Journal: International Journal of Nanomedicine

Article Title: Short communication: selective cytotoxicity of curcumin on osteosarcoma cells compared to healthy osteoblasts

doi: 10.2147/IJN.S55505

Figure Lengend Snippet: Decreased osteosarcoma cell densities after 24 hours of curcumin treatment. Notes: * P <0.05 compared to 5 μM; # P <0.01 compared to 10 μM; ** P <0.005 compared to 0 μM (ie, dimethylsulfoxide only) sample. Data are expressed as standard error of the mean and N=3.

Article Snippet: MG-63 osteosarcoma cells (ATCC-CRL-1427), Eagle’s Minimum Essential Medium, dimethyl sulfoxide (DMSO), and phosphate buffered saline were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques:

Journal: EMBO Reports

Article Title: An hepatitis B and D virus infection model using human pluripotent stem cell-derived hepatocytes

doi: 10.1038/s44319-024-00236-0

Figure Lengend Snippet: ( A ) HDV infection (MOI = 5 Int. Units/cell) of HLCs incubated with or without 500 nM entry inhibitor bulevirtide (BLV) was assessed by immunofluorescence staining (IF) against the HDV antigen (HDAg, green) 5 days post-infection (p.i.). GT: genotype. Scale bar = 200 μm. Images are representative of three independent HLC differentiations. ( B , C ) HLCs, differentiated (d)HepaRG, and Huh7 NTCP cells were infected with HDV (MOI = 5 Int. Units/cell) with or without 500 nM BLV. HDV infection was analyzed by ( B ) counting HDAg-positive cells using CellProfiler or by ( C ) quantifying HDV genome copies by RT-qPCR. IU infectious unit. Dashed line: limit of quantification (LOQ). N = 5 biological replicates from two independent experiments. ( D , E ) HLCs were transduced with or without AAV6 (MOI = 10 4 viral genomes, vg/cell) encoding for YFP or NTCP two days before HDV infection. ( D ) NTCP was stained with Atto-MyrB-565 (magenta) two days post transduction. Scale bar = 50 μm. (E) [ 3 H]-taurocholate uptake was determined 2 days post transduction in HLCs ( + YFP or NTCP) or HepG2 cells ( + NTCP) treated or not with 1 μM BLV. CPM[ 3 H]: Tritium scintillation counts per minute. n.a: not assessed. N = 6 biological replicates from two independent HLC differentiations, N = 4 biological replicates (HepG2) from two independent experiments. ( F , G ) Two days post transduction, mature HLCs YFP/NTCP were infected with HDV (MOI = 5) and analyzed by ( F ) counting HDAg-positive cells or ( G ) quantifying HDV genome copies 5 days p.i. N = 6 biological replicates from two independent HLC differentiations. ( H ) HLCs were infected with the indicated HDV genotype (MOI = 15 Int. Units/cell for GTs 1 T, 1E, 4, 6, 7, 8; MOI = 30 Int. Units/cell for GTs 3 & 5) and 5 days p.i., HDV genome copies were quantified using RT-qPCR. MOIs used were based on different infectious titers of the genotypes obtained on Huh7 NTCP cells to reach similar infection efficiency of HLCs. N = 3 biological replicates. ( I – K ) HDV infection (MOI = 5) of HLCs incubated with or without 500 nM BLV was analyzed over time by quantifying (I) HDV genome copies ( N = 4 biological replicates from two independent HLC differentiations). ( J ) HDV genomic RNA (gRNA) and antigenomic RNA (agRNA) ( N = 6 biological replicates from two independent HLC differentiations), as well as ( K ) large (L-HDAg) and small (S-HDAg) HDV antigen expression by Western blot. n.d.: not detected. Data information: In ( B , C , E – J ) data are presented as mean ± SD. ( B , C ) Statistical analysis was performed by multiple comparisons of ordinary one-way ANOVA. Statistical significance in ( B ) was tested between HDV-infected DMSO-treated HLC and dHepaRG ( P = 0.1384); between HDV-infected DMSO-treated HLC and Huh7 NTCP ( P < 0.0001) and between HDV-infected DMSO-treated dHepaRG and Huh7 NTCP ( P < 0.0001). Statistical significance in ( C ) was tested between HDV-infected DMSO-treated HLC and dHepaRG ( P = 0.9402); between HDV-infected DMSO-treated HLC and Huh7 NTCP ( P = 0.0022) and between HDV-infected DMSO-treated dHepaRG and Huh7 NTCP ( P = 0.0037). Statistical significance in ( E ) was tested between DMSO-treated HLC and HLC YFP (P > 0.9999), between DMSO-treated HLC and HLC NTCP ( P = 0.0003), between DMSO-treated HLC YFP and HLC NTCP ( P = 0.0002) and between DMSO-treated HepG2 and HepG2 NTCP ( P < 0.0001) by multiple comparisons of two-way ANOVA. ( F , G ) Statistical analysis was performed by multiple comparisons of ordinary one-way ANOVA. Statistical significance in ( F ) was tested between HDV-infected HLC Mock and HLC YFP ( P = 0.977) and between HDV-infected HLC mock and HLC NTCP ( P = 0.0015). Statistical significance in ( G ) was tested between HDV-infected HLC mock and HLC YFP ( P = 0.9713) and between HDV-infected HLC Mock and HLC NTCP ( P < 0.0001). Statistical significance in ( H ) was tested among the HLC infected with different HDV genotypes ( P < 0.0001) by ordinary one-way ANOVA. Statistical significance in ( I ) was tested between day 1- and day 3-harvested HDV-infected HLC ( P = 0.0464) by an unpaired two-tailed t test. Statistical significance in ( J ) was tested between day 1- and day 3-harvested HDV-infected HLC ( P = 0.0129) by an unpaired two-tailed t test. **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05, n.s. non-significant. .

Article Snippet: For the secondary infection, 1 × 10 5 Huh7 NTCP cells were seeded in 24-well plates and inoculated with approximately one-fifth of the culture supernatant from HDV-infected HLCs in DMEM containing 4% PEG and 2% DMSO (Carl Roth) after 24 h. 16 to 24 h post-infection, cells were washed twice with PBS and replenished with fresh DMEM supplemented with 2% DMSO.

Techniques: Infection, Incubation, Immunofluorescence, Staining, Quantitative RT-PCR, Transduction, Expressing, Western Blot, Two Tailed Test

Journal: EMBO Reports

Article Title: An hepatitis B and D virus infection model using human pluripotent stem cell-derived hepatocytes

doi: 10.1038/s44319-024-00236-0

Figure Lengend Snippet: ( A ) HLCs were infected with HDV (MOI = 5 Int. Units/cell) and the next day transduced with AAV6-YFP or AAV6-HBsAg. SN: supernatant. Nine days post transduction, HLCs were stained for HBsAg (red) and nuclei (blue). Images are representative of two independent differentiations. Scale bars = 50 μm. ( B ) HBsAg was quantified in the supernatant by ELISA collected at the end of indicated time periods. Int. Unit international unit. N = 6 biological replicates from two independent HLC differentiations. ( C ) Progeny HDV from HLCs harvested at the indicated time points was diluted 1:5 and used to infect Huh7 NTCP cells with or without 500 nM BLV. Infected Huh7 NTCP cells were fixed and stained for HDAg to quantify HDV infections. IU infectious unit. N = 6 biological replicates from 2 independent experiments. ( D ) HLCs were infected with HDV (MOI = 5 Int. Units/cell), transduced with AAV6-HBsAg and incubated with or without 500 nM BLV (between D0-D1 p.i.) or 2 μM Lonafarnib (LNF; between D0-D5 p.i.). HDV infection was quantified by counting HDAg-positive HLCs five days p.i. N = 6 biological replicates from two independent HLC differentiations. ( E ) The supernatant from these HLCs was diluted 1:5 to infect Huh7 NTCP cells which were analyzed for HDV infection by HDAg staining five days p.i. N = biological replicates. N = 6 biological replicates from two independent HLC differentiations. ( F ) Wild-type HLCs were infected with HDV (MOI = 5 Int. Units/cell) and the next day transduced with AAV6-HBsAg. Two days post-infection, they were dissociated and co-cultured with ZsGreen expressing HLCs in the presence or absence of BLV. Eight days later, cells were fixed, stained, and imaged for HDAg (magenta), ZsGreen (cyan) and nuclei (DAPI, blue). Scale bar = 50 μm. ( G ) Experimental setup. HLCs were infected with HDV (MOI = 5 Int. Units/cell) and the next day transduced with AAV6-HBsAg. After removal of the inoculum on day 2 p.i., HLCs were incubated with drugs, which were replenished every four days. Ten days p.i., HLCs were fixed to analyze HDV infections and their culture supernatant was harvested for titration of HDV progenies on Huh7 NTCP cells. ( H ) Relative HDV infection events normalized to vehicle DMSO-treated cells were quantified by counting HDAg-positive HLCs 10 days p.i. N = 8 biological replicates from three independent HLC differentiations. ( I ) The supernatant from HLCs was diluted 1:5 to infect Huh7 NTCP cells, which were then analyzed for HDV infection by HDAg staining 5 days p.i. N = biological replicates. N = 8 biological replicates from three independent HLC differentiations. Data information: In ( B – E , H , I ) data are presented as mean ± SD and statistical analysis was performed by multiple comparisons of ordinary one-way ANOVA. Statistical significance in ( B ) was tested between HLC Mock and HLC HBsAg (days 3–6; P < 0.0001) and between HLC YFP and HLC HBsAg (days 3–6; P < 0.0001). Statistical significance in ( C ) was tested between HLC YFP and HLC HBsAg (days 3–6; P = 0.0010) and between HLC HBsAg and BLV-treated HLC HBsAg (days 3–6; P = 0.0010). Statistical significance in ( D ) was tested between DMSO- and LNF-treated HDV-infected HLC ( P = 0.0021) and between DMSO- and BLV-treated HDV-infected HLC ( P = 0.0003). Statistical significance in ( E ) was tested between DMSO- and LNF-treated HDV-infected HLC ( P < 0.0001) and between DMSO- and BLV-treated HDV-infected HLC ( P < 0.0001). Statistical significance in ( H ) was tested between DMSO- and 500 nM BLV-treated HDV-infected HLC ( P = 0.0071), between DMSO- and 10 nM BLV-treated HDV-infected HLC ( P = 0.3508), between DMSO- and 2 μM LNF-treated HDV-infected HLC ( P = 0.8643) and between DMSO- and 20 nM LNF-treated HDV-infected HLC ( P = 0.9944). Statistical significance in ( I ) was tested between DMSO- and 500 nM BLV-treated HDV-infected HLC ( P < 0.0001), between DMSO- and 10 nM BLV-treated HDV-infected HLC ( P < 0.0001), between DMSO- and 2 μM LNF-treated HDV-infected HLC ( P < 0.0001) and between DMSO- and 20 nM LNF-treated HDV-infected HLC ( P < 0.0001). **** P < 0.0001; *** P < 0.001; ** P < 0.01, n.s. non-significant. .

Article Snippet: For the secondary infection, 1 × 10 5 Huh7 NTCP cells were seeded in 24-well plates and inoculated with approximately one-fifth of the culture supernatant from HDV-infected HLCs in DMEM containing 4% PEG and 2% DMSO (Carl Roth) after 24 h. 16 to 24 h post-infection, cells were washed twice with PBS and replenished with fresh DMEM supplemented with 2% DMSO.

Techniques: Infection, Transduction, Staining, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture, Expressing, Titration

Journal: EMBO Reports

Article Title: An hepatitis B and D virus infection model using human pluripotent stem cell-derived hepatocytes

doi: 10.1038/s44319-024-00236-0

Figure Lengend Snippet: Reagents and tools table

Article Snippet: For the secondary infection, 1 × 10 5 Huh7 NTCP cells were seeded in 24-well plates and inoculated with approximately one-fifth of the culture supernatant from HDV-infected HLCs in DMEM containing 4% PEG and 2% DMSO (Carl Roth) after 24 h. 16 to 24 h post-infection, cells were washed twice with PBS and replenished with fresh DMEM supplemented with 2% DMSO.

Techniques: Recombinant, Immunofluorescence, Western Blot, Sequencing, Membrane, Knock-Out, Transfection, Reverse Transcription, SYBR Green Assay, Protease Inhibitor, Diagnostic Assay, Software, Microscopy, Imaging

Journal: International Journal of Molecular Sciences

Article Title: S mall M olecule Ar ranged T hermal P roximity C o a ggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses

doi: 10.3390/ijms23105605

Figure Lengend Snippet: Melting curve data of the CETSA experiments shown in Figure 1 (n: number of replicates). Thermal shifts induced by the inhibitors are reported as Δ T m to DMSO as the vehicle control.

Article Snippet: 40 μL of 10 mM stock solutions of the three different kinase inhibitors AMG-548 (Tocris, cat. no. 3920), SB203580 (Tocris, cat. no. 1202), or ERK 11e (VX-11e; Tocris, cat. no. 4465) in dimethyl sulfoxide (DMSO) were added separately to three of the aliquots to obtain a final concentration of 20 μM per compound.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: S mall M olecule Ar ranged T hermal P roximity C o a ggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses

doi: 10.3390/ijms23105605

Figure Lengend Snippet: ITDR-CETSA experiments for a compound concentration range between 0.3 nM and 20 µM as well as without compound (only DMSO) at a constant treatment temperature of 51 °C. ( a ) Immunoblots for one of the replicates of ITDR-CETSA experiments for living cells and cell extract, respectively. ( b ) Dose–response curves (solid lines) derived from integrated, normalized, and fitted signal intensities from the immunoblots. Datapoints and error bars represent means over the four replicates (n = 4) +/− one standard deviation. The fitted pEC50 values are summarized in . Data for ERK 11e are shown in .

Article Snippet: 40 μL of 10 mM stock solutions of the three different kinase inhibitors AMG-548 (Tocris, cat. no. 3920), SB203580 (Tocris, cat. no. 1202), or ERK 11e (VX-11e; Tocris, cat. no. 4465) in dimethyl sulfoxide (DMSO) were added separately to three of the aliquots to obtain a final concentration of 20 μM per compound.

Techniques: Concentration Assay, Western Blot, Derivative Assay, Standard Deviation