Journal: Molecular Imaging and Biology

Article Title: Development of New CD38 Targeted Peptides for Cancer Imaging

doi: 10.1007/s11307-024-01901-5

Figure Lengend Snippet: Radiolabeling and cellular uptake studies ( a ) Representative chromatograph of the 64 Cu labeled CD38-peptide (NODAGA-PEG4-SL022-GGS) under two different conditions; ( b ) In vitro blocking study of 64 Cu-NODAGA-PEG4-SL022-GGS in MM.1S-GFP-CBR-WT cells with unlabeled NODAGA-PEG4-SL022-GGS, daratumumab and isatuximab respectively.

Article Snippet: Nonspecific binding was blocked by incubating cells with 1% BSA, 0.1% Tween 20 in DPBS for 30 min. After overnight incubation at 4 °C with anti-CD38 mouse polyclonal antibody (# PA5-95,840, Invitrogen, ThermoFisher Scientific, Waltham, MA, 1:100 dilution) or anti-CD31 mouse monoclonal antibody (JC/70A #MA5-13,188, Invitrogen, ThermoFisher Scientific, Waltham, MA, 1:50 dilution), cells were washed and incubated for 1 h at room temperature with Alexa Fluor 594- or 647-conjugated secondary goat anti-mouse IgG1 (cross-adsorbed, # A-21240, Invitrogen, ThermoFisher Scientific, Waltham, MA, 1:50 dilution) or F(ab')2-goat anti-rabbit IgG (H + L) (cross adsorbed, # A48285, Invitrogen, ThermoFisher Scientific, Waltham, MA, 1:100 dilution).

Techniques: Radioactivity, Labeling, In Vitro, Blocking Assay

Journal: Molecular Imaging and Biology

Article Title: Development of New CD38 Targeted Peptides for Cancer Imaging

doi: 10.1007/s11307-024-01901-5

Figure Lengend Snippet: a CD38 & CD31 expression in MM.1S-GFP-CBR-WT cells. CD38 (left column): Immunocytochemistry analysis of CD38 expression in MM.1S-CBR-GFP cells with the CD38 1° antibody followed by the AlexaFluor 647 conjugated goat anti-rabbit IgG secondary antibody. Nuclei were counter stained with Hoechst 33,342. The sections were imaged with GFP filter pair (410/520) for endogenous GFP expression, Cy5 (620/705) for CD38 with AlexaFlour 647-conjugated secondary antibody, and Hoechst 33,342 (370/405) to stain nuclei. CD31 (right column): Immunohistochemistry analysis of CD31 expression in MM.1S-CBR-GFP cells with CD31 antibody followed with AlexaFluor 647 conjugated goat anti-rabbit IgG secondary antibody. Nuclei were counter-stained with Hoechst 33,342. The sections were imaged with GFP filter pair (410/520) to visualize endogenous GFP expression, Cy5 (620/705) for CD31 expression with AlexaFlour 647-conjugated secondary antibody, Hoechst 33,342 (370/405) to stain nuclei; b Characterization of the disseminated MM.1S-CBR-GFP-WT tumor model. In vivo BLI showing presence of disseminated MM.1S-CBR-GFP-WT in both femurs and tibia at 5 weeks after tumor implantation; c MM.1S-CBR-GFP-WT tumor burden additionally evidenced in the femur via GFP intracellular expression seen with epifluorescence microscopy of unstained femur section, scale bar – 20 µm; d Histological assessment of the femur with H&E staining shown at 10x, 20x, 40 × at scale of 200 µm, 100 µm, 20 µm, respectively.

Article Snippet: Nonspecific binding was blocked by incubating cells with 1% BSA, 0.1% Tween 20 in DPBS for 30 min. After overnight incubation at 4 °C with anti-CD38 mouse polyclonal antibody (# PA5-95,840, Invitrogen, ThermoFisher Scientific, Waltham, MA, 1:100 dilution) or anti-CD31 mouse monoclonal antibody (JC/70A #MA5-13,188, Invitrogen, ThermoFisher Scientific, Waltham, MA, 1:50 dilution), cells were washed and incubated for 1 h at room temperature with Alexa Fluor 594- or 647-conjugated secondary goat anti-mouse IgG1 (cross-adsorbed, # A-21240, Invitrogen, ThermoFisher Scientific, Waltham, MA, 1:50 dilution) or F(ab')2-goat anti-rabbit IgG (H + L) (cross adsorbed, # A48285, Invitrogen, ThermoFisher Scientific, Waltham, MA, 1:100 dilution).

Techniques: Expressing, Immunocytochemistry, Staining, Immunohistochemistry, In Vivo, Tumor Implantation, Epifluorescence Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Phenotype and Metabolism of Peritoneal Macrophages in Mucin-2 Knockout Mice and Partial Restoration of Their Functions In Vitro After L-Fucose Treatment

doi: 10.3390/ijms26010013

Figure Lengend Snippet: Addition of fucose to the cell culture medium affected the production of cytokines, ATP and Ca 2+ levels, and surface and intracellular expression of CD38 of peritoneal macrophages derived from C57BL/6 and Muc2 −/− mice. ( A ). Inflammatory cytokine levels in cell culture medium of peritoneal macrophages from the two mouse strains with and without the addition of 0.1% L-fucose; the median, min, and max for each cytokine is presented in pg/mg protein. ( B ). ATP levels in the peritoneal macrophages of two mouse strains incubated with and without 0.1% L-fucose. ( C ). Ca 2+ levels in the peritoneal macrophages of the two mouse strains incubated with and without 0.1% L-fucose. ( D ). Percentage of peritoneal macrophages with surface CD38 expression from the two mouse strains incubated with and without 0.1% L-fucose. ( E ). Percentage of peritoneal macrophages with intracellular CD38 expression from the two mouse strains incubated with and without 0.1% L-fucose. “C57BL/6” vs. “ Muc2 −/− “ and “with 0.1% L-fucose” vs. “without 0.1% L-fucose”: * p < 0.05, ** p < 0.01, and *** p < 0.001. Two-way PERMANOVA test.

Article Snippet: After centrifugation, the cells were resuspended in 100 μL of permeabilization buffer and incubated with Pacific Blue–anti-CD45 (BioLegend, San Diego, CA, USA, Rat IgG2b, k, clone S18009F), PE-anti-CD11b (BioLegend, USA, Rat IgG2b, k, clone M1/70), and FITC-anti-CD38 (Invitrogen, Waltham, MA, USA, clone 90) for 60 min at 4 °C in the dark.

Techniques: Cell Culture, Expressing, Derivative Assay, Incubation

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A Western blot analysis confirmed the overexpression of CD38 in A549-CD38 cells compared with A549-Plvx cells. B Relative ROS levels in A549-Plvx and A549-CD38 cells ( n = 3). C and D NAD + and NADP + levels in A549-Plvx and A549-CD38 cells were determined by peak areas from the metabolomics analysis ( n = 3). E Relative ROS levels of A549 cells treated with or without 100 nM FK866 for 12 h ( n = 3). F A549 cells were treated with or without 100 nM FK866 for 12 h. Protein levels of DHFR and Actin (loading control) were analyzed by western blot. Graphs represent the quantification of the blots ( n = 3). G The relative transcription levels of DHFR in untreated and 100 nM FK866-treated A549 cells ( n = 3). H The relative ROS levels in A549-CD38 cells treated with or without 1 mM NMN for 12 h ( n = 3). I A549-Plvx and A549-CD38 cells were treated with or without 1 mM NMN. Protein levels of DHFR and Actin (loading control) were analyzed by western blot. Graphs represent the quantification of the blots ( n = 3). J The relative transcription levels of DHFR in A549-Plvx and A549-CD38 cells treated with or without 1 mM NMN ( n = 3). Data were shown as mean ± SD and analyzed by Student’s t -test or one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Western Blot, Over Expression, Control

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A Relative ROS levels were measured in A549 cells treated with different concentrations of H 2 O 2 (0, 0.05, 0.1, 0.5, 1, 2 mM) for 12 h ( n = 3). B A549 cells were treated with different concentrations of H 2 O 2 (0, 0.05, 0.1, 0.5, 1, 2 mM) for 12 h. Protein levels of DHFR and Actin (loading control) were analyzed by western blot. Graphs represent the quantification of the blots ( n = 3). C The transcription levels of DHFR in A549 cells treated with different concentrations of H 2 O 2 (0, 0.05, 0.1, 0.5, 1, 2 mM) for 12 h ( n = 3). D Western blot images of DHFR and Actin (loading control) in untreated and 1 mM H 2 O 2 -treated A549 cells pre-treated with 6 mM NAc for 6 h or not. Graphs represent the quantification of the blots ( n = 3). E Western blot images of DHFR and Actin (loading control) in untreated and 1 mM H 2 O 2 -treated A549 cells co-treated with 100 nM Baf-A1 or not. Graphs represent the quantification of the blots ( n = 3). F Western blot images of DHFR and Actin (loading control) in untreated and 1 mM H 2 O 2 -treated A549 cells pre-treated with 10 μM MG132 for 4 h or not. Graphs represent the quantification of the blots ( n = 3). G Western blot images of DHFR and Actin (loading control) in DMSO, NAc (6 mM, 6 h), Baf-A1 (100 nM, 6 h), and MG132 (10 μM, 4 h)-treated A549-CD38 cells. Graphs represent the quantification of the blots ( n = 3). Data were shown as mean ± SD and analyzed by one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Control, Western Blot

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A and B The survival rate of A549-Plvx and A549-CD38 cells treated with different concentrations of Erastin ( A ) or RSL3 ( B ) ( n = 3). C and D The survival rate of A549-Plvx and A549-CD38 cells treated with Erastin (5, 2.5 μM) or RSL3 (2.5, 1.25 μM) with or without 1 mM NMN co-treatment for 12 h ( n = 3). E and F The survival rate of A549 cells under Erastin or RSL3 treatment co-treated with or without 100 nM methotrexate (MTX) ( n = 3). G and H The survival rate of A549-CD38 cells under Erastin or RSL3 treatment before or after replenishing DHFR ( n = 3). I The survival rate of A549-CD38 cells treated with RSL3 (0.625 μM, 12 h), RSL3 (0.625 μM) + Baf-A1 (100 nM, co-treatment with RSL3 for 12 h), and RSL3 (0.625 μM) + MG132 (10 μM, pre-treatment for 4 h) ( n = 3). J The survival rate of A549-CD38 cells treated with DMSO (Control), Baf-A1 (100 nM), and MG132 (10 μM, pre-treatment for 4 h) for 12 h ( n = 3). Data were shown as mean ± SD and analyzed by Student’s t -test or one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Control

Journal: Cell Death & Disease

Article Title: Oxidative degradation of dihydrofolate reductase increases CD38-mediated ferroptosis susceptibility

doi: 10.1038/s41419-022-05383-7

Figure Lengend Snippet: A Western blot analysis of CD38, DHFR, and Actin (loading control) in BMDMs from young and aged mice, respectively. Graphs represent the quantification of the blots ( n = 3). B The relative ROS levels of BMDMs from young and aged mice ( n = 3). C and D The mRNA expression levels of cd38 and dhfr in BMDMs from young and aged mice, respectively ( n = 3). E and F The survival rate of BMDMs from young and aged mice under Erastin ( E ) or RSL3 ( F ) treatment ( n = 3). G The survival rate of BMDMs from young and aged mice under Erastin (20 μM), Erastin (20 μM) + NMN (1 mM), Erastin (20 μM) + DFO (50 μM), Erastin (20 μM) + Fer-1 (1 μM) treatment for 12 h ( n = 3). H The survival rate of BMDMs from young and aged mice under RSL3 (0.625 μM) or + RSL3 (0.625 μM) + NMN (1 mM), RSL3 (0.625 μM) + DFO (50 μM), RSL3 (0.625 μM) + Fer-1 (1 μM) treatment for 12 h ( n = 3). I The survival rate of BMDMs from aged mice under RSL3 (0.625 μM, 12 h), RSL3 (0.625 μM) + Baf-A1 (100 nM, co-treatment with RSL3 for 12 h), and RSL3 (0.625 μM) + MG132 (10 μM, pre-treatment for 4 h) ( n = 3). J The survival rate of BMDMs from aged mice under DMSO (Control), Baf-A1 (100 nM), and MG132 (10 μM, pre-treatment for 4 h) treatment for 12 h ( n = 3). Data were shown as mean ± SD and analyzed by Student’s t -test or one-way ANOVA test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies for β-Actin (ABclonal, Woburn, MA), DHFR (Abcam, Cambridge, UK), CD38-human, CD38-mouse, Flag (Cell Signaling Technology, Danvers, MA), and secondary anti-rabbit HRP-IgG antibodies (Cell Signaling Technology, Danvers, MA) were used for detecting target proteins.

Techniques: Western Blot, Control, Expressing