Journal: Signal Transduction and Targeted Therapy

Article Title: Analogous comparison unravels heightened antiviral defense and boosted viral infection upon immunosuppression in bat organoids

doi: 10.1038/s41392-022-01247-w

Figure Lengend Snippet: Bat organoids elicited a more robust antiviral response to Poly(I:C) treatment. Bat and human intestinal organoids were sheared mechanically and treated with 10 μg/ml Poly(I:C) or mock-treated with DMSO. a , b Induction of IFNs (left) and ISGs (right) in bat intestinal organoids ( a ) and human intestinal organoids ( b ) at the indicated hours after Poly(I:C) treatment. Results show the log2-fold change of GAPDH-normalized expression level in the treated organoids relative to mock-treated organoids. Data represent the mean and s.d. of a representative experiment in organoids from a bat and human donor, n = 3. c Culture media from the treated or mock-treated human intestinal organoids at the indicated hours were applied to ELISA to measure concentrations of IFNL1 and IFNL3. Data represent the mean and s.d. of a representative experiment in one line of human organoids, n = 3. Two-tailed unpaired Student’s t test. d Poly(I:C) treated or mock-treated human intestinal organoids were collected at the indicated hours post-treatment and subjected to Western blot to detect human ISG15. e , f Bat and human intestinal organoids were sheared and incubated with 10 μg/ml Poly(I:C) Fluorescein in triplicate for 2 h and 6 h. The organoids were then dissociated and applied to flow cytometry to detect the percentage at 2 h post treatment ( e ) and mean fluorescence intensity (MFI, f ) of Fluorescein-positive cells at 2 and 6 h post treatment. g , h Induction of TNF-a and IL6 ( g ) and IP10 ( h ) in bat and human intestinal organoids at the indicated hours after treatment. Results show the fold change of GAPDH-normalized expression level in Poly(I:C)-treated organoids relative to mock-treated organoids. Data represent the mean and s.d. of a representative experiment in organoids from a bat and human donor, n = 3

Article Snippet: Cell-free media were harvested from treated and mock-treated human intestinal organoids for measuring the amount of human IFNL1 and IFNL3 using ELISA kits (R&D Systems, DY7246, D28B00).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot, Incubation, Flow Cytometry, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Analogous comparison unravels heightened antiviral defense and boosted viral infection upon immunosuppression in bat organoids

doi: 10.1038/s41392-022-01247-w

Figure Lengend Snippet: TLR3 and RLR signaling pathways mediate antiviral responses in bat and human organoids. a A schematic graph outlines the experimental procedure for panels b – g . Bat and human intestinal organoids were pretreated with BX795 (0, 0.1 and 1 μM) or CYT387 (0, 0.1, and 1 μg/ml) overnight. After mechanical shearing, the organoids were incubated with or without 10 μg/ml Poly(I:C), together with the initial concentrations of BX795 or CYT387. At the indicated hours after Poly(I:C) treatment, the organoids were harvested and subjected to RT-qPCR assay to examine mRNA expression levels of bat and human IFNL1 and IFNL3; cell-free media from human organoids were applied to ELISA to detect IFNL1 and IFNL3. Data represent the mean and s.d. of a representative experiment in organoids from a bat and human donor, n = 3. Ordinary one-way ANOVA with Tukey’s multiple comparison test. b , c Induction of IFNL1 (left) and IFNL3 (right) in bat intestinal organoids treated with BX795 ( b ) and CYT387 ( c ). Results show the fold change of GAPDH-normalized expression level in the Poly(I:C)-treated organoids relative to mock-treated organoids. d , e Induction of IFNL1 and IFNL3 in human intestinal organoids treated with BX795 ( d ) and CYT387 ( e ). IFNL1 ( f ) and IFNL3 ( g ) secretion from the human organoids at 4 h post-stimulation. h A schematic graph describes the experimental procedure for panels i and j . Bat and human intestinal organoids were pretreated with 1 μg/ml CYT387 or DMSO overnight. After mechanical shearing, the organoids were incubated with 1 μg/ml CYT387 or DMSO with or without 10 μg/ml Poly(I:C) for 4 h. Cell-free media were then harvested and subjected to PRM-MS to analyze bat IFNL3 ( i ) and ISG15 of bat and human ( j ). The dotted lines represent the detection limit. Data represent the mean and s.d. of a representative experiment in organoids from a bat and human donor, n = 3. Two-tailed unpaired Student’s t test

Article Snippet: Cell-free media were harvested from treated and mock-treated human intestinal organoids for measuring the amount of human IFNL1 and IFNL3 using ELISA kits (R&D Systems, DY7246, D28B00).

Techniques: Protein-Protein interactions, Incubation, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test

Journal: Oncotarget

Article Title: Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

doi:

Figure Lengend Snippet: A . RPMI-8226, ALMC-2, MM.1S, and U266 cells were incubated for 48 hours in the presence or absence of lovastatin ( Lov , 2-50 μM) or DGBP (2-50 μM). Immunoblot analysis of p62, Atg3 and β-tubulin (loading control) is shown. The relative intensity ( Rel Int) values underneath the p62 and Atg3 blots represent relative levels as determined by densitometric analysis of Atg3 or p62 (relative to tubulin) of treated cells compared to control cells. The gels are representative of 3-4 independent experiments. B . ALMC-2 cells were incubated for 48 hours in the presence of 10 μM lovastatin ( Lov ) and/or mevalonate ( Mev , 5 mM), FPP (10 μM), or GGPP (10 μM) for 48 hrs. Densitometric analysis of p62 or Atg3 levels (normalized to β-tubulin levels) for the combination treatments normalized to untreated cells (control) is shown. Data are displayed as average +/− standard deviation (n=3 independent experiments). * denotes p-value <0.05 from two-sided t-testing. C . RPMI-8226 and ALMC-2 cells were incubated for 48 hours in the presence or absence of 10 μM lovastatin ( Lov ), 10 μM DGBP, 10 μM GGTI-2133 ( GGTI ), 10 mM 3-PEHPC ( 3P ), or the combination of 10 μM GGTI-2133 and 10 mM 3-PEHPC ( GGTI + 3P ). Immunoblot analysis of p62, Atg3 and β-tubulin (loading control) is shown. The gels are representative of 3-4 independent experiments. D . RPMI-8226 and ALMC-2 cells were incubated for 48 hours in the presence or absence of 10 μM lovastatin ( Lov ), 10 μM DGBP, 10 mM 3-PEHPC ( 3P ), or 5 nM bafilomycin A1 ( Baf ) with or without protease inhibitors (10 μg/mL E64d and pepstatin A, prot ). Immunoblot analysis of LC3 and β-tubulin (loading control) is shown. The gels are representative of 3 independent experiments. E . Intracellular lambda light chain levels were measured via ELISA. Data are expressed as a percentage of control (mean +/− standard deviation of 3 independent experiments). Two-sided t-testing was performed to compare the IBP inhibitor alone vs. in combination with the protease inhibitors. No significant differences between the treatment groups were observed.

Article Snippet: A human lambda light chain kit (E80-116, Bethyl Laboratories) was used to quantify intracellular monoclonal protein levels.

Techniques: Incubation, Western Blot, Control, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

doi:

Figure Lengend Snippet: RPMI-8226 cells were incubated with 2 nM bafilomycinA1, 10 μM lovastatin, or 5 mM 3-PEHPC for 48 hours. Staining was performed as described in Materials and Methods, using antibodies directed against lambda light chain (red) and PDI as an ER marker (green) in A . and RCAS1 as a Golgi marker (green) in B . DAPI was used for nuclear staining (blue). Colocalization was determined using the RGB plot profile tool in ImageJ and indicated by similarity of the patterns of red and green peaks. Scale bar 10μm.

Article Snippet: A human lambda light chain kit (E80-116, Bethyl Laboratories) was used to quantify intracellular monoclonal protein levels.

Techniques: Incubation, Staining, Marker

Journal: Oncotarget

Article Title: Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

doi:

Figure Lengend Snippet: A . RPMI-8226 cells were incubated with 2 nM bafilomycin A1 ( Baf ), 10 μM lovastatin ( Lov ), or 5 mM 3-PEHPC ( 3P ) for 48 h or 10 nM bortezomib for 24 h. Staining was performed as described in Materials and Methods, using Proteostat (Enzo Life Sciences, Inc.) (green) to visualize aggresomes and an antibody directed against lambda light chain (red). DAPI was used for nuclear staining (blue). Colocalization was determined using the RGB plot profile tool in ImageJ. Scale bar 10 μm. B . The average number of aggresomes per cell was quantified using the GFP-LC3 macro for ImageJ (mean ± SEM). **** denotes P <0.0001 from unpaired two-tailed t- test comparing treated with control cells.

Article Snippet: A human lambda light chain kit (E80-116, Bethyl Laboratories) was used to quantify intracellular monoclonal protein levels.

Techniques: Incubation, Staining, Two Tailed Test, Control

Journal: Oncotarget

Article Title: Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

doi:

Figure Lengend Snippet: U266 cells were treated with 2 nM bafilomycin A1 ( Baf ), 10 μM lovastatin ( Lov ) or 10 mM 3-PEHPC for 48 hours before processing for staining with LC3. A . The average number of LC3 puncta per cell and B . the average size of each punctate structure in pixels was quantified using the GFP-LC3 macro for ImageJ (mean ± SEM) (n>175). The **** denotes p <0.0001 from unpaired two-tailed t- test comparing treated with control cells. C . Representative confocal maximum projections are shown for each treatment with LC3 in green and DAPI staining the nucleus in blue. D . RPMI-8226 cells were incubated with 2 nM bafilomycin A1, 10μM lovastatin, 5mM 3-PEHPC or a co-treatment with bafilomycin A1 ( Baf ) and lovastatin ( Lov ) for 48 hours. Staining was performed as described in Materials and Methods, using CytoID (Enzo Life Sciences, Inc., Farmingdale, NY) to visualize autophagosomes and an antibody directed against lambda light chain. DAPI was used for nuclear staining. Colocalization was determined using the RGB plot profile tool in ImageJ. Scale bar 10μm.

Article Snippet: A human lambda light chain kit (E80-116, Bethyl Laboratories) was used to quantify intracellular monoclonal protein levels.

Techniques: Staining, Two Tailed Test, Control, Incubation