Journal: Bioscience Reports

Article Title: Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells

doi: 10.1042/BSR20190477

Figure Lengend Snippet: ( A ) HepG2 cells were incubated with 30 μM berberine at 37°C for 0.5, 1, 2, 4, or 6 h, respectively. ( B ) HepG2 cells were incubated with 0.9, 3, 9, 30, 90, or 300 μM berberine at 37°C for 0.5 h, respectively. ( C ) HepG2 cells were incubated with 0.9, 3, 9, 30, 90, or 300 μM berberine at 4 or 37°C for 0.5 h, respectively. After incubation, the cells were washed three times with chilled PBS and harvested by trypsinisation. The cells were stored in 0.5 ml chilled PBS and the accumulated berberine in the cells was semi-quantified by flow cytometry analysis with excitation at 488 nm and emission at 530 nm.

Article Snippet: The concentration of berberine and Rho123 (a organic cation used to reflex the MMP in the study) in the cells were semi-quantified by flow cytometry analysis (Becton-Dickenson FACSCaliburTM system, New Jersey, U.S.A.) according to a previous report [ ].

Techniques: Incubation, Flow Cytometry

Journal: Bioscience Reports

Article Title: Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells

doi: 10.1042/BSR20190477

Figure Lengend Snippet: ( A ) HepG2 cells were incubated with HBSS or HBSS-K containing 10 μM berberine at 37°C for 0.5 h, respectively. ( B ) HepG2 cells were incubated with 10 μM berberine or in the presence of cimetidine (0.3, 1 mM, Cim) or rifampicin (10 μM, Rif) at 37°C for 0.5 h, respectively. After incubation, the cells were washed three times with chilled PBS and harvested by trypsinisation. The cells were stored in 0.5 ml chilled PBS and the accumulated berberine in the cells was quantified by flow cytometry analysis with excitation at 488 nm and emission at 530 nm. ** indicates P <0.01 versus control or between 0.3 or 1 mM cimetidine-treated groups.

Article Snippet: The concentration of berberine and Rho123 (a organic cation used to reflex the MMP in the study) in the cells were semi-quantified by flow cytometry analysis (Becton-Dickenson FACSCaliburTM system, New Jersey, U.S.A.) according to a previous report [ ].

Techniques: Incubation, Flow Cytometry, Control

Journal: Bioscience Reports

Article Title: Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells

doi: 10.1042/BSR20190477

Figure Lengend Snippet: HepG2 cells were pre-incubated with drug-free HBSS or HBSS containing 0.125, 0.25, 0.5, 1, or 2 μM CCCP at 37°C for 20 min, then equal volume of HBSS containing 20 μM berberine or 6 μg/ml Rho123 was added and the cells were further incubated at 37°C for 0.5 h. After incubation, the cells were washed three times with chilled PBS and harvested by trypsinisation. The cells were stored in chilled PBS and the concentration of berberine in the cells was quantified by flow cytometry analysis with excitation at 488 nm and emission at 530 nm. * indicates P <0 . 05 while ** indicates P <0.01 versus groups that treated without CCCP.

Article Snippet: The concentration of berberine and Rho123 (a organic cation used to reflex the MMP in the study) in the cells were semi-quantified by flow cytometry analysis (Becton-Dickenson FACSCaliburTM system, New Jersey, U.S.A.) according to a previous report [ ].

Techniques: Incubation, Concentration Assay, Flow Cytometry

Journal: Bioscience Reports

Article Title: Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells

doi: 10.1042/BSR20190477

Figure Lengend Snippet: HepG2 cells were incubated with 10 μM berberine and with or without 0.125 μM CCCP, 0.3 or 1 mM cimetidine (Cim), or the HBSS-K solution 37°C for 0.5 h, respectively. After incubation, the cells were washed three times with chilled PBS and harvested by trypsinisation. The cells were stored in 0.5 ml chilled PBS and the concentration of berberine in the cells was quantified by flow cytometry analysis with excitation at 488 nm and emission at 530 nm. ( A ) Synergistic inhibition of CCCP and HBSS-K on berberine uptake in the HepG2 cells; ( B ) synergistic inhibition of HBSS-K and cimetidine on berberine uptake in the HepG2 cells; ( C ) influence of CCCP on the inhibition of HBSS-K in terms of berberine uptake in the HepG2 cells; ( D ) inhibition of the combination of CCCP, HBSS-K, and cimetidine on berberine uptake in the HepG2 cells.* indicates P <0 . 05 while ** indicates P <0.01 versus control or between groups indicated by the lines.

Article Snippet: The concentration of berberine and Rho123 (a organic cation used to reflex the MMP in the study) in the cells were semi-quantified by flow cytometry analysis (Becton-Dickenson FACSCaliburTM system, New Jersey, U.S.A.) according to a previous report [ ].

Techniques: Incubation, Concentration Assay, Flow Cytometry, Inhibition, Control

Journal: Bioscience Reports

Article Title: Mitochondrial membrane potential played crucial roles in the accumulation of berberine in HepG2 cells

doi: 10.1042/BSR20190477

Figure Lengend Snippet: HepG2 cells were pre-incubated with 10 μM berberine at 37°C for 2 h, then the cells were incubated with drug-free HBSS or HBSS containing CCCP (0.25 μM), or verapamil (100 μM), or CCCP (0.25 μM) plus verapamil (100 μM) at 37°C for 0.5, 1, 2, 4 h, respectively. After incubation, the cells were washed three times with chilled PBS and harvested by trypsinisation. The cells were stored in 0.5 ml chilled PBS and the accumulated berberine in the cells was quantified by flow cytometry analysis with excitation at 488 nm and emission at 530 nm. The amount of berberine was normalised to that at zero time point. * indicates P <0.05 while ** indicates P <0.01 versus verapamil treated groups.

Article Snippet: The concentration of berberine and Rho123 (a organic cation used to reflex the MMP in the study) in the cells were semi-quantified by flow cytometry analysis (Becton-Dickenson FACSCaliburTM system, New Jersey, U.S.A.) according to a previous report [ ].

Techniques: Incubation, Flow Cytometry

Journal: Molecular Therapy Oncolytics

Article Title: Development of a bicistronic anti-CD19/CD20 CAR construct including abrogation of unexpected nucleic acid sequence deletions

doi: 10.1016/j.omto.2023.07.001

Figure Lengend Snippet: Expression of two functional CARs by bicistronic CAR constructs (A) A diagram of the STD construct is shown. The first CAR of STD had a CD8α SS (CD8α SS), the Hu19 scFv, CD8-HTM (CD8α), a CD28 domain, and a CD3ζ domain. Next, were F2A and furin sequences (F2A). The second CAR had a GM-CSF-receptor-α SS (GM-CSFr SS), the Hu20 scFv, CD8-HTM, a 4-1BB domain, and a CD3ζ domain. (B) Human PBMC were stimulated with anti-CD3 in IL-2-containing media. Two days later, transductions were conducted. Five days after transductions, flow cytometry was conducted to assess CAR expression. Plots gated on live CD3 + lymphocytes show combined expression of anti-CD19 CARs and anti-CD20 CARs on T cells transduced with Hu1928-Hu20BB-Original (Original) or STD. (C) Four experiments with cells from different donors were conducted as in (B). Statistical comparison was by two-tailed, paired t test. (D) Anti-CD19-CAR and (E) anti-CD20-CAR antibody staining was assessed on CD4 + and CD8 + T cells transduced with STD or LONG as described in (B). Untransduced T cells are also shown. Plots are gated on live CD3 + lymphocytes. Percentages of (F) CD4 + and (G) CD8 + T cells that stained with the anti-CD19-CAR antibody were compared. T cells were transduced and flow cytometry was performed as in (D). Percentages of (H) CD4 + and (I) CD8 + T cells that stained with the anti-CD20-CAR antibody were compared. T cells were transduced and flow cytometry was performed as in (E). (J) CD3 + CD4 + and (K) CD3 + CD8 + T cells expressing STD or LONG were cultured for 4 h with st486 cells in the presence of an antibody against CD107a. Cells were assessed by flow cytometry for CD107a expression on live T cells. (L) CD3 + CD4 + and (M) CD3 + CD8 + T cells were assessed for CD107a expression as in (J) and (K), except st486-CD19neg target cells were used. For (F–M), statistical comparisons were two-tailed paired t tests; n = 4 different donors. N.S., not statistically significant. Throughout the figure, dots connected by lines represent results of the same patient’s cells transduced with the indicated constructs.

Article Snippet: Flow cytometry analysis for all experiments was performed with FlowJo (Tree Star, Inc.).

Techniques: Expressing, Functional Assay, Construct, Flow Cytometry, Transduction, Comparison, Two Tailed Test, Staining, Cell Culture

Journal: Molecular Therapy Oncolytics

Article Title: Development of a bicistronic anti-CD19/CD20 CAR construct including abrogation of unexpected nucleic acid sequence deletions

doi: 10.1016/j.omto.2023.07.001

Figure Lengend Snippet: Lengthening the linker of the Hu20 scFv affects function of CAR T cells (A) T cells transduced with the indicated CAR constructs or left untransduced were cultured overnight with target cells. IFN-γ ELISA was performed on culture supernatants; n = 4 different donors. CD19-K562 target cells expressed CD19. CD20-K562, st486-CD19neg, and Toledo-CD19neg expressed CD20. st486 and Toledo expressed CD19 and CD20. CCRF-CEM and NGFR-K562 were CD19 and CD20 negative. (B) Supernatants from the same CAR T cell plus target cell cultures in A were assessed by ELISA for IL-2; n = 5 donors except n = 4 for Toledo and Toledo-CD19neg. For (A and B), statistics were two-tailed, ratio paired t tests; ∗p < 0.05, ∗∗p < 0.001. For (A and B), cytokine values were normalized for CAR expression by dividing cytokine values by the fraction of T cells expressing both CARs in the constructs. (C) T cells expressing either STD or LONG were cultured for 4 h with either st486-CD19neg or NGFR-K562 target cells. Cells were stained with the anti-CD20-CAR antibody and analyzed by flow cytometry to assess CAR expression. Plots are gated on CD4 + or CD8 + live (7-aad-negative), CD3 + lymphocytes. (D) Annexin V staining of the same cells as (C). Plots are gated on live anti-CD20-CAR + CD4 + or anti-CD20-CAR + CD8 + T cells. (E) CD4 + T cells were analyzed as shown in (C). Antigen-specific CAR expression decrease of Hu20-CARs was quantified as the percent anti-CD20-CAR + cells with st486-CD19neg stimulation divided by the percent anti-CD20-CAR + cells with NGFR-K562 stimulation (%CAR + with st486-CD19neg/NGFR-K562 stim). (F) CD8 + T cells from the same cultures as in (E) were analyzed as in (E). (G) Specific %annexin V + cells are shown for the same anti-CD20-CAR + CD4 + T cells in (E). (H) Specific %annexin V + cells are shown for the same anti-CD20-CAR + CD8 + T cells in (F). For (G and H), specific %annexin V + was calculated as %annexin V + CAR + T cells with st486-CD19neg stimulation minus the %annexin V + CAR + T cells with NGFR-K562 stimulation. For (E–H), n = 6 different donors; statistics are two-tailed paired t tests. Dots connected by lines represent results of the same patient’s cells transduced with indicated constructs. p < 0.05 was considered statistically significant.

Article Snippet: Flow cytometry analysis for all experiments was performed with FlowJo (Tree Star, Inc.).

Techniques: Transduction, Construct, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Expressing, Staining, Flow Cytometry

Journal: Molecular Therapy Oncolytics

Article Title: Development of a bicistronic anti-CD19/CD20 CAR construct including abrogation of unexpected nucleic acid sequence deletions

doi: 10.1016/j.omto.2023.07.001

Figure Lengend Snippet: Antigen-specific CAR T cell function (A) CAR expression on T cells transduced with the indicated constructs was assessed by flow cytometry five days after transduction. Hu19-CD828Z was stained by the anti-CD19-CAR antibody. Hu20-CD8BBZ-L was stained by the anti-CD20-CAR antibody. Plots are gated on live CD3 + cells. Similar results were obtained with nine donors. (B) With CD19 + , CD20-negative CD19-K562 target cells, IFN-γ production was statistically lower for Hu20-CD8BBZ-L T cells compared with Hu19-CD828Z T cells and LONG T cells. IFN-γ production was statistically lower with T cells expressing Hu19-CD828Z than T cells expressing Hu20-CD8BBZ-L or LONG when T cells were stimulated with these CD20 + , CD19-negative/dim target cells: CD20-K562, st486-CD19neg, and st486. There was not a statistically significant difference in IFN-γ production when Hu19-CD828Z or LONG were cultured with CD19-K562. There was also not a statistically significant difference in IFN-γ production when Hu20-CD8BBZ-L or LONG were cultured with either CD20-K562, st486, or st486-CD19neg. Compared with T cells expressing the other CAR constructs, Hu20-CD8BBZ-L T cells had higher antigen-independent IFN-γ release against negative-control target cells NGFR-K562 and CCRF-CEM. Asterisks indicate CAR types with IFN-γ release that was statistically different at the p < 0.01 level when compared with the IFN-γ release of the other two CAR types. Comparison was by two-tailed, ratio paired t test. p < 0.05 was considered statistically significant. There was no correction for multiple comparisons. Bars represent mean + SEM; n = 5 donors. Values are pg/mL of IFN-γ normalized for CAR expression. (C) NSG mice were injected with CD20 + , CD19-negative CD20-MM.1S cells. Seven days later, when palpable tumors were present, mice were left untreated or received intravenous injections of 3 × 10 6 CAR + human T cells of the indicated types. Values are mean tumor volume ± SEM; n = 5 mice per group. (D) Kaplan-Meier plots of the same mice as (C). By log rank test, survival was longer for both Hu20-CD8BBZ-L and LONG versus each of the other three groups. p < 0.003 for all comparisons. (E) NSG mice were injected with NALM6 cells. When palpable tumors were present 6 days later, mice received the indicated number of LONG CAR + T cells or were untreated. Values are mean tumor volume ± SEM; n = 5 mice per group. (F) Kaplan-Meier plots of mice from (E). Survival of all LONG groups was longer than survival of untreated mice. p < 0.005 by log rank test for all comparisons.

Article Snippet: Flow cytometry analysis for all experiments was performed with FlowJo (Tree Star, Inc.).

Techniques: Cell Function Assay, Expressing, Transduction, Construct, Flow Cytometry, Staining, Cell Culture, Negative Control, Comparison, Two Tailed Test, Injection