Journal: bioRxiv

Article Title: KRAS inhibition activates an actionable CD24 ‘don’t eat me’ signal in pancreas cancer

doi: 10.1101/2023.09.21.558891

Figure Lengend Snippet: A, IC 50 values for AMG 510 in two mice KRAS G12C cell lines was determined by MTT assay. B , CyTOF immune profiling by viSNE of KRAS G12C mouse pancreatic tumors with vehicle or AMG 510 treatment. C , Representative viSNE plots of macrophage populations in KRAS G12C mouse pancreatic tumor with vehicle or AMG 510 treatment. D, IHC of xenograft tumors showing phosphor-ERK was reduced whereas CD8 + T cells and F4/80 + macrophages were increased in AMG 510-treated tumors compared to vehicle-treated group. E, CyTOF analysis demonstrates that CD24 intensity was upregulated in cancer cells after AMG 510 treatment. Left , quantification of CD24 intensity. Right , representative viSNE plots of CD24 staining in tumors with vehicle or AMG 510 treatment. Data, mean ± SEM; n = 3. F, Median of membrane CD24+ in 49725 K C PC cancer cells treated with vehicle or AMG 510 was measured by flow cytometry. Data, mean ± SEM. G, Median of membrane CD24+ (top) or CD47+ (bottom) in 50760 K C PC cancer cells treated with vehicle or AMG 510 was measured by flow cytometry. Data, mean ± SEM. H, Kinase array analysis indicated that AMG 510 treatment in 50760 K C PC cancer cells results in decrease of p-ERK and p-c-Jun and increase of p-STAT3 (Y705). I, Western blots of whole-cell lysates from vehicle, AMG 510 or AMG 510 plus C188-9 treated 50760 K C PC cancer cell lines.

Article Snippet: For nude mice CD24 antibody treatment, mice were injected intraperitoneally 100 μg of CD24 antibody (ATG-031; cat#HY-P99176; MCE) twice a week.

Techniques: MTT Assay, Staining, Membrane, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: KRAS inhibition activates an actionable CD24 ‘don’t eat me’ signal in pancreas cancer

doi: 10.1101/2023.09.21.558891

Figure Lengend Snippet: A, B, Left , Growth curves of xenograft tumors ( A ,49725; B , 50760) show change in tumor volume over vehicle or AMG 510 treatment. Data, mean ± SEM. Right , Waterfall plot of vehicle- and AMG 510-treated tumors showing a change in tumor volume after treatment compared with baseline at day 0. Each bar represents a single tumor. C, Quantification of the immune cell populations by CyTOF immune profiling. Data, mean ± SEM; n = 3. The statistical difference between vehicle and AMG 510 treated groups was determined by two-tailed t tests. D, Quantification of macrophage populations by CyTOF analysis. Data, mean ± SEM; n = 3. E , Representative viSNE plots of p-SHP2+ macrophage populations in KRAS G12C mouse pancreatic tumor with vehicle or AMG 510 treatment. F , Quantification of p-SHP2+ macrophage populations in E . Data, mean ± SEM; n = 3. G , Median of membrane CD24+ in 50760 K C PC cancer cells treated with vehicle or AMG 510 was measured by flow cytometry. Data, mean ± SEM; the statistical difference between experimental groups was determined by two-tailed t tests. H , qRT-PCR for CD24 expression in 50760 K C PC cancer cells treated with vehicle or AMG 510. Data, mean ± SEM; n = 3. The statistical difference was determined by two-tailed t tests. I , Western blots of whole-cell lysates from vehicle or AMG 510 treated 50760 K C PC cancer cell lines. J , Median of membrane CD24+ in 50760 K C PC cancer cells treated with vehicle, AMG 510 or AMG plus C188-9 was measured by flow cytometry. Data, mean ± SEM; the statistical difference between experimental groups was determined by two-tailed t tests. K , qRT-PCR for CD24 expression in 50760 K C PC cancer cells treated with vehicle, AMG 510 or AMG plus C188-9. Data, mean ± SEM; n = 3. The statistical difference was determined by two-tailed t tests. L , In vitro phagocytosis of mouse 50760-GFP PDAC cells cocultured with macrophage cell line RAW264.7 in the presence of AMG 510, anti-CD24 mAb, or dual treatment vs. IgG control. Phagocytosis was measured as the number of F4/80+, GFP+ macrophages, quantified as a percentage of the total F4/80+ macrophages. Data, mean ± SEM; n = 3. Statistics with significance were indicated n.s. not significant; * P <0.05; ** P <0.01; ***, P < 0.001, **** P <0.0001.

Article Snippet: For nude mice CD24 antibody treatment, mice were injected intraperitoneally 100 μg of CD24 antibody (ATG-031; cat#HY-P99176; MCE) twice a week.

Techniques: Two Tailed Test, Membrane, Flow Cytometry, Quantitative RT-PCR, Expressing, Western Blot, In Vitro, Control

Journal: bioRxiv

Article Title: KRAS inhibition activates an actionable CD24 ‘don’t eat me’ signal in pancreas cancer

doi: 10.1101/2023.09.21.558891

Figure Lengend Snippet: A, B, Top, Growth curves of xenograft K C PC tumors ( A ,49725; B , 50760) show change in tumor volume over vehicle, anti-CD24, AMG 510 or anti-CD24 + AMG 510 treatment. Data, mean ± SEM. Bottom , Waterfall plot of vehicle-, anti-CD24-, AMG 510- or anti-CD24 + AMG 510-treated tumors showing a change in tumor volume after treatment compared with baseline at day 0. Each bar represents a single tumor. C , In vivo phagocytosis. Mouse 50760-GFP PDAC cells were injected into the right flank of C57BL/6 mice and treated with IgG control or AMG 510+ anti-CD24 mAb for 14 days. Mice tumor tissues were collected for flow cytometry. Phagocytosis was measured as the number of F4/80+, GFP+ macrophages, quantified as a percentage of the total F4/80+ macrophages. Data, mean ± SEM; n = 4. D , Mice tumor tissues collected in C were measured by flow cytometry for F4/80 and p-SHP2. Percentage of p-SHP2+ macrophages were compared in IgG control or AMG 510+ anti-CD24 mAb groups. Data, mean ± SEM; n = 4. E , Growth curves of xenograft K C PC tumor (49725) show changes in tumor volume over vehicle, vehicle + anti-CSF1R, anti-CD24 + AMG 510 (combination), or anti-CSF1R + combination treatment. Data, mean ± SEM. F , Median of membrane CD24+ in K D PC cancer cells (55582) treated with vehicle or MRTX1133 was measured by flow cytometry. Data, mean ± SEM; the statistical difference between experimental groups was determined by two-tailed t tests. G , qRT-PCR for CD24 expression in K D PC cancer cells (55582) treated with vehicle or MRTX1133. Data, mean ± SEM; n = 3. The statistical difference was determined by two-tailed t tests. H , Left , Growth curves of xenograft K D PC tumor (55582) show change in tumor volume over vehicle, anti-CD24, MRTX1133 or anti-CD24 + MRTX1133 treatment. Data, mean ± SEM. Right , Waterfall plot of vehicle, anti-CD24, MRTX1133 or anti-CD24 + MRTX1133-treated tumors showing a change in tumor volume after treatment compared with baseline at day 0. Each bar represents a single tumor. I , Top , Growth curves of human KRAS G12D tumors (AsPC1) show change in tumor volume over vehicle, anti-CD24, MRTX1133 or anti-CD24 + MRTX1133 treatment. Data, mean ± SEM. Bottom, Waterfall plot of vehicle, anti-CD24, MRTX1133 or anti-CD24 + MRTX1133-treated tumors showing a change in tumor volume after treatment compared with baseline at day 0. Each bar represents a single tumor. Statistics with significance were indicated n.s. not significant; * P <0.05; ** P <0.01; ***, P < 0.001, **** P <0.0001.

Article Snippet: For nude mice CD24 antibody treatment, mice were injected intraperitoneally 100 μg of CD24 antibody (ATG-031; cat#HY-P99176; MCE) twice a week.

Techniques: In Vivo, Injection, Control, Flow Cytometry, Membrane, Two Tailed Test, Quantitative RT-PCR, Expressing

Journal: Biology

Article Title: Effect of Maternal Hyperglycemia on Fetal Pancreatic Islet Development.

doi: 10.3390/biology14060728

Figure Lengend Snippet: Figure 3. Islet of Langerhans basal membrane protein immunostaining. (A,C,E,G): Immunohis- tochemistry of pan-laminin, laminin alpha 1 and gamma 1 chains and alpha 3 integrin. Islet of Langerhans—dashed line. Brown staining indicates protein-of-interest staining. Scale bar: 20.0 µm. (B,D,F,H): Quantification of stained area related to total islet area for each analyzed protein. Data presented as mean ± standard error of the mean, * p < 0,02 and *** p < 0.0001 vs. control group by Student’s t-test.

Article Snippet: For protein detection, sections were incubated with specific primary rabbit antibodies: pan-laminin (ab1575; Abcam, diluted 1:500, Cambridge, UK), alpha 1 laminin (sc H-300 Santa Cruz H-300: SC-5582 diluted 1:50, Santa Cruz, CA, USA), gamma 1 laminin (sc5584; Santa Cruz, diluted 1:50), alpha 3 integrin (600-401 R13, Rockland diluted 1:200, Baltimore, MD, USA), anti-PCNA (ab2426 Abcam 1: 200), anti-glucagon (# A0565 1:350) and anti-insulin (# A0564 1:250).

Techniques: Membrane, Immunostaining, Staining, Control

Journal: Antioxidants

Article Title: Oxidative Stress Plays an Important Role in Glutamatergic Excitotoxicity-Induced Cochlear Synaptopathy: Implication for Therapeutic Molecules Screening

doi: 10.3390/antiox13020149

Figure Lengend Snippet: BDNF, NT3, and BDNF mimetics boost synapse regeneration or repair. ( A – C ): Maximum intensity projections of confocal z-stacks of IHCs of the cochlear explants labeled with anti-CtBP2 (green), anti-PSD95 (red), and anti-VGLUT3 (blue) ( A ) and quantifications of synapses (paired CtBP2-PSD95) per IHC in cochlear regions coding 16 KHz ( B ) and 32 KHz ( C ). The cochlear samples were imaged in the regions coding 16 KHz and 32 kHz from explants treated with either medium alone for 2 h + 48 h (Ctrl + 48 h), medium containing kainate for 2 h and then medium alone for 48 h (KA + 48 h), medium containing 0.5 mM kainate for 2 h + NT3 for 48 h (KA + NT3 48 h), or kainate for 2 h+ BDNF for 48 h (KA + BDNF 48 h). Scale bar: ( A ) = 15 µm. ( D – I ): Quantification of synapses (paired CtBP2-PSD95) per IHC in cochlear regions coding 16 KHz ( D , F , H , J ) and 32 KHz ( E , G , I , K ). In ( D , E ), the samples were collected from explants exposed to medium alone for 2 h + 48 h (Ctrl + 48 h); medium containing kainate for 2 h, then medium alone for 48 h (KA + 48 h); medium containing kainate for 2 h, then medium containing LM22A-4 at 1, 5, and 10 µM for 48 h (KA + LM22A-4 48 h). In ( F , G ), the samples were collected from explants exposed to medium alone for 2 h + 48 h (Ctrl + 48 h); medium containing kainate for 2 h, then medium alone for 48 h (KA + 48 h); medium containing kainate for 2 h, then medium containing CF3CN at 1, 5, and 10 µM for 48 h (KA + CF3CN 48 h). In ( H , I ), the samples were collected from explants exposed to medium alone for 2 h + 48 h (Ctrl + 48 h); medium containing kainate for 2 h, then medium alone for 48 h (KA + 48 h); medium containing kainate for 2 h, then medium containing R13 at 1, 5, and 10 µM for 48 h (KA + R13 48 h). ( J , K ): Comparison of the regenerative efficacy of BDNF, NT3, and different BDNF mimetics at their effective concentration in the 16 and 32 kHz coding regions. All data are expressed as mean ± SEM (n = 6–10 cochleae per condition). One-way ANOVA test was followed by Dunn’s test: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: R13, a TrkB ligand prodrug, was purchased from Axon Medchem (#3775, Groningen, Netherlands).

Techniques: Labeling, Comparison, Concentration Assay