Journal: Molecular cell

Article Title: Early Scanning of Nascent Polypeptides inside the Ribosomal Tunnel by NAC.

doi: 10.1016/j.molcel.2019.06.030

Figure Lengend Snippet: Figure 3. N-bNAC Probes Nascent Chains inside the Ribosomal Tunnel (A) cryo-EM reconstruction of C. elegans NAC∙60S. (Left) Cross section of the map to better visualize the NAC density in the tunnel is shown. (Right) Close-up view of the NAC density in the tunnel that reaches the constriction formed by the extension of uL22 is shown. The resolution of the NAC density inside the ribosomal tunnel is 6–8 A˚ . The map was low-pass filtered to 4.2-A˚ resolution. The residues R126 of uL22 and R28 of eL39 shown in the stick model were substituted with Bpa in the analyses shown in (C) and Figures S5A–S5C. (B) Site-specific photo-crosslinking of different Bpa-NAC variants (see Figure S4A) to stalled ribosome-nascent chain complexes (RNCs) car- rying in vitro translated, S35-labeled NCs with defined length (10–50 aas; translated substrate = human glucose-6-phosphate isomerase [GPI]). Autoradiograph images are shown. The peptidyl- tRNA (NC-tRNA) and position-specific NC-tRNA- NAC crosslinks (arrowheads) are indicated. (C) Engineered yeast 60S ribosomes carrying Bpa at tunnel-wall-lining position R126 of FLAG-uL22, indicated in (A), were photo-crosslinked to purified human and C. elegans (C.e.) NAC. WT-NAC as well as NAC deletion mutants lacking the N-terminal aNAC domain (C. elegans DN1–53 and human DN1–67) were used. FLAG, aNAC, and bNAC im- munoblots are shown. Red arrowheads indicate bNAC-specific crosslinks. Asterisk on FLAG blot marks a NAC-independent intra-60S ribosomal crosslink. See also Figures S5A–S5C. (D) Human NAC carrying Bpa at position 2 of bNAC (b-X2) was photo-crosslinked to puromycin- washed empty 60S or untreated full 80S. eL22, uL22, and eL39 immunoblots are shown. See also Figures S4 and S5.

Article Snippet: Antibodies used throughout this study were FLAG (F7425, Sigma), uL16 (AP17603a, Abgent), eL19 (sc-100830, Santa Cruz), uL22 (14121-1-AP, Proteintech), eL22 (25002-1-AP, Proteintech), eL39 (14990-1-AP, Proteintech), uL4 (sc-100838, Santa Cruz), Gapdh (sc-137179, Santa Cruz), Actin (sc-47778, Santa Cruz), human aNAC (40-1000, Invitrogen), human bNAC (ab66940, Abcam), Puromycin (MABE343, Merck), HSP4/GRP78 (PA5-22967, Thermo Scientific), Sec61a (sc-393182, Santa Cruz), and PDI3 (kind gift of Antony Page, University of Glasgow, Scotland (Eschenlauer and Page, 2003)).

Techniques: Cryo-EM Sample Prep, In Vitro, Labeling, Autoradiography, Western Blot

Journal: Molecular cell

Article Title: Early Scanning of Nascent Polypeptides inside the Ribosomal Tunnel by NAC.

doi: 10.1016/j.molcel.2019.06.030

Figure Lengend Snippet: Figure 5. Sensing of De Novo Synthesized Nascent Chains by NAC Ribosome binding of NAC is mediated by a ribosome-binding regulatory arm (N-aNAC) and a translation sensor domain (N-bNAC). N-aNAC directly contacts the ribosome close to the tunnel exit but also possesses a ribosome binding inhibitory element that interacts with eL19. The empty tunnel of idle and early-stage translating ribosomes is sensed by N-bNAC, which inserts deeply into the ribosomal tunnel up to the constriction formed by uL22. In the tunnel-inserted conformation, NAC blocks the premature, unproductive ribosome association of Sec61 and likely of other cotranslational protein biogenesis factors, including RAC and SRP (left, early state). During polypeptide elongation, N-bNAC senses short nascent chains and is partially pushed out of the ribosomal tunnel, which likely repositions the NAC domain outside the tunnel (dotted arrows) to facilitate the early recruitment of other protein biogenesis factors, like SRP (middle, intermediate state). Once the N-bNAC tail is completely pushed out of the tunnel, it can relocate to an alternate binding site on the ribosome surface involving eL22 and eL31 (right, late stage). At this stage, NAC may orchestrate cotranslational protein folding and targeting processes by regulating the specific binding of other chaperones and targeting factors. MTS, mitochondrial targeting sequence; SS, endoplasmic reticulum signal sequence; TMD, transmembrane domain.

Article Snippet: Antibodies used throughout this study were FLAG (F7425, Sigma), uL16 (AP17603a, Abgent), eL19 (sc-100830, Santa Cruz), uL22 (14121-1-AP, Proteintech), eL22 (25002-1-AP, Proteintech), eL39 (14990-1-AP, Proteintech), uL4 (sc-100838, Santa Cruz), Gapdh (sc-137179, Santa Cruz), Actin (sc-47778, Santa Cruz), human aNAC (40-1000, Invitrogen), human bNAC (ab66940, Abcam), Puromycin (MABE343, Merck), HSP4/GRP78 (PA5-22967, Thermo Scientific), Sec61a (sc-393182, Santa Cruz), and PDI3 (kind gift of Antony Page, University of Glasgow, Scotland (Eschenlauer and Page, 2003)).

Techniques: Synthesized, Binding Assay, Sequencing

Journal: Cell Reports Methods

Article Title: Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV

doi: 10.1016/j.crmeth.2022.100236

Figure Lengend Snippet:

Article Snippet: 155Gd_PD-1 , Fluidigm , Cat# 3155009B, RRID: AB_2811087.

Techniques: Virus, Recombinant, Electroporation, Antibody Labeling, Labeling, Software

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schematic illustration of US-triggered deep-tissue activation of SPINs to release immunomodulators. b Schematic illustration of sonodynamic activation of SPINs to debulk tumor, enhance tumor immunogenicity, and release immunomodulators in situ as well as synergetic action of IDO inhibition and PD-L1 blocking on enhancing antitumor immunity with alleviated irAEs relative to free-drug administration.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Activation Assay, In Situ, Inhibition, Blocking Assay

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Chemical structures of amphiphilic semiconducting polymeric modulators and schematic illustration of their self-assembly and surface modification to form SPINs. b The molar ratios of each component in different SPINs. c Zeta potentials and hydrodynamic sizes of different SPINs in 1× PBS buffer (pH = 7.4) ( n = 4). d Photographs of erythrocytes after incubation with 1× PBS buffer (negative control), 1% Triton X-100 (positive control), and 1× PBS buffer containing SPINs at the concentration of 100 µg/mL for 2 h, followed by centrifugation. e Hemolysis percentages of erythrocytes after incubation with SPINs at different concentrations for 2 h ( n = 4). f Schematic illustration of US irradiation of SPIN D2 solutions covered with a pork tissue. g ESR spectra of 1 O 2 for SPIN D2 (20 µg/mL) after US irradiation (1.2 W/cm 2 , 3 min) without or with coverage of pork tissues at different thicknesses. h Release profiles of aPD-L1 and NLG919 from SPIN D2 (40 µg/mL) after US irradiation for different time ( n = 4). i PD-L1/PD-1 binding activity assay after treatment with free aPD-L1 or SPIN D2 (40 µg/mL) with or without US irradiation ( n = 4). SPIN D2 – US versus SPIN D2 + US: P < 0.0001. Statistical significance was calculated via a two-tailed Student’s t test. *** P < 0.001. In ( g – i ), the power intensity of US irradiation was 1.2 W/cm 2 (1.0 MHz, 50% duty cycle). Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Modification, Incubation, Negative Control, Positive Control, Concentration Assay, Centrifugation, Irradiation, Binding Assay, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schedule for the establishment of primary and distant tumors, triple systemic injection of SPINs (0.2 mL, 0.6 mg/mL) via tail vein, US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min), and analysis of immune responses. b , c Relative tumor volumes of primary ( b ) and distant ( c ) tumors of Panc02 tumor-bearing C57BL/6 mice ( n = 6) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), or SPIN D2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). SPIN D2 + US versus drug + US: P < 0.0001 for primary tumors ( b ); SPIN D2 + US versus drug: P < 0.0001 for distant tumors ( c ). d Survival curves of Panc02 tumor-bearing C57BL/6 mice ( n = 10) receiving different treatments as indicated. e Schematic illustration of treatment of rechallenged tumor mouse models using SPINs. f Growths of rechallenged tumors in Panc02 tumor-bearing mice after injection of saline or SPIN D2 (0.2 mL, 0.6 mg/mL) with US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min) ( n = 5). Saline versus SPIN D2 + US: P < 0.0001. g The survival curves of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge ( n = 10). h Flow cytometry analysis of populations of effector memory T cells in the spleen of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge ( n = 4). Saline versus SPIN D2 + US: P = 0.0059. i Differentially expressed gene numbers in tumor tissues of mice after different treatments. j Relative expression of Carl , Hmgb1-ps1 , Hmgb1-ps2 , Cd80 , Cd86 , Cd40 , Pdcd1 , Cd3e , Cd8a , Ifng , Gzmb , Cxcl1 , Cxcl2 , Cxcl9 , Cxcl10 , Cxcl11 , Ccl4 , Ccl5 , Il1b , Il2 , Il6 , Il7 , Il15 , Ido1 , and Cd274 in tumors of Panc02 tumor-bearing mice after different treatments (the experiment was repeated independently five times with similar results). k Unsupervised hierarchical clustering of relative gene expression in tumors of Panc02 tumor-bearing C57BL/6 mice after different treatments ( n = 5). Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Injection, Irradiation, Flow Cytometry, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schematic of sono-immunotherapy of subcutaneous pancreatic mouse tumors covered with 5-cm tissue. b , c Relative tumor volumes of primary ( b ) and distant ( c ) tumors of Panc02 tumor-bearing C57BL/6 mice ( n = 5) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), SPIN 0 or SPIN D2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). The primary tumors were covered with 5-cm tissue under US irradiation. SPIN D2 + US versus SPIN 0 + US: P < 0.0001 for primary tumors ( b ); SPIN D2 + US versus SPIN 0 + US: P < 0.0001 for distant tumors ( c ). d Survival curves of Panc02 tumor-bearing C57BL/6 mice ( n = 10) after different treatments for 60 days. e Schematic of US-mediated deep-tissue sonodynamic therapy of orthotopic pancreatic rabbit tumors. f Radiolabeling stability of 131 I-SPIN 0 after storage in saline or 50% serum at 37 °C for different time ( n = 3). g , h SPECT imaging ( g ) and signal intensity ( h ) of orthotopic pancreatic rabbit tumors after systemic injection of 131 I-SPIN 0 (1.0 mL, 1.5 mg/mL) for different time ( n = 4). The white dotted circle indicated tumors. i Computed tomography (CT) imaging of orthotopic pancreatic rabbit tumors after systemic injection of saline or SPIN 0 (1.0 mL, 1.5 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 30 min). The white dotted circle indicated tumors. j Tumor volume of orthotopic pancreatic rabbit tumors ( n = 3) after treatments as indicated for different days. Saline + US versus SPIN 0 + US: P = 0.0108. k H&E staining images of orthotopic pancreatic rabbit tumors after different treatments. The experiment was repeated independently three times with similar results. l Survival curves of orthotopic pancreatic tumor-bearing rabbits ( n = 4) after different treatments for 20 days. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; * P < 0.05, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Injection, Irradiation, Radioactivity, Single Photon Emission Computed Tomography, Imaging, Computed Tomography, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a , b Flow cytometry analysis of percentages of CD3 + CD4 + Th cells ( a ), and CD3 + CD8 + CTLs ( b ) in blood of mice ( n = 4) at 30 day after systemic administrations of saline, SPIN 0 , SPIN D2 (0.2 mL, 1.2 mg/mL) or free-drug mixture (8 mg/kg body weight for NLG919 and aPD-L1) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). Saline – US versus drug − US: P = 0.0023; saline − US versus drug + US: P = 0.0006; drug + US versus SPIN D2 + US: P = 0.0071 for CD3 + CD4 + Th cells ( a ); saline − US versus drug − US: P = 0.0004; saline − US versus drug + US: P = 0.0001; drug + US versus SPIN D2 + US: P = 0.0093 for CD3 + CD8 + CTLs ( b ). c , d Flow cytometry analysis of percentages of CD3 + CD4 + Th cells ( c ), and CD3 + CD8 + CTLs ( d ) in spleen of mice ( n = 4) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0008; saline − US versus drug + US: P = 0.0005; drug + US versus SPIN D2 + US: P = 0.0015 for CD3 + CD4 + Th cells ( c ); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P = 0.0002; drug + US versus SPIN D2 + US: P = 0.0049 for CD3 + CD8 + CTLs ( d ). e Representative H&E staining images of liver after 30 days of treatments in different groups (white arrows indicate the infiltrated lymphocytes). The experiments were repeated independently three times with similar results. f Heatmap to show relative fold of cytokine levels in serum of mice after different treatments for 30 days relative to those in saline control group. g , h Serum levels of ALT ( g ) and AST ( h ) in mice ( n = 5) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0010; saline − US versus drug + US: P = 0.0020; drug + US versus SPIN D2 + US: P = 0.0054 for ALT ( g ); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P < 0.0001; drug + US versus SPIN D2 + US: P = 0.0013 for AST ( h ). i Summary comparison of the antitumor immunity and irAEs between SPIN D2 -mediated sono-immunotherapy and free-drug treatment. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Flow Cytometry, Irradiation, Staining, Two Tailed Test