Journal: Science Advances

Article Title: Anti-EGFR VHH-armed death receptor ligand–engineered allogeneic stem cells have therapeutic efficacy in diverse brain metastatic breast cancers

doi: 10.1126/sciadv.abe8671

Figure Lengend Snippet: ( A ) Representative microphotograph of low and high (the insets) magnifications of H&E and immunohistochemistry of EGFR and DR4/5 in patient samples of TNBC- BM. Scale bars, 100 μm (main images) and 10 μm (insets). ( B ) Schematic showing the construction of anti-EGFR VHH-DR L (E V DR L ) and anti-EGFR scFv-DR L (E S DR L ) proteins. ( C ) Cell viability of BLBC-BM lines after 72-hour treatment with control media (Ctrl), E S DR L , or E V DR L . ( n = 3, technical replicates). ( D ) WB showing cleavage of caspases and poly(ADP-ribose) polymerase (PARP) in BLBC-BM lines after 8-hour treatment with Ctrl, E S DR L , or E V DR L ( n = 3, technical replicates). (Loading control–adjusted ratios are provided under blots; only cleaved part was quantified). ( E ) Cell viability of 18 BC cell lines after 72-hour treatment with different concentrations of Ctrl, E V , DR L , or E V DR L ( n = 3, technical replicates). ( F ) Correlation between cell surface DR4/5 expression and growth inhibition effect of DR L at the time point of 24 hours. ( G ) Correlation between cell surface EGFR expression and growth inhibition ratio between DR L and E V DR L at the time point of 24 hours. ( H ) Correlation between cell surface DR5 and EGFR expression and the growth inhibition efficacy of E V DR L . ( I ) WB showing phosphorylation of EGFR and its downstream elements in BLBC-BM lines with EGF treatment after pretreatment with various concentrations of E V DR L ( n = 3, technical replicates). ( J ) WB showing cleavage of caspases and PARP in BLBC-BM lines after 8-, 16-, and 24-hour treatment with Ctrl or E V DR L ( n = 3, technical replicates). ( K ) Caspase-Glo 3/7 assay of BLBC-BM lines after 8-hour treatment with Ctrl or E V DR L ( n = 3, technical replicates).

Article Snippet: Antibodies against β-actin (#4970), phospho-AKT (Ser 473 , #4060), AKT (#9272), caspase-7 (#9492), caspase-8 (#9746), caspase-9 (#9508), cleaved caspase-3 (#9661), EGFR (#2646 and #4267), phospho-EGFR (Tyr 1068 , #3777), cleaved poly(ADP-ribose) polymerase (PARP; #9541), phospho-p44/42 mitogen-activated protein kinase (MAPK) (ERK1/2) (Thr 202 /Tyr 204 , #9101), p-44/42 MAPK (ERK1/2) (#9102), Fas-associated death domain protein (#2782), Bcl-2 (#2872), Bcl-xL (#2764), XIAP (#2042), cIAP2 (#3130), phospho–signal transducers and activators of transcription 3 (STAT3) (Tyr 705 , #9145), STAT3 (#4904), HER2 (#2242), horseradish peroxidase (HRP) anti-rabbit (#7074), Rab5 (#46449), Rab7 (#95746) (Cell Signaling Technology), anti–nuclear factor κB (#ab16502), anti-TRAIL (#ab9959), anti-CD31 (#ab28364), HRP anti-mouse (#ab205719) (Abcam), anti–α-tubulin (#T5168), anti-Vinculin (#V4505), NeuN (#MAB377), glial fibrillary acidic protein (GFAP) (#MAB3402) (Sigma-Aldrich), Alexa Fluor 488 anti-EGFR antibody (#352908), anti-DR4 (#1139), anti-DR5 (#2019) (ProSci), anti-DR4 (#sc-32255), anti-DR5 (#sc-166624), anti-cIAP1 (#sc-271419), normal mouse IgG (#sc-2025) (Santa Cruz), anti-Ki-67 (#180191Z), anti-GFAP (#180063), Alexa Fluor anti-rabbit 405 (#A-31556), Alexa Fluor anti-rabbit 488 (#A-11008), Alexa Fluor anti-mouse 555 (#A-21422), Alexa Flour anti-rabbit 647 (#A-21244), Phycoerythrin (PE) anti-DR4 (#12-6644-42), PE anti-DR5 (#12-9908-42), PE mouse IgG isotype (#12-4714-42) (Invitrogen), Cetuximab (ImClone Systems), Erlotinib (#SYN-1039, Selleck Chemicals), human recombinant EGF (R&D Systems), PE anti-EGFR (#352903, BioLegend), and IBA1 (#019-19741, FUJIFILM).

Techniques: Immunohistochemistry, Control, Expressing, Inhibition, Phospho-proteomics, Caspase-Glo Assay

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Naringenin suppresses epithelial ovarian cancer by inhibiting proliferation and modulating gut microbiota.

doi: 10.1016/j.phymed.2022.154401

Figure Lengend Snippet: Fig. 4. Naringenin suppressed EOC through the PI3K signaling pathway in vitro. Western blotting analysis of lysates (40 μg) of EOC cells treated with naringenin at the indicated concentrations for 24 h (a, b, i, j). Membranes were incubated with antibodies against EGFR (c, f), PI3K (d, g), CCND1 (e, h) and p-PI3K (k, l). All data are expressed as the mean ± SD of values from experiments performed in triplicate. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to ctrl groups.

Article Snippet: Tissues were stained with primary antibodies against EGFR (1:50, A00023-2, Boster, Wuhan, China), PI3K (1:50, bs-4160R, Bioss, Beijing, China) and CCND1 (1:50, BM4272, Boster, Wuhan, China) for immunohistochemical analysis.

Techniques: In Vitro, Western Blot, Incubation

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Naringenin suppresses epithelial ovarian cancer by inhibiting proliferation and modulating gut microbiota.

doi: 10.1016/j.phymed.2022.154401

Figure Lengend Snippet: Fig. 6. Naringenin suppressed EOC through the PI3K signaling pathway in vivo. HE staining (a) and IHC staining for EGFR (b, f), PI3K (c, g) and CCND1 (d, h) in tumors and quantification (n=5) (Nar p.o. means Nar p.o., per os; ns means not statistically significant, * p < 0.05; ** p < 0.01; *** p < 0.001 compared to untreated; # p < 0.05 Nar p.o., per os compared to Nar i.p.).

Article Snippet: Tissues were stained with primary antibodies against EGFR (1:50, A00023-2, Boster, Wuhan, China), PI3K (1:50, bs-4160R, Bioss, Beijing, China) and CCND1 (1:50, BM4272, Boster, Wuhan, China) for immunohistochemical analysis.

Techniques: In Vivo, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a Relative expression of IEC markers from different branches, including enteroendocrine cells ( Neurog3 , Insm , and Pax4 ), secretory lineage ( Muc2 , Gfi1 , Foxa2 , Reg3a , and Lyz ), M cells ( Spib ), Tuft cells ( Dclk ), stem cells ( Olfm4 , Lgr5 ), and absorptive cells ( Si ) in mouse organoids with rSPINK4 stimulation. The degree of color depth indicates the abundance of expression positively. b Levels of phosphorylated and total EGFR in NCM460 and HT-29 cells with 5 ng/mL rSPINK4 stimulation for 0–60 min. c Direct downstream molecules of the EGFR pathway detected using western blotting with rSPINK4 stimulation for 0–60 min. d Typical fluorescent images of intestinal organoids from WT and KO mice including MUC2 (green), E-cadherin (red), and DAPI (blue), with rSPINK4 (100 ng/mL) and AG1478 (10 μM) stimulation under inflammatory conditions (50 ng/mL TNF-α treatment). e Quantitative analysis of weight loss with SPINK4 and gefitinib intervention following DSS administration in the control group drinking water (control, n = 4); DSS and PBS administration group (DSS + PBS, n = 6); DSS and rSPINK4 administration group (DSS + rSPINK4, n = 5); and DSS, rSPINK4, and gefitinib administration group (DSS + rSPINK4 + gefitinib, n = 4). f Representative canonical endoscopic images with the lesion circled with white arrows. g Quantification of colonic length. h The levels of the phosphorylated and total EGFR were determined in WT and KO mouse tissues (upper panel) and rSPINK4 treatment group (lower panel). Data are presented as the mean ± SEM. All tests were two-sided. Statistical significance was calculated using unpaired Student’s t test ( h ). Besides, Kruskal–Wallis test ( h ) and one-way ANOVA ( b , e , g ) were performed for multiple comparison; n = 3–5 biologically independent experiments ( b , g , h ). Source data are provided as a Source Data file.

Article Snippet: The organoids generated from both human and mouse tissues were cultured in the medium without EGF, which synergistically affects the EGFR pathway, and supplemented with 100 ng/mL rSPINK4 and 10 μM AG1478 (MCE, USA) for 48 h. The inflammatory condition could be developed using 50 ng/mL TNF-α.

Techniques: Expressing, Western Blot, Control, Comparison

Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a The FLAG-SPINK4 protein, which exists in the concentrated medium of SPINK4- overexpressing cells, is involved in the EGFR protein from cell lysis combined with EGFR antibody. IgG is the contrast to EGFR antibody. b The binding test of rSPINK4 protein and EGFR in membrane extraction under the incubation in vitro. c rSPINK4 coprecipitates with rEGFR (extracellular part) mutually in the in vitro pull-down assay controlled by IgG. d Representative immunofluorescent image of rSPINK4 and EGFR localization (EGFR: green, rSPINK4: red, DAPI: blue). e The curve of released heat and change in △H are performed when the rEGFR-His protein (6 μM) is titrated by rSPINK4-His protein (30 μM) via the ITC assay. f The pull-down assay of rEGF and rEGFR (extracellular part) in a SPINK4-dose-dependent manner. The concentration of rSPINK4 is shown. g A typical binding curve between biotinylated EGF and EGFR was performed using AlphaLISA binding assay. h Image profile of the interaction between rSPINK4 and the deleted extracellular domain of EGFR (△1: the deletion of 57–168 aa, △2: the deletion of 177–338 aa, △3: the deletion of 361–481 aa, △4: the deletion of 505–637 aa, ED: whole extracellular EGFR, NC: vector). i Interaction between mutant rSPINK4 (wt: whole sequence of SPINK4, mut1: mutation sites: C65A, C68A, and C86A, mut2: mutation sites: Q48A and M49A, NC: vector) and extracellular EGFR in vitro. j Predictive interactive model of SPINK4 and extracellular EGFR (pink: SPINK4, the tail is at N-terminal; red: EGFR subdomain including 57–168 aa; green: EGFR subdomain including 177–338 aa; yellow: EGFR subdomain including 361–481 aa; orange: EGFR subdomain including 505–637 aa). Data are presented as the mean ± SEM. The Statistical test was two-sided using one-way ANOVA ( g ); n = 6 biologically independent experiments ( g ). Source data are provided as a Source Data file.

Article Snippet: The organoids generated from both human and mouse tissues were cultured in the medium without EGF, which synergistically affects the EGFR pathway, and supplemented with 100 ng/mL rSPINK4 and 10 μM AG1478 (MCE, USA) for 48 h. The inflammatory condition could be developed using 50 ng/mL TNF-α.

Techniques: Lysis, Binding Assay, Membrane, Extraction, Incubation, In Vitro, Pull Down Assay, Isothermal Titration Calorimetry, Concentration Assay, Plasmid Preparation, Mutagenesis, Sequencing