Journal: Nature Metabolism

Article Title: PSD3 downregulation confers protection against fatty liver disease

doi: 10.1038/s42255-021-00518-0

Figure Lengend Snippet: Downregulation of endogenous PSD3 expression using siRNA in primary human hepatocytes cultured in 2D and 3D. For 2D culture, after attachment of cells in collagen-coated plates, cells were incubated with regular growth medium supplemented with 10 µM OA and transfected with negative control SCR siRNA or PSD3 siRNA for 48 h. a , b , Intracellular neutral fat content was visualized by ORO staining and quantified by Biopix iQ software v.2.3.1 in primary human hepatocytes carrying ( a ) the 186L allele ( n = 5) or ( b ) the 186T allele ( n = 4). Average PSD3 downregulation efficiency was ~80% as evaluated by quantitative retro transcription PCR analysed by the 2 − ΔΔCt method and western blotting for both donor types. For 3D culture of primary human hepatocytes, spheroids were generated by seeding 2,000 cells per well in a 96-well round-bottom flask, along with transfection mix in 100 µl of medium. For the generation of 186T allele spheroids, 5 nM of FMK-Z-VAD was added to support spheroid formation. After 24 h, additional growth medium was added to give a total volume of 200 µl per well. Fifty per cent of the total media was replenished with fresh media every 48 h. After 7 days of formation, spheroids were collected and 8-µM sections were subjected to ORO staining to visualize intracellular neutral fat content. c , d , Nuclei were stained with 4,6-diamidino-2-phenylindole and ORO staining was quantified by Image J, normalized to number of nuclei of primary human hepatocyte spheroids carrying ( c ) the 186L allele ( n = 4) and ( d ) the 186T allele ( n = 4). Average gene knockdown efficiency was ~50–60% as evaluated by quantitative retro transcription PCR analysed by the 2 −ΔΔCt method for both donor types. Cellular ATP levels (marker of viability) remained stable between the negative control SCR and PSD3 siRNA groups. Data are shown as mean ± s.d. of the reported independent experiments. Two-sided P values were calculated by Mann–Whitney non-parametric test comparing SCR siRNA versus PSD3 siRNA. RU, relative units; CNX, calnexin.

Article Snippet: In experimental conditions, 24 h after seeding, cells were transiently transfected with 30 nM SCR siRNA (AM4611; ThermoFisher Scientific), human PSD3 siRNA (mix of s23653, s23654 and s23655 for primary and Huh7 cells;, ThermoFisher Scientific), rat Psd3 siRNA (mix of s157480, s157481 and s157482 for McA-RH7777 cells; ThermoFisher Scientific) or human ARF6 siRNA (mix of s1565, s1566 and s1567 for Huh7 cells; ThermoFisher Scientific) by Lipofectamine 3000 transfection reagent (L3000-075; ThermoFisher Scientific) according to the manufacturer’s instructions, and grown in serum-free regular medium supplemented with 10 μM (primary cells), 50 μM (McA-RH7777) or 25 μM (Huh7) OA for 48 h. Intracellular neutral fat content was visualized by ORO staining and images were acquired using an Axio KS 400 Imaging System and AxioVision v.4.8 software (Zeiss) at ×100 magnification.

Techniques: Expressing, Cell Culture, Incubation, Transfection, Negative Control, Staining, Software, Western Blot, Generated, Knockdown, Marker, MANN-WHITNEY

Journal: Nature Metabolism

Article Title: PSD3 downregulation confers protection against fatty liver disease

doi: 10.1038/s42255-021-00518-0

Figure Lengend Snippet: Human hepatoma Huh7 cells were transiently transfected with negative control SCR siRNA, PSD3 siRNA or ARF6 siRNA. Forty-eight hours after transfection, the cell lysates were incubated with GGA3 PBD agarose beads that selectively isolate and pull down endogenous active ARF6 (ARF6–GTP). a , After precipitation, active ARF6–GTP was detected by immunoblotting using an anti-ARF6 antibody provided in the kit. Cells transfected with ARF6 siRNA were used as a control. The experiment was performed independently six times with similar results. Representative blot presented. b , Knockdown efficiency with ~60% reduction for PSD3 and ~75% for ARF6 as evaluated by quantitative retro transcription PCR analysed by the 2 − ΔΔCt method ( n = 6). c , Relative ARF6–GTP (active) calculated as GTP–ARF6/calnexin (CNX) ( n = 6). Data are shown as mean ± s.d. of the reported independent experiments. Two-sided P values calculated by Mann–Whitney non-parametric test. RU, relative units.

Article Snippet: In experimental conditions, 24 h after seeding, cells were transiently transfected with 30 nM SCR siRNA (AM4611; ThermoFisher Scientific), human PSD3 siRNA (mix of s23653, s23654 and s23655 for primary and Huh7 cells;, ThermoFisher Scientific), rat Psd3 siRNA (mix of s157480, s157481 and s157482 for McA-RH7777 cells; ThermoFisher Scientific) or human ARF6 siRNA (mix of s1565, s1566 and s1567 for Huh7 cells; ThermoFisher Scientific) by Lipofectamine 3000 transfection reagent (L3000-075; ThermoFisher Scientific) according to the manufacturer’s instructions, and grown in serum-free regular medium supplemented with 10 μM (primary cells), 50 μM (McA-RH7777) or 25 μM (Huh7) OA for 48 h. Intracellular neutral fat content was visualized by ORO staining and images were acquired using an Axio KS 400 Imaging System and AxioVision v.4.8 software (Zeiss) at ×100 magnification.

Techniques: Transfection, Negative Control, Incubation, Western Blot, Control, Knockdown, MANN-WHITNEY

Journal: Molecular Therapy Oncolytics

Article Title: Intratumoral delivery of a novel oncolytic adenovirus encoding human antibody against PD-1 elicits enhanced antitumor efficacy

doi: 10.1016/j.omto.2022.04.007

Figure Lengend Snippet: AdC68-spE1A-αPD-1 induced tumor cell death in vitro (A–I) HFL-1 (A), HCT-8 (B), A549 (C), Siha (D), NCI-H508 (E), Huh7 (F), MC38 (G), CT26.WT (H), and YUMM5.2 (I) were infected with adenoviruses at indicated MOIs for 72 h. The viability of different cells was measured by CCK-8 assay. Data are shown as mean ± SEM.

Article Snippet: Human embryonic kidney (HEK) 293 cells, human normal lung fibroblast cell line (HFL-1), human colon adenocarcinoma cell line (HCT-8), human colorectal carcinoma cell line (NCI-H508), human hepatocarcinoma cell line (Huh7), and murine melanoma cell line (YUMM5.2) were purchased from ATCC (Manassas, VA, USA).

Techniques: In Vitro, Infection, CCK-8 Assay

Journal: Molecular Therapy Oncolytics

Article Title: Intratumoral delivery of a novel oncolytic adenovirus encoding human antibody against PD-1 elicits enhanced antitumor efficacy

doi: 10.1016/j.omto.2022.04.007

Figure Lengend Snippet: AdC68-spE1-αPD-1 induced tumor cell death by activating apoptotic and autophagic pathways NCI-H508 and Huh7 cells were infected with the indicated adenoviruses at 20 MOIs. The cells were collected at 24 and 48 hpi to conduct the western blot assay under denaturing condition. The proteins p62, LC3 Ι / Π , cleaved caspase-3, cleaved PARP, p53, p21, Mcl-1, CyclinD1, and CyclinE1 were analyzed. Actin was used as a loading control.

Article Snippet: Human embryonic kidney (HEK) 293 cells, human normal lung fibroblast cell line (HFL-1), human colon adenocarcinoma cell line (HCT-8), human colorectal carcinoma cell line (NCI-H508), human hepatocarcinoma cell line (Huh7), and murine melanoma cell line (YUMM5.2) were purchased from ATCC (Manassas, VA, USA).

Techniques: Infection, Western Blot

Journal: PLoS Pathogens

Article Title: Characterization of miR-122-independent propagation of HCV

doi: 10.1371/journal.ppat.1006374

Figure Lengend Snippet: (A) Subgenomic HCV replicon RNA was electroporated into Huh7-122KO and Huh7-122KOR cells, or into Huh7-122KO cells together with control- or miR-122-mimic, and G418-resistant colonies were stained with crystal violet at 21 days post-transduction. (B) Expression of miR-122 in Huh7-122KO cells electroporated with either control-mimic or miR-122-mimic at 72 h post-electroporation. Relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control. (C) Each of the three clones derived from each type of replicon cells was subjected to qRT-PCR after extraction of total RNA (top) and to immunoblotting by using anti-NS5A and β-actin (middle). The relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control (bottom). (D) Intracellular HCV-RNA replication in Huh7-122KO-SGR cells (#1, #3, #5) and Huh7-122KOR-SGR cells (#1, #5, #6) in the presence of 20 nM of either LNA-control or LNA-miR122 was determined by qRT-PCR. (E) The Ago2 complex was immunoprecipitated from Huh7-122KO-SGR#1 and Huh7-122KOR-SGR#1 cells by using either anti-IgG or anti-Ago2 mouse antibody. The HCV-RNA associated with Ago2 was determined by qRT-PCR and Ago2 was detected by immunoblotting. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P < 0.05; **P < 0.01) versus the results for the control.

Article Snippet: Human hepatocellular carcinoma cell line Huh7, human embryonic kidney cell line 293T and human endometrial adenocarcinoma cell line Hec1B were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB0403, JCRB9068 and JCRB1193).

Techniques: Control, Staining, Transduction, Expressing, Electroporation, Quantitative RT-PCR, Clone Assay, Derivative Assay, Extraction, Western Blot, Immunoprecipitation, Standard Deviation

Journal: PLoS Pathogens

Article Title: Characterization of miR-122-independent propagation of HCV

doi: 10.1371/journal.ppat.1006374

Figure Lengend Snippet: (A) HCV was inoculated into Huh7-122KO and Huh7-122KOR cells at an MOI of 3, and intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) were determined by qRT-PCR and focus formation assay, respectively. (B) HCV-RNA replication was inhibited by the treatment with IFNα, BILN, BMS790052, PSI7977 and anti-CD81 antibody. (C) HCV replication in Huh7-122KO cells was resistant to treatment with an miR-122 inhibitor, LNA (HCV-RNA replication: left; infectious titer: right). Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P < 0.05; **P < 0.01) versus the results for the control.

Article Snippet: Human hepatocellular carcinoma cell line Huh7, human embryonic kidney cell line 293T and human endometrial adenocarcinoma cell line Hec1B were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB0403, JCRB9068 and JCRB1193).

Techniques: Quantitative RT-PCR, Tube Formation Assay, Standard Deviation, Control

Journal: PLoS Pathogens

Article Title: Characterization of miR-122-independent propagation of HCV

doi: 10.1371/journal.ppat.1006374

Figure Lengend Snippet: (A) Huh7-122KO (left) and Huh7-122KOR cells (right) infected with HCV at an MOI of 10 or 0.1, respectively, were fixed at 72 hpi and stained with antibodies to NS5A protein (green). The boxes in the top panels were magnified (bottom panel). Numbers of foci per infectious particle (B) and numbers of cells per focus (C, average of 75 foci) in Huh7-122KO (white bar) and Huh7-122KOR cells (gray bar) upon infection with HCV are shown. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P < 0.01) versus the results for the control. (D) Focus formation in Huh7-122KO (left) and Huh7-122KOR cells (right) infected with HCV at an MOI of 0.1 or 10, respectively. Each cell was fixed at the indicated time post-infection and stained with appropriate antibodies to dsRNA (red) and NS5A (green). Cell nuclei were stained with DAPI (blue).

Article Snippet: Human hepatocellular carcinoma cell line Huh7, human embryonic kidney cell line 293T and human endometrial adenocarcinoma cell line Hec1B were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB0403, JCRB9068 and JCRB1193).

Techniques: Infection, Staining, Standard Deviation, Control

Journal: PLoS Pathogens

Article Title: Characterization of miR-122-independent propagation of HCV

doi: 10.1371/journal.ppat.1006374

Figure Lengend Snippet: (A) HCV was inoculated into Huh7-122KO (#2), Huh7-122KO-cured (#3 or #5), or 751-122KO (#1, #2 or #3) cells, and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined by qRT-PCR and focus formation assay, respectively, at 72 hpi. (B) Infectious titer in the culture medium on serial passage of each 751-122KO cell clone. (C) HCV and HCV 122KO were inoculated into 751-122KO and Huh7.5.1 cells and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined at 72 hpi. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P < 0.05; **P < 0.01) versus the results for the control. (D) Nuclear translocation of IPS-GFP (arrows) in Huh7.5.1 and 751-122KO cells upon infection with HCV and HCV 122KO (left panels). The numbers of cells having translocated GFP in their nuclei through propagation of HCV were counted and the infection ratios at 24 hpi (right top) and 72 hpi (right bottom) were determined.

Article Snippet: Human hepatocellular carcinoma cell line Huh7, human embryonic kidney cell line 293T and human endometrial adenocarcinoma cell line Hec1B were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB0403, JCRB9068 and JCRB1193).

Techniques: Quantitative RT-PCR, Tube Formation Assay, Standard Deviation, Control, Translocation Assay, Infection

Journal: PLoS Pathogens

Article Title: Characterization of miR-122-independent propagation of HCV

doi: 10.1371/journal.ppat.1006374

Figure Lengend Snippet: (A) Mutation of G28A in the 5’UTR of HCV was identified in all independently isolated HCV propagated in the three 751-122KO cell clones (751-122KO#1~#3). Arrows indicate the position of nt28 in the 5’UTR of HCV. Each RNA base is represented as a colored peak: A, green; U, red; G, black; and C, blue. (B) Frequency and distribution of SNV in HCV independently cultured in Huh7.5.1 (JFH-P5; top) and 751-122KO cell clones (bottom). Six independently isolated HCV 122KO viruses were obtained from three wells for each of two 751-122KO cell clones (751-122KO#1 and #2). Each sequence read was mapped to pHH-JFH1-E2p7NS2mt. Arrows indicate the detected substitutions.

Article Snippet: Human hepatocellular carcinoma cell line Huh7, human embryonic kidney cell line 293T and human endometrial adenocarcinoma cell line Hec1B were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB0403, JCRB9068 and JCRB1193).

Techniques: Mutagenesis, Isolation, Clone Assay, Cell Culture, Sequencing

Journal: PLoS Pathogens

Article Title: Characterization of miR-122-independent propagation of HCV

doi: 10.1371/journal.ppat.1006374

Figure Lengend Snippet: (A) Infectious titer in the culture medium on serial passage of 751-122KO#1 or Huh7.5.1 cells. Red circles indicate the passage in 751-122KO cells, and the other circles indicate the passage in Huh7.5.1 cells. Three independent passages (#4–6, #4–8, #7–8) are shown. (B) Nuclear translocation of IPS-GFP (arrows) in Huh7.5.1 and 751-122KO cells upon infection with Con1C3/JFH and Con1C3/JFH 122KO . (C) Con1C3/JFH and Con1C3/JFH 122KO were inoculated into 751-122KO#1 and Huh7.5.1 cells, and the levels of intracellular HCV-RNA replication were determined. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P < 0.01) versus the results for the control. (D) 293T-CLDN cells infected with either Con1C3/JFH or Con1C3/JFH 122KO were treated with IFNα and BILN and then the intracellular HCV-RNA level was determined at 12, 24 and 48 hpi. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P < 0.01) versus the results for the control.

Article Snippet: Human hepatocellular carcinoma cell line Huh7, human embryonic kidney cell line 293T and human endometrial adenocarcinoma cell line Hec1B were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB0403, JCRB9068 and JCRB1193).

Techniques: Translocation Assay, Infection, Standard Deviation, Control

Journal: PLoS Pathogens

Article Title: Characterization of miR-122-independent propagation of HCV

doi: 10.1371/journal.ppat.1006374

Figure Lengend Snippet: (A) Infectious titers in the culture media upon serial passage of three clones each of 751-122KO-shlacZ, 751-122KO-shXrn1 or 751-122KO-shXrn1/Xrn2 cells (#1~#3). (B) Colony formation in Huh7-122KO and Huh7-122KOR cells upon electroporation with the wild type and G28A-mutated JFH-SGR RNA (upper). The numbers of colonies of each cell type were quantified (bottom). Culture supernatants of 751-122KO and Huh7.5.1 cells co-electroporated with the wild type and G28A-mutated JFH1 HCV-RNA were harvested at each passage, and the infectious titers (C) and the sequences of viral RNA (D) were determined. Each RNA base is represented as a colored peak: A, green; U, red; G, black; and C, blue. Variations in the wild type and G28A mutant at passages 1 and 4 are shown (D, bottom). Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P < 0.05; **P < 0.01) versus the results for the control.

Article Snippet: Human hepatocellular carcinoma cell line Huh7, human embryonic kidney cell line 293T and human endometrial adenocarcinoma cell line Hec1B were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB0403, JCRB9068 and JCRB1193).

Techniques: Clone Assay, Electroporation, Mutagenesis, Standard Deviation, Control

Journal: PLoS Pathogens

Article Title: Characterization of miR-122-independent propagation of HCV

doi: 10.1371/journal.ppat.1006374

Figure Lengend Snippet: (A) Intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) of Huh7-122KO and Huh7-122KOR cells infected with either HCV or HCV 122KO in the presence of either control-LNA or LNA-miR-122 were determined at 72 hpi. (B) Ago2 complexes in 751-122KO and Huh7.5.1 cells infected with HCV were immunoprecipitated by either anti-IgG or anti-Ago2 mouse antibody at 12 dpi. Levels of Ago2 and HCV-RNA in the precipitates were determined by immunoblotting and qRT-PCR, respectively. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P < 0.05; **P < 0.01) versus the results for the control.

Article Snippet: Human hepatocellular carcinoma cell line Huh7, human embryonic kidney cell line 293T and human endometrial adenocarcinoma cell line Hec1B were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB0403, JCRB9068 and JCRB1193).

Techniques: Infection, Control, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: PLoS Pathogens

Article Title: Characterization of miR-122-independent propagation of HCV

doi: 10.1371/journal.ppat.1006374

Figure Lengend Snippet: Huh7 cells (5x10 5 cells) were infected with HCV or HCV 122KO and harvested at 72 hpi for polysome analysis. A254 absorbance (top), distribution of HCV-RNA (middle) and β-actin mRNA levels (bottom) were determined.

Article Snippet: Human hepatocellular carcinoma cell line Huh7, human embryonic kidney cell line 293T and human endometrial adenocarcinoma cell line Hec1B were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB0403, JCRB9068 and JCRB1193).

Techniques: Infection