars represent standard deviation. ( H ) Trypan blue exclusion analysis of the percent viability in wild-type MEFs or wild-type LCLs exposed to 10 µM CDDP, CDDP plus 2 µM ferrostatin-1 (Fer-1), or CDDP plus 20 µM zVAD-fmk. The data represent the average of three independent experiments. Error bars represent standard deviation. ( I ) Proposed model depicting the relative abilities of wild-type p53 and S47 to induce senescence, apoptosis, and ferroptosis and suppress spontaneous tumor initiation.

Article Snippet: Primary antibodies used for Western blotting included p53 (ab6) (Calbiochem, OP43), p53 Ser-46-P (Abcam, ab122898), p53 Ser-15-P (Cell Signaling, 9284), MDM2 (ab1 and ab2) (Calbiochem, OP46T and OP115), p21 (ab6) (Calbiochem, OP79), cleaved lamin A (Cell Signaling, 2035), cleaved caspase-3 (Cell Signaling, 9061), GAPDH (14C10) (Cell Signaling, 2118), GPX4 (Abcam, 125066), and GLS2 (Abcam, ab113509).

Techniques: Western Blot, Infection, Control, Trypan Blue Exclusion Assay, Standard Deviation, Quantitative RT-PCR

Journal: Nature medicine

Article Title: SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

doi: 10.1038/nm.4163

Figure Lengend Snippet: SAMHD1-mediated HIV-1 restriction in cells does not involve RNase activity. (a–e) Both D137N and Q548A mutants of SAMHD1 restrict HIV-1 infection in PMA-differentiated U937 cells by decreasing viral cDNA synthesis, but not viral genomic RNA (gRNA). U937 cell lines stably expressing WT and mutant SAMHD1 were differentiated with PMA (30 ng/ml) for 20 h. The reverse transcriptase inhibitor nevirapine (NVP) was used as a control. (a) Lysates from PMA-differentiated cells were collected for immunoblotting to confirm SAMHD1 expression and T592-phosphorylated SAMHD1 (phospho-T592). GAPDH was used as a loading control. Blot is representative of three independent experiments. (b) PMA-differentiated cells were infected with single-cycle HIV-1-Luc/VSV-G at a multiplicity of infection (MOI) of 1. At 24 h postinfection (h.p.i.), luciferase assay was used to measure HIV-1 infectivity. Graph depicts relative percentage of luciferase per 10 μg of protein, with the vector set as 100%. Data are presented as mean ± s.e.m. of six independent experiments with two biological replicates per experiment. Statistical analysis was performed using the one-way ANOVA with Dunnett’s correction, ***P < 0.001. (c) Decreased dNTP levels of PMA-differentiated U937 cell lines expressing WT or mutant SAMHD1. Data are presented as means ± s.e.m. of two independent experiments with two biological replicates per experiment. The average concentrations of dNTPs (left) and dCTP (right) in different cell lines are shown. Statistical analysis was performed using the Kruskal–Wallis one-way ANOVA. (d) SAMHD1 does not degrade HIV-1 gRNA in PMA-differentiated U937 cells. The levels of HIV-1 gRNA were measured at 1, 3 and 6 h.p.i., as described at an MOI of 1. Data are presented as mean ± s.e.m. of three independent experiments with two biological replicates per experiment and depicted as relative to HIV-1 gRNA, with the time point 1 h.p.i. set as 1. RNA samples without reverse transcription were used as negative controls and showed no detection of HIV-1 gRNA (data not shown). (e) The expression of WT or mutant SAMHD1 reduces HIV-1 late reverse transcription (RT) products in PMA-differentiated U937 cells at 12 and 24 h.p.i. The levels of HIV-1 late RT products were measured by qPCR. Data are presented as means ± s.e.m. (n = 4 experiments for 12 h.p.i. and n = 6 experiments for 24 h.p.i.). Statistical analysis was performed using the one-way ANOVA with Dunnett’s correction, ***P < 0.001, **P < 0.01, *P < 0.05. (f–h) Characterization of SAMHD1 enzymatic activities in vitro. (f) The purity of recombinant WT SAMHD1 from different batches of preparations (sample 1 and sample 2), as demonstrated by analytical size-exclusion chromatography and SDS–PAGE. SAMHD1 (150 μl) at 2 mg/ml was applied to a Superdex 200 10/300 GL column. Fraction numbers on the SDS–PAGE indicate elution volumes. (g) Moderate exonuclease activities on both ssDNA and ssRNA were observed for sample 1; only background-level activities were detected for sample 2. Ctrl indicates a control without protein. The nuclease-activity assays were performed at 37 °C for 1 h, with 1 μM of SAMHD1 and 1 μM of ssDNA or ssRNA in the presence of 5 mM Mg2+. (h) The dNTPase activity of SAMHD1 proteins (0.5 μM) was assayed with 1 mM dGTP from 5–15 min. The amount of dG products generated in the reactions was quantified by HPLC. Error bars represent s.e.m. from triplicate experiments.

Article Snippet: Rabbit antibodies specific for T592 phospho-SAMHD1 (ProSci, #8005) were used at a dilution of 1:1,000, as previously described 22 .

Techniques: Activity Assay, Infection, cDNA Synthesis, Stable Transfection, Expressing, Mutagenesis, Reverse Transcription, Control, Western Blot, Luciferase, Plasmid Preparation, In Vitro, Recombinant, Size-exclusion Chromatography, SDS Page, Generated

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Luciferase gene expression results of polyplexes formulated with pDNA and each of the five different polymers in HeLa cells. Luciferase expression was measured 48 hours after transfection; all data are normalized to the cells only control. Cells were transfected with JetPEI (PEI) at an N/P ratio of 5. All other polymers were used at an N/P ratio of 20. Statistical analysis for expression efficiency was performed using the Tukey-Kramer HSD method on the log of the data (n = 6). Bars with different letters are statistically significant from each other (p < 0.05). For the cell viability data, T443 is significantly lower than A442 and PEI (p < 0.05, denoted with similar asterisks), and PEI, T443 and A442 were all statistically significant from the cells only and DNA only controls (p < 0.05) according to the Tukey-Kramer HSD method (n = 6).

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Luciferase, Gene Expression, Expressing, Transfection, Control

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: (a) Mitochondrial membrane potential measured by DiIC(1)5 fluorescence normalized to the cells only control and (b) phosphatidylserine (PS) exposure measured by Annexin V binding assay in HeLa cells transfected with polyplexes formed using PEI, T4, and A442. The data in (b) is presented as the percent increase in PS exposure compared to the cells only control. Letters represent statistical analysis; bars with different letters are statistically significant (p < 0.05) according to the Tukey-Kramer HSD method (n = 3).

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Membrane, Fluorescence, Control, Binding Assay, Transfection

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Confocal microscopy images of HeLa cells 30 minutes after being transfected with polyplexes formed with Cy5-labeled pDNA and the following polymers: (a) pDNA only; (b) JetPEI (PEI); (c) T46; (d) T412; (e) T443; (f) A442. Colors represent the following; green = WGA AF488-labeled cytosol; blue = DAPI-labeled nucleus; magenta = Cy5-labeled pDNA. Scale bar = 20 µm. Arrows denote sites of polyplex internalization. (g) Manders coefficients between each polyplex type (formed with Cy5-pDNA) and AlexaFluor-488-conjugated Wheat Germ Agglutinin-labeled plasma membrane. Bars with different letters are statistically significant from each other (p < 0.05) according to the Tukey-Kramer HSD method (n = 3).

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Confocal Microscopy, Transfection, Labeling, Clinical Proteomics, Membrane

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Measurement of propidium iodide fluorescence in HeLa cells after transfection with each polyplexes type at time points of 30 mins and 4 hours as measured via flow cytometry. Data is presented as the percent increase in PI positive cells as normalized to the cells only control.

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Fluorescence, Transfection, Flow Cytometry, Control

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Confocal images of HeLa cells fixed 4 hours after transfection. Cells were transfected with the following: (a) DNA only; (b) JetPEI (PEI); (c) T46; (d) T412; (e) T443; (f) A442. Colors represent the following; blue = nucleus; green = nuclear lamin; magenta = pDNA. Scale bar = 20 µm. White arrows indicate sites of deep nuclear envelope indentation.

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Transfection

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Confocal microscopy images of the nuclei in whole HeLa cells transfected with polyplexes formed with Cy5-pDNA and (a) JetPEI (PEI); (b) T46; (c) T412; (d) T443; (e) A442. The nucleus is shown as blue, nuclear lamin is shown as green, and the polyplexes are shown as magenta. Scale bar = 5 µm.

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Confocal Microscopy, Transfection

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Trypan blue exclusion assay performed on nuclei treated with polyplexes for 4 hours. Nuclei were isolated from HeLa cells and treated with (a) pDNA only; (b) JetPEI (PEI); (c) T46; (d) T412; (e) T443; or (f) A442. Scale bar = 400 µm. (g) Nuclei were counted and analyzed for pixel intensity using ImageJ and the statistics were calculated using JMP. Roughly 300 nuclei were analyzed for each polyplex, and the Tukey-Kramer HSD test reveal each set of data points to be statistically significant from one another.

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Trypan Blue Exclusion Assay, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Potassium Iodide Induces Apoptosis in Salivary Gland Cancer Cells

doi: 10.3390/ijms26115199

Figure Lengend Snippet: KI reduces the viability of SLC5A5-expressing SGC cells. ( A , B ) The fold change in SLC5A5 expression was determined by qPCR in ( A ) human submandibular SG epidermoid carcinoma (A253; n = 5), myeloid hematopoietic (HL-6; n = 2, U937; n = 2), human, and murine endothelial, respectively (HUVEC; n = 2, BMEC1; n = 3), as well as in (HT1080; n = 3) fibrosarcoma cells and ( B ) murine lymphoblast (32D; n = 2) cells, fibroblasts (MS-5; n = 2, 3T3; n = 2), and submandibular SG adenocarcinoma (WR21; n = 3) cells. SLC5A5 gene expression levels were normalized to BETA-ACTIN expression in the same samples, and fold changes were adjusted relative to the expression levels in control samples. ( C , D ) The iodine concentration was assessed in cell lysates of A253 ( C ) and WR21 ( D ) cells 48 h after KI treatment at the indicated concentrations ( n = 4/5 per group). ( E ) Representative light microscopy images of murine WR21 and human A253 SGC cells 48 h after incubation with or without KI (100 μM; scale bar = 100 μm). ( F ) The viability rate of A253 cells treated with the indicated KI concentrations for 48 h was determined by trypan blue exclusion ( n = 8 for the control (co) group and n = 3 for KI 25, 50, 100, and 200 μM groups). ( G , H ) The absolute number of viable and control (co) A253 ( G ) and WR21 cells ( H ) after 48 h in culture, following the addition of KI (100 μM), was determined using the trypan blue exclusion assay ( n = 10 and 5/group for A253 cells and n = 4, 3/group for WR21 cells). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using a one-way ANOVA test (to determine the effects of two independent variables on a dependent variable) or Student’s t -test (to compare the performance of two groups under different conditions), with mean ± SD depicted.

Article Snippet: The basic media for the human submandibular SG epidermoid carcinoma (A253) cells (Cat. HTB-41, ATCC, Manassas, VA, USA) was McCoy’s 5A (Modified) Medium (Cat. 16600082, Gibco, Grand Island, NY, USA); for U-937 human histiocytic lymphoma cells (Cat. CRL-1593.2, ATCC, Manassas, VA, USA), it was RPMI-1640 medium (Cat. 11875093, Gibco, Grand Island, NY, USA); for HL-60 human promyeloblast cells (Cat. CCL-240, ATCC, Manassas, VA, USA), it was IMDM (Cat. 12440053, Gibco, Grand Island, NY, USA); for HUVEC human umbilical venule endothelial cells (Cat. CRL-1730, ATCC, Manassas, VA, USA), it was F-12K Medium (Cat. 21127022, Gibco, Grand Island, NY, USA) supplemented with heparin (Cat. H3393, Sigma, Saint Louis, MO, USA) and ECGS (Cat. CB-40006, Fisher Scientific, Waltham, MA, USA); for BMEC human bone marrow microvascular endothelial cell (Cat. CRL-3421, ATCC, Manassas, VA, USA), it was MCDB-131 medium (Cat. 10372-019, Gibco, Grand Island, NY, USA); for HT-1080 human fibrosarcoma cells (Cat. CCL-121, ATCC, Manassas, VA, USA), it was MEM (Cat. 137-17215, Wako, Osaka, Japan); for WR21 mouse submandibular SG adenocarcinoma cells (Cat. CRL-2189, ATCC, Manassas, VA, USA) and for murine NIH/3T3 fibroblast (Cat. CRL-1658, ATCC, Manassas, VA, USA), it was D-MEM medium (Cat. 044-29765, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); for 32D mouse lymphoblast cells (Cat. CRL-11346, ATCC, Manassas, VA, USA), it was RPMI 1640 (Cat. 11875093, Gibco, Grand Island, NY, USA); and for MS5 murine stromal cells (kindly provided by Dr. MAS Moore, Sloan Kettering Cancer Center, New York, NY, USA), it was IMDM (Cat. 12440053, Gibco, Grand Island, NY, USA).

Techniques: Expressing, Gene Expression, Control, Concentration Assay, Light Microscopy, Incubation, Trypan Blue Exclusion Assay

Journal: International Journal of Molecular Sciences

Article Title: Potassium Iodide Induces Apoptosis in Salivary Gland Cancer Cells

doi: 10.3390/ijms26115199

Figure Lengend Snippet: KI treatment induces apoptosis in SGC cells involving the mitochondria-associated molecule BAX. ( A ) A representative histogram illustrates the cell cycle analysis of control and KI-treated A253 and WR21 cells after a 48 h incubation period, assessed by flow cytometry ( n = 3/group). ( B ) Representative light microscopic images depict human A253 cells treated with KI at 100 μM for 48 h. Scale bar = 100 μm. The panel on the right illustrates the cell size ratio between treated and untreated cells ( n = 49 and 38 cells/group). ( C ) Flow cytometric analysis evaluates early (AnnexinV+PI-) and late (AnnexinV+PI+) apoptosis in KI-treated or control A253 cells. Means represents three independently conducted experiments. The left panel displays FACS plots of control and KI-treated cells stained with Annexin V-FITC and PI. The right panel presents the statistical analysis of two independent experiments. ( D ) Caspase 3/7 activity in KI-treated and control cells after a 48 h incubation using the Caspase 3/7 activity assay. Signals were normalized to control samples ( n = 2/group). ( E ) Fold changes in BCL-2 expression were determined by qPCR in human SGC cell A253 after 48 h of treatment with 100 µM KI. Target gene expression levels were normalized to BETA-ACTIN expression in the same samples, and fold changes were adjusted relative to control expression levels ( n = 11/5 per group). ( F , G ) Representative Western blot ( F ) and relative band intensity ( G ) of Bcl-2 and β-actin (internal control) in control and KI-treated (KI 100 µM) A253 cells. Band intensities were calculated by normalizing Bcl-2 expression to β-actin, with comparisons made to the corresponding controls (uncropped Western blot images as ( n = 2 per group). ( H ) BAX expression was determined by qPCR in human SGC cell A253 after 48 h of treatment with 100 µM KI. Target gene expression levels were normalized to BETA-ACTIN expression in the same samples, and fold changes were adjusted relative to control expression levels ( n = 4/5 per group). ( I , J ) Representative Western blot ( I ) and relative band intensity ( J ) of BAX and GAPDH (internal control) in control and KI-treated (KI 100 µM) A253 cells. Band intensities were calculated by normalizing BAX expression to GAPDH, with comparisons made to the corresponding controls (uncropped Western blot images as ( n = 2 per group). ( K , L ) Representative Western blot ( K ) and relative band intensity ( L ) of p53 and GAPDH (internal control) in control and KI-treated (KI 100 µM) A253 cells. Band intensities were calculated by normalizing p53 quantity to GAPDH ( K ), compared to the corresponding controls (uncropped Western blot images presented as ; n = 2 per group. * p < 0.05, ** p < 0.05, **** p < 0.0001 using a one-way ANOVA test (for determination of the effects of two independent variables on a dependent variable) or Student’s t -test (to compare the performance of two groups under different conditions) with mean ± SD depicted.

Article Snippet: The basic media for the human submandibular SG epidermoid carcinoma (A253) cells (Cat. HTB-41, ATCC, Manassas, VA, USA) was McCoy’s 5A (Modified) Medium (Cat. 16600082, Gibco, Grand Island, NY, USA); for U-937 human histiocytic lymphoma cells (Cat. CRL-1593.2, ATCC, Manassas, VA, USA), it was RPMI-1640 medium (Cat. 11875093, Gibco, Grand Island, NY, USA); for HL-60 human promyeloblast cells (Cat. CCL-240, ATCC, Manassas, VA, USA), it was IMDM (Cat. 12440053, Gibco, Grand Island, NY, USA); for HUVEC human umbilical venule endothelial cells (Cat. CRL-1730, ATCC, Manassas, VA, USA), it was F-12K Medium (Cat. 21127022, Gibco, Grand Island, NY, USA) supplemented with heparin (Cat. H3393, Sigma, Saint Louis, MO, USA) and ECGS (Cat. CB-40006, Fisher Scientific, Waltham, MA, USA); for BMEC human bone marrow microvascular endothelial cell (Cat. CRL-3421, ATCC, Manassas, VA, USA), it was MCDB-131 medium (Cat. 10372-019, Gibco, Grand Island, NY, USA); for HT-1080 human fibrosarcoma cells (Cat. CCL-121, ATCC, Manassas, VA, USA), it was MEM (Cat. 137-17215, Wako, Osaka, Japan); for WR21 mouse submandibular SG adenocarcinoma cells (Cat. CRL-2189, ATCC, Manassas, VA, USA) and for murine NIH/3T3 fibroblast (Cat. CRL-1658, ATCC, Manassas, VA, USA), it was D-MEM medium (Cat. 044-29765, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); for 32D mouse lymphoblast cells (Cat. CRL-11346, ATCC, Manassas, VA, USA), it was RPMI 1640 (Cat. 11875093, Gibco, Grand Island, NY, USA); and for MS5 murine stromal cells (kindly provided by Dr. MAS Moore, Sloan Kettering Cancer Center, New York, NY, USA), it was IMDM (Cat. 12440053, Gibco, Grand Island, NY, USA).

Techniques: Cell Cycle Assay, Control, Incubation, Flow Cytometry, Staining, Activity Assay, Expressing, Targeted Gene Expression, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Potassium Iodide Induces Apoptosis in Salivary Gland Cancer Cells

doi: 10.3390/ijms26115199

Figure Lengend Snippet: The influence of KI on EGF signaling in cancerous and normal SG cells. ( A ) The fold change in IL-10 expression levels in human SGC cells A253 after 48 h of treatment with 100 µM KI was measured using qPCR. The target gene expression was normalized to BETA-ACTIN expression in the same samples, and fold changes were calculated relative to the control samples ( n = 10/6 per group). ( B ) IL-10 levels in the culture medium of A253 cells, both treated and untreated, were assessed after 48 h of incubation with 100 µM KI using ELISA ( n = 3/group). ( C , D ) Representative Western blot ( C ) and relative band intensity of TNF-α and NF-κB ( D ), along with β-actin (internal control), in A253 cells that were untreated and treated with 100 µM KI. Band intensities were determined by normalizing the quantity of TNF-α and NF-κB to β-actin compared to the corresponding controls (uncropped Western blot images can be found in –C ( n = 2/group). ( E ) Kaplan–Meier plots displaying the overall survival of HNSC patients with high (red line) and low (blue) EGF mRNA expression ( EGF Log-rank p = 0.00078, HR (high) = 1.6, p (HR) = 0.00087, n (high) = 257, n (low) = 256). Patient stratification based on EGF levels was conducted using the GEPIA datasets http://gepia.cancer-pku.cn/ (accessed on 23.12.2024). TPM refers to transcripts per million. ( F ) Fold changes in expression levels of EGF-b , EGFR , and PI3K in submandibular SG tissues from mice drinking regular water (control) or water with KI (1 mg/mL) ( n = 3/group, except for EGFR (KI group), n = 7). ( G , H ) Fold changes in mEGF and mEGFR expression levels in WR21 ( G ) and A253 ( H ) cells after 48 h of treatment with 100 µM KI were determined using qPCR. Target gene expression levels were normalized to BETA-ACTIN expression in the same samples, and fold changes were calculated relative to the expression levels in control samples (WR21 mEGF n = 6/group; EGFR = 3/group; A253 huEGF n = 9 and 4/group; huEGFR n = 10 and 5/group). ( I , J ) Representative Western blot ( I ) and relative band intensity of EGFR ( J ) and GAPDH (internal control) in A253 cells, both control and treated with 100 µM KI. Band intensities were calculated by normalizing EGFR quantity to GAPDH compared to the corresponding controls. Uncropped Western blot images are in ; n = 2/group). ( K ) Fold changes in expression levels of PI3K in A253 cells after 48 h of treatment with 100 µM KI were determined using qPCR. The target gene expression levels were normalized to BETA-ACTIN expression in the same samples, and fold changes were normalized relative to the expression levels in control samples ( n = 5 and n = 3 per group). ( L – N ) Representative Western blot ( L ) and the relative band intensity of AKT ( M ), pAKT ( N ), and GAPDH (internal control) in A253 cells, both control and treated with 100 µM KI. Band intensities were calculated by normalizing AKT and pAKT quantity to GAPDH, compared to the respective controls (uncropped Western blot images are shown in ( –C). * p < 0.05 using Student’s t -test (to compare the performance of two groups under different conditions) with mean ± SD depicted.

Article Snippet: The basic media for the human submandibular SG epidermoid carcinoma (A253) cells (Cat. HTB-41, ATCC, Manassas, VA, USA) was McCoy’s 5A (Modified) Medium (Cat. 16600082, Gibco, Grand Island, NY, USA); for U-937 human histiocytic lymphoma cells (Cat. CRL-1593.2, ATCC, Manassas, VA, USA), it was RPMI-1640 medium (Cat. 11875093, Gibco, Grand Island, NY, USA); for HL-60 human promyeloblast cells (Cat. CCL-240, ATCC, Manassas, VA, USA), it was IMDM (Cat. 12440053, Gibco, Grand Island, NY, USA); for HUVEC human umbilical venule endothelial cells (Cat. CRL-1730, ATCC, Manassas, VA, USA), it was F-12K Medium (Cat. 21127022, Gibco, Grand Island, NY, USA) supplemented with heparin (Cat. H3393, Sigma, Saint Louis, MO, USA) and ECGS (Cat. CB-40006, Fisher Scientific, Waltham, MA, USA); for BMEC human bone marrow microvascular endothelial cell (Cat. CRL-3421, ATCC, Manassas, VA, USA), it was MCDB-131 medium (Cat. 10372-019, Gibco, Grand Island, NY, USA); for HT-1080 human fibrosarcoma cells (Cat. CCL-121, ATCC, Manassas, VA, USA), it was MEM (Cat. 137-17215, Wako, Osaka, Japan); for WR21 mouse submandibular SG adenocarcinoma cells (Cat. CRL-2189, ATCC, Manassas, VA, USA) and for murine NIH/3T3 fibroblast (Cat. CRL-1658, ATCC, Manassas, VA, USA), it was D-MEM medium (Cat. 044-29765, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); for 32D mouse lymphoblast cells (Cat. CRL-11346, ATCC, Manassas, VA, USA), it was RPMI 1640 (Cat. 11875093, Gibco, Grand Island, NY, USA); and for MS5 murine stromal cells (kindly provided by Dr. MAS Moore, Sloan Kettering Cancer Center, New York, NY, USA), it was IMDM (Cat. 12440053, Gibco, Grand Island, NY, USA).

Techniques: Expressing, Targeted Gene Expression, Control, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Potassium Iodide Induces Apoptosis in Salivary Gland Cancer Cells

doi: 10.3390/ijms26115199

Figure Lengend Snippet: KI induces ROS generation in SGC cells. ( A ) Representative images of fluorescent 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA)-stained control and KI-treated A253 and WR21 cells after a 48 h incubation. Bright green fluorescence indicates ROS-producing cells. Scale bar = 20 μm. ( B ) ROS production was assessed by quantifying DCFH-DA fluorescence and normalizing it to total protein in control ( n = 4/group) and KI-treated ( n = 5/group) cells. ( C ) The relative optical density values from the CCK-8 assay reflect the viability of A253 cells under various treatment conditions: untreated ( n = 16/group), treated with KI ( n = 8/group), treated with N-acetyl-l-cysteine (NAC) ( n = 16/group), and treated with a combination of KI and NAC ( n = 8/group). * p < 0.05, *** p < 0.001 using a one-way ANOVA test (to determine the effects of two independent variables on a dependent variable) or Student’s t -test (to compare the performance of two groups under different conditions), with mean ± SD depicted.

Article Snippet: The basic media for the human submandibular SG epidermoid carcinoma (A253) cells (Cat. HTB-41, ATCC, Manassas, VA, USA) was McCoy’s 5A (Modified) Medium (Cat. 16600082, Gibco, Grand Island, NY, USA); for U-937 human histiocytic lymphoma cells (Cat. CRL-1593.2, ATCC, Manassas, VA, USA), it was RPMI-1640 medium (Cat. 11875093, Gibco, Grand Island, NY, USA); for HL-60 human promyeloblast cells (Cat. CCL-240, ATCC, Manassas, VA, USA), it was IMDM (Cat. 12440053, Gibco, Grand Island, NY, USA); for HUVEC human umbilical venule endothelial cells (Cat. CRL-1730, ATCC, Manassas, VA, USA), it was F-12K Medium (Cat. 21127022, Gibco, Grand Island, NY, USA) supplemented with heparin (Cat. H3393, Sigma, Saint Louis, MO, USA) and ECGS (Cat. CB-40006, Fisher Scientific, Waltham, MA, USA); for BMEC human bone marrow microvascular endothelial cell (Cat. CRL-3421, ATCC, Manassas, VA, USA), it was MCDB-131 medium (Cat. 10372-019, Gibco, Grand Island, NY, USA); for HT-1080 human fibrosarcoma cells (Cat. CCL-121, ATCC, Manassas, VA, USA), it was MEM (Cat. 137-17215, Wako, Osaka, Japan); for WR21 mouse submandibular SG adenocarcinoma cells (Cat. CRL-2189, ATCC, Manassas, VA, USA) and for murine NIH/3T3 fibroblast (Cat. CRL-1658, ATCC, Manassas, VA, USA), it was D-MEM medium (Cat. 044-29765, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); for 32D mouse lymphoblast cells (Cat. CRL-11346, ATCC, Manassas, VA, USA), it was RPMI 1640 (Cat. 11875093, Gibco, Grand Island, NY, USA); and for MS5 murine stromal cells (kindly provided by Dr. MAS Moore, Sloan Kettering Cancer Center, New York, NY, USA), it was IMDM (Cat. 12440053, Gibco, Grand Island, NY, USA).

Techniques: Staining, Control, Incubation, Fluorescence, CCK-8 Assay

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A) Domain structures of ADAR1. (B-C) Expression of human ADAR1 N-terminal truncation mutants (B) and purification of ADAR1-S126 indicated by a dashed box in the chromatogram, the estimated molecular weight of MBP tagged ADAR1-S126 homodimer has been indicated, and purity of the protein was checked with SDS-PAGE gel (C). W: Washed, including proteins that do not bind to the amylose resin. E: Elution, including proteins that bind to the amylose resin and can be eluted with the buffer containing maltose. B: Bound, including proteins that bind to the amylose resin but cannot be eluted. (D-F) Representative gels showing RNA editing activities of ADAR1 against GLI RNA variants (V7-V12). (E) Normalized editing activity of (D). (F) The secondary structures of RNA variants were tested in (D), with the editable A highlighted in red. (G-H) Representative gels showing RNA editing activities of ADAR1 against GLI RNA variants (V13-V58) (G) and (V59-V63) (H). (I) Normalized editing activity of (H). (J) The secondary structures of RNA variants were tested in (H), with the editable A highlighted in red. (E) and (I). Values represent the mean of three independent experiments, with error bars indicating standard deviation. 120 nM ADAR1 and 25 nM each RNA were used.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Expressing, Purification, Molecular Weight, SDS Page, Activity Assay, Standard Deviation

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A) Diagram illustrating the coupled enzymatic assay utilized for ADAR1 activity assessment. Bands of substrate and cleavage products are indicated. The RNA diagram represents the secondary structure of the GLI-V11 RNA used in our experiments. (B) Representative gels showing RNA editing activities of ADAR1 against GLI RNA variants (V1-V6) (C) Normalized editing activity of (B). N.D. indicates not determined. (D) The secondary structures of RNA variants were tested in (B) with the editable A highlighted in red. (E-H) ADAR1 editing activity on GLI RNA variants with diverse sequences and lengths. In (E-G), RNA variant backbones are shown as secondary structures. The editable A sites are highlighted in red, while mutated bases are enclosed in dashed boxes and labeled in orange. In (H), lengths of the 5ʹ or 3ʹ arms of the RNA backbones are indicated, with mutated RNA variants of different lengths denoted in parentheses. (I) Summary of the RNA sequence and length preference for ADAR1 editing. The secondary structures and folds of the GLI-V1 and HT-V1 RNAs were displayed and followed by the summary of RNA sequence and length preferences for ADAR1 editing. The editing site A is highlighted in red. Optimal editing lengths for the 5ʹ or 3ʹ arm and sequence preferences surrounding editing sites are indicated. Values represent the mean of three independent experiments, with error bars indicating standard deviation. 120 nM ADAR1 or mutants and 25 nM each RNA were used. See also Figures S1 and S2.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Enzymatic Assay, Activity Assay, Variant Assay, Labeling, Sequencing, Standard Deviation

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A-C) Representative gels showing RNA editing activities of ADAR1 against HT RNA variants (V1-V16) (A). The secondary structures of HT-V1 to V4 are shown in (A) and (B), with the editable A highlighted in red. The lengths of the 5ʹ or 3ʹ arms of the RNA backbones are indicated, with mutated RNA variants of different lengths denoted in parentheses in (B). (C) Normalized editing activity of (A). (D) Representative gels showing RNA editing activities of ADAR1 against HT RNA variants (V17-V26). (E) Normalized editing activity of (D). HT-V6 RNA backbone is shown as a secondary structure. The editable A sites are highlighted in red, while mutated bases are enclosed in dashed boxes and labeled in orange in (E). Values represent the mean of three independent experiments, with error bars indicating standard deviation. 120 nM ADAR1 and 25 nM each RNA were used.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Activity Assay, Labeling, Standard Deviation

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A-B) Two views of the cryo-EM maps of the ADAR1-GLI (A) or ADAR1-HT (B) complex. The secondary structure of GLI-V11 or HT-V2 RNA is displayed in the top panel of (A) or (B), the modified A (8-aza) in GLI-V11 or HT-V2 RNA is denoted as "X" and highlighted in red. The editing strand of GLI-V11 or HT-V2 is colored in green or blue, respectively, while the non-editing strand of each is colored in yellow and orange, the disorder sequences in GLI-V11 or HT-V2 are colored in grey. Monomer A is colored in cyan, and monomer B is colored in magenta. (C-D) Two views of the atomic model of the ADAR1-GLI complex (C) or ADAR1-HT complex (D). The models are color-coded as in (A) and (B). See also Figures S3-S7 and Tabel S1.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Cryo-EM Sample Prep, Modification

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A), (D), and (G) Gold-standard Fourier shell correlation (FSC) curves of ADAR1-GLI complex (A), ADAR1-HT complex (D), and ADAR1-HT complex 2 (G) between the two half maps, with indicated resolution at FSC = 0.143 shown in blue. FSC curves between the refined model and the cryo-EM map, with indicated resolution at FSC = 0.5, are shown in red, respectively. (B), (E), and (H) The angular distribution of particles used in the final 3D reconstruction of ADAR1-GLI complex (B), ADAR1-HT complex (E), and ADAR1-HT complex 2 (H). (C), (F), and (I) Local resolution estimation of the cryo-EM density map of ADAR1-GLI complex (C), ADAR1-HT complex (F), and ADAR1-HT complex 2 (I).

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A) Structural comparison of the monomer A and B from ADAR1-GLI complex. The shift of E1008 residue is indicated with a red arrow. (B) Structural comparison of the ADAR1-GLI and ADAR2-RNA complex (PDB 8E0F), the monomer A from two structures aligned. Only the deaminase domain and RNA in the ADAR2-RNA complex are shown for clarity and colored in grey. The zoom-in view of the flexible loop in ADAR1 and ADAR2 is shown on the right panel. (C-D) The electrostatic potential surface of ADAR1-GLI (C) and ADAR1-HT complex (D). (E-F) Structural comparison of the 3-bp "CCC:AAA" bubble on the 5’ arm of the GLI RNA (E) and the short 3-bp “GCU:UGA” 5’ duplex of the HT RNA with regular A-form dsRNA (F).

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Comparison, Residue

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A) Cryo-EM density maps of the ADAR1-GLI complex. (B) The local density map of the catalytic sites in the ADAR1-GLI complex. (C) The local density map of the dimerization interface of ADAR1-GLI complex. (D) The local density map of the two Zn 2+ binding motifs in the ADAR1-GLI complex. (E-F) The local density map of IHP and its contacted residues in the monomer A (E) and monomer B (F) in the ADAR1-GLI complex. (G) The cryo-EM density map of GLI-V11 RNA, with a zoom-in view of the 3 bp CCC:AAA bubble is shown in the right panel. (H) Cryo-EM density maps of the ADAR1-HT complex. (I) The local density map of the catalytic sites in the ADAR1-HT complex. (J) Cryo-EM density map of HT-V2 RNA in ADAR1-HT complex. (K) Cryo-EM density maps of the ADAR1-HT complex 2. (I) The local density map of the RBD3 and linker. (M) Cryo-EM density map of HT-V2 RNA in ADAR1-HT complex 2.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A) Structural comparison of the ADAR1-GLI and ADAR1-HT complex, with the monomer A aligned. The ADAR1-GLI complex and HT-V2 RNA in the ADAR1-HT complex are color-coded as in and , respectively. The ADAR1 dimer of the ADAR1-HT complex was colored in grey. (B-C) The zoom-in views of the RNA recognition site in ADAR1-GLI (B) and ADAR1-HT complexes (C). (D-E) Overview of the molecular interactions between ADAR1 and GLI-V11(D) and HT-V2 (E) RNA. (I) (F) Editing activity of ADAR1 wild type (WT) or mutants with different RNA substrates. Secondary structures of RNA substrates are displayed, with the editing site A highlighted in red. (G-H) Nucleotide recognition by E1008 in the ADAR1-GLI complex (G) and the ADAR1-HT complex (H). (I) Editing activity of ADAR1 E1008 mutants with four HT-RNA variants with different sequence contexts around the editing site. (F) and (I) Values represent the mean of three independent experiments, with error bars indicating standard deviation. 100 nM ADAR1 or mutants and 25 nM each RNA were used. See also Figures S6-S10.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Comparison, Activity Assay, Sequencing, Standard Deviation

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A-F) Representative gels of the RNA editing activities of ADAR1 RNA binding or dimerization-related mutants against GLI-V11 (A), GLI-V32 (B), HT-V6 (C), HT-V2 (D), HT-V5 (E), and HT-V16 (F) RNAs. (G-I) Representative gels of the RNA editing activities of ADAR1 E1008 mutants against HT-RNA variants. (J) Normalized editing activity as shown in (I). (K-P) Representative gels of the RNA editing activities of AGS-associated mutants against GLI-V11 (K), GLI-V32 (I), HT-V6 (M), HT-V2 (N), HT-V5 (O), and HT-V16 (P) RNAs. 100 nM ADAR1 or mutants and 25 nM each RNA were used. The values represent the mean of three independent experiments, with the error bars representing the standard deviation.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: RNA Binding Assay, Activity Assay, Standard Deviation

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A) Recognition of the flipped 8-aza in the active site, the hydrogen bond interactions are shown as red dashed lines. (B-C) Structural modeling of replacing the orphan base in ADAR1 GLI (B) or HT (C) complex. (D-E) Structural modeling of replacing 5ʹ-adjacent “A” on the non-editing strand in ADAR1 GLI (D) or HT (E) complex. Clashes were defined as the hydrogen bond interaction shorter than 2.5 Å in (B-E).

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques:

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A) The dimerization interface between monomer A and monomer B in the ADAR1-GLI complex, the monomer A and monomer B are color-coded as in , while the interfaces on monomer A and B are colored in orange and wheat. The zoom-in view of the interactions between the two key residues (W1022 and D1023) from monomer A and their interacting residues in monomer B are shown on the right panel. (B) The molecular contacts between monomer A and monomer B. VDW: van der Waals interactions. (C) Editing activity of W1022A and D1023A against different RNA substrates. Values represent the mean of three independent experiments, with error bars representing the standard deviation.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Activity Assay, Standard Deviation

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A) Mapping of AGS-associated mutations onto the ADAR1-GLI complex. (B) Editing activity of ADAR1 AGS-associated mutants with different RNA substrates. Values represent the mean of three independent experiments, with error bars indicating standard deviation. 100 nM ADAR1 or mutants and 25 nM each RNA were used. (C) Heatmap of RNA editing rates for WT and AGS-associated ADAR1 mutants across 37 selected RNAs, as determined by RNA-Seq. The name, for example, CCNI_971, indicates editing at position A971 in the CCNI mRNA. Among the tested groups, the G1007R mutant shows the most severe defects in RNA editing. (D) Editing activity of wild-type (WT) ADAR1 and AGS-associated mutants on selected dsRNAs identified from RNA-Seq data. Secondary structures of the RNAs are shown, with the editing site adenosine (A) highlighted in red. The black dotted line indicates 10% editing activity as a reference. See also Figures S10–S13.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Activity Assay, Standard Deviation, RNA Sequencing Assay, Mutagenesis

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A-I) Representative EMSA gel images of WT and ADAR1-associated mutants complex with GLI-V11, HT-V5, HT-V6, or HT-V16. (J) The secondary structures of GLI-V11, HT-V5, HT-V6, and HT-V16. (K-N) Calculation of binding affinity between WT or ADAR1-associated mutant with HT-V5 (K), HT-V16 (L), GLI-V11 (M), and HT-V6 (N) based on data from (A-I). The concentration of protein is serially diluted to 150, 120, 90, 60, and 30 nM. The concentration of each RNA variant is 5 nM. Bound and unbound fractions were quantified by densitometry. At least two times each experiment was repeated independently with similar results (A-I).

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Binding Assay, Mutagenesis, Concentration Assay, Variant Assay

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A-D) SDS-PAGE analysis of purified ADAR1 proteins after size-exclusion column purification, the truncation proteins without MBP tag were indicated. (F) Size-exclusion chromatography profiles of purified ADAR1 AGS-associated mutants as well as dimerization defect mutants with W1022A or D1023A mutation. Dashed lines indicate the elution peaks corresponding to the protein fractions.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: SDS Page, Purification, Size-exclusion Chromatography, Mutagenesis

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A) Overall RNA editing events observed in WT ADAR1, AGS-associated mutants, and the empty vector control groups from RNA-Seq analysis. Editing sites were selected based on a minimum of 5 reads and an editing rate exceeding 1%. (B) RNA expression levels of ADAR1, ADAR2, ADAR3, and GLI in each transfection group. Expression levels were normalized to GAPDH RNA. (C–D) Secondary structures of RNAs which were excluded from activity comparisons as the editing site was located outside dsRNA regions (B) or positioned on large bulges or near branching structures (D). The editing site A is highlighted in red. (E-F) The editing activity of WT ADAR1 and AGS-associated mutants was evaluated on the remaining dsRNAs (E) and with the G1007R mutant showing high background editing (>10%) for five RNA substrates (F) identified from RNA-Seq. Secondary structures of the RNAs are shown, with the editing site adenosine A highlighted in red. The black dotted line indicates 10% editing activity as a reference. (G) Validation of A-to-G RNA editing in GLI RNA by Sanger sequencing. The editing site is labeled, and the difference in peak change from adenosine (A) to guanosine (G, editing product) is related to the editing activity of each transfection group. (H) Normalized editing activity, as shown in (G). Values represent the mean of three independent experiments, with error bars indicating the standard deviation.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Plasmid Preparation, Control, RNA Sequencing Assay, RNA Expression, Transfection, Expressing, Activity Assay, Mutagenesis, Sequencing, Labeling, Standard Deviation

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A) Diagram of ADAR1 truncation mutants. (B) Editing activity of ADAR1 truncation mutants against three HT RNA variants. Secondary structures of RNA substrates are indicated on the top panel, with the editing site A highlighted in red. Values represent the mean of three independent experiments, with error bars representing standard deviation. 100 nM ADAR1 or truncation mutants and 25 nM each RNA were used. (C) The cryo-EM map of the ADAR1-HT complex 2. The structure is color-coded similarly as in . (D) Structural comparison of the HT-RNA in ADAR1-HT complex and ADAR1-HT complex 2. Monomer A from the two structures is aligned. In the ADAR1-HT complex, only the HT-RNA is shown for clarity, and colored in light blue (editing strand) and wheat (non-editing strand), respectively. The movement of HT-RNA is indicated with a red arrow. See also Figures S14-S15.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Activity Assay, Standard Deviation, Cryo-EM Sample Prep, Comparison

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A-C) Representative gels of the RNA editing activities of ADAR1 domain truncation mutants against HT-V5 (A), HT-V6 (B), and HT-V16 (C). 100 nM ADAR1 or mutants and 25 nM each RNA were used. (D) Representative gels of the RNA editing activities of ADAR1 or deaminase domain alone (D-801 and D-833) against GLI-V11, HT-V5, HT-V6, and HT-V16. The concentration of protein was indicated, the concentration of each RNA is 25 nM. (E) Normalized editing activity as shown in (D). (F) Representative EMSA gel images of R3D and deaminase domain alone (D-801 and D-833) complex with GLI-V11, HT-V5, HT-V6, or HT-V16. The concentration of R3D is serially diluted to 300, 200 and 100 nM, while the concentration of D-801 or D-833 is serially diluted to 3000, 1800, 900, and 450 nM. The concentration of each RNA variant is 25 nM. (G) Normalized RNA binding affinity as shown in (F). (H) Representative gels of the RNA editing activities of R3D and R3D-EAA mutants against the indicated RNAs. (I) Representative EMSA gel images of R3D complex with HT-V5, HT-V6, or HT-V16. The concentration of R3D is serially diluted to 300, 270, 240, 210, 180, 150, 120, 90, 60, and 30 nM. The concentration of each RNA variant is 10 nM. (J) Calculation of binding affinity between R3D and HT-V5, HT-V6, or HT-V16. Bound and unbound fractions were quantified by densitometry. (K) Representative EMSA gel images of R3D or R3D-EAA mutant complex with HT-V5. The protein concentration is serially diluted to 200, 160, 120, 80, and 40 nM. The concentration of HT-V5 is 10 nM. In (A-C), the deaminase domain alone (D-801 and D-833) with the MBP tag was used, while in (D) and (F), the MBP tag was removed. At least three times each experiment was repeated independently with similar results (A-D, and H). At least two times each experiment was repeated independently with similar results (F, I, and K).

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Concentration Assay, Activity Assay, Variant Assay, RNA Binding Assay, Binding Assay, Mutagenesis, Protein Concentration

Journal: bioRxiv

Article Title: Biochemical Profiling and Structural Basis of ADAR1-Mediated RNA Editing

doi: 10.1101/2025.01.02.631069

Figure Lengend Snippet: (A) Structural comparison of ADAR1-RNA complex 2 and ADAR2-RNA complex (PDB 8E0F), with the monomer A from two structures aligned. The RBD3 from monomer A of ADAR1-RNA complex 2 was colored in dark green, while the RBD2 from monomer B of ADAR2-RNA complex was colored in light green. (B) Structural comparison of ADAR1-RNA complex and ADAR2-RNA complex (PDB 8E0F), with the monomer A from two structures aligned. The structure of RBD3 of ADAR1 (PDB 7ZJ1) (colored in dark green) was aligned with the RBD2 (colored in light green) from monomer B of ADAR2-RNA complex.

Article Snippet: The gene of human ADAR1-p150 (pmGFP-ADAR1-p150) was ordered from Addgene (plasmid #117927).

Techniques: Comparison

Journal: bioRxiv

Article Title: Oncogenic Mutant p53 Sensitizes Non-Small Cell Lung Cancer Cells to Proteasome Inhibition via Oxidative Stress-Dependent Induction of Mitochondrial Apoptosis

doi: 10.1101/2024.02.22.581532

Figure Lengend Snippet: A. 20S proteasome activity was determined in the indicated cell lines using a fluorometric proteosome activity assay. All pairwise comparisons were made using student’s t-test. B. (Left) Basal levels of 20S proteasome activity in lysates of H1975 Control and p53 KO cells. ( Right ) Cell lysates of H1975-Control and H1975-p53KO (p53KO) cell lines generated using CRISPR/Cas9 were immunoblotted with p53 and GAPDH antibodies. C. The indicated cells were treated with vehicle or increasing concentrations of bortezomib (BTZ; left) or carfilzomib (CFZ; right) for 72 h, and cell viability was determined by the MTT assay. D . H1975-Control and H1975-p53KO cells were treated with vehicle (-) or BTZ (5 nM) for 48 h and cell viability was determined by Trypan-blue exclusion assay. E. ( Left ) H460 and H460-p53KO cells were treated with vehicle (-) or BTZ (5 nM) for 72 h and cell viability was determined by WST-1 assay. ( Right ) Cell lysates of H460 and H460-p53KO cells were immunoblotted with p53 and GAPDH antibodies. F. ( Left ) H460-p53KO cells stably expressing GFP or p53R273H cDNA were treated with increasing concentrations of BTZ for 72 h and cell viability was determined by WST-1 assay. ( Right ) Cell lysates of H460-p53KO cells stably expressing GFP or p53R273H cDNA were immunoblotted with p53 and GAPDH antibodies. * p <0.05, ** p <0.01, *** p <0.005, ns indicates p >0.05. Error bars indicate +/- 1.0 S.D.

Article Snippet: Lentiviral short-hairpin RNA (shRNA) vectors, shNOXA (TCRN0000338867), shNOXA#5 TCRN0000338864), shNRF2 (TCRN0000273494), shATF3 (TCRN0000329690), and shATF3#3 (TCRN000013568) were purchased from Sigma Aldrich (St. Louis, MO, USA). shControl (1864), GFP (35637) and p53R273H expression vectors (22934) were purchased from Addgene (Watertown, MA, USA).

Techniques: Activity Assay, Control, Generated, CRISPR, MTT Assay, Trypan Blue Exclusion Assay, WST-1 Assay, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Distinct non-coding RNA cargo of extracellular vesicles from M1 and M2 human primary macrophages

doi: 10.1101/2022.08.19.504493

Figure Lengend Snippet: Extracellular vesicles were isolated by size exclusion chromatography (SEC) and characterised by nanoparticle tracking analysis (NTA), electron microscopy, western blotting, and ELISA. a) Representative EV count measured by NTA (solid blue line; right Y axis) in the first eight SEC fractions overlaid with the protein concentration in 26 SEC fractions (dashed orange line; left Y axis) showing separation of EVs from soluble protein. b) Representative image of silver staining for the 26 SEC fractions. c) Total particle count measured by NTA in fractions 1-3, per million PBMC seeded. Wilcoxon matched-pairs signed rank test, p≤0.01, n=12. d) Median size of EVs from M1 and M2 cells, measured by NTA. Wilcoxon matched-pairs signed rank test, p≥0.01, n=12. e) Representative size profile of M1 (red) and M2 (blue) macrophage EVs measured by NTA. f) Transmission electron micrographs of EVs from M1 and M2 macrophages. g) ELISA for the canonical EV surface markers CD63 and HLA-A in the first five SEC fractions. h) Western blot for the luminal EV marker GAPDH and the negative EV marker Calnexin.

Article Snippet: Gene expression was determined using TaqMan Gene Expression Assays containing a FAM dye labelled probe (Applied Biosystems) duplexed with VIC dye labelled probe against GAPDH (Hs99999905_m1, Applied Biosystems) as housekeeping expression control.

Techniques: Isolation, Size-exclusion Chromatography, Electron Microscopy, Western Blot, Enzyme-linked Immunosorbent Assay, Protein Concentration, Silver Staining, Transmission Assay, Marker