Journal: Archives of Dermatological Research

Article Title: EGFR influences the resistance to targeted therapy in BRAF V600E melanomas by regulating the ferroptosis process

doi: 10.1007/s00403-025-03895-8

Figure Lengend Snippet: Construction of A375 cells with high EGFR expression and validation of their drug resistance in vitro. A, B,C . High expression of EGFR was confirmed by fluorescence detection (green is GFP), PCR and Western blot (WB) in A375 EGFR+ stable cell line, * indicates P < 0.05; D, E, F .Comparison of drug resistance of A375 cells (NC group) and A375 EGFR + cells (EGFR + group) to vemurafenib, trametinib and SCH772984. * P < 0.05.Drug response curves are shown as mean ± SEM of three replicates and are representative of at least three independent experiments. NC group (A375): human BRAFV600E melanoma A375 cells; EGFR + group (A375EGFR+): A375 cells overexpressing EGFR; treated groups NC-V and EGFR+-V with the BRAF inhibitor Vemurafenib (MCE, HY-12057); NC-T and EGFR+-T treated with the MEK inhibitor Trametinib (MCE, HY-10999); and NC-S and EGFR+-S treated with the ERK inhibitor SCH772984 (MCE, HY-50846)

Article Snippet: NC group (A375): human BRAFV600E melanoma A375 cells; EGFR + group (A375EGFR+): A375 cells overexpressing EGFR; treated groups NC-V and EGFR+-V with the BRAF inhibitor Vemurafenib (MCE, HY-12057); NC-T and EGFR+-T treated with the MEK inhibitor Trametinib (MCE, HY-10999); and NC-S and EGFR+-S treated with the ERK inhibitor SCH772984 (MCE, HY-50846) Fig. 5 Measurement of the IC50 of ferroptosis-induced BRAFV600E melanoma cells for vemurafenib.

Techniques: Expressing, Biomarker Discovery, In Vitro, Fluorescence, Western Blot, Stable Transfection, Comparison

Journal: Archives of Dermatological Research

Article Title: EGFR influences the resistance to targeted therapy in BRAF V600E melanomas by regulating the ferroptosis process

doi: 10.1007/s00403-025-03895-8

Figure Lengend Snippet: Measurement of the IC50 of ferroptosis-induced BRAFV600E melanoma cells for vemurafenib. A375 and A375EGFR + cells were pretreated with DMSO and 5 μm artesunate for 8 h, respectively, to calculate the IC50 values of vemurafenib. A , C. IC50 determination for A375 cells induced with vemurafenib; B. D. IC50 determination for A375 EGFR+ cells induced with vemurafenib; E.F. Percentages of viable cells calculated relative to DMSO. Drug response curves are shown as mean ± SEM of three replicates and are representative of at least three independent experiments

Article Snippet: NC group (A375): human BRAFV600E melanoma A375 cells; EGFR + group (A375EGFR+): A375 cells overexpressing EGFR; treated groups NC-V and EGFR+-V with the BRAF inhibitor Vemurafenib (MCE, HY-12057); NC-T and EGFR+-T treated with the MEK inhibitor Trametinib (MCE, HY-10999); and NC-S and EGFR+-S treated with the ERK inhibitor SCH772984 (MCE, HY-50846) Fig. 5 Measurement of the IC50 of ferroptosis-induced BRAFV600E melanoma cells for vemurafenib.

Techniques:

Journal: Advanced Science

Article Title: Scaffolding Protein ENH Promotes Tumor Angiogenesis and Growth Through Macrophage Recruitment and Polarization

doi: 10.1002/advs.202416476

Figure Lengend Snippet: CCL5 is essential for ENH‐induced TAMs recruitment, tumor growth, and angiogenesis. A)Schematic for CCL5 signaling blocking in C57BL/6 mice implanted subcutaneously with LLC tumor cells. B) LLC‐Vector and LLC‐ENH‐OV tumor growth in mice treated with or without maraviroc ( n = 6). C) Weight of dissected tumors obtained from mice of indicated groups ( n = 6). D) Representative images of IF staining for CD31 and IHC staining for F4/80 in LLC‐Vector and LLC‐ENH‐OV tumors sections treated with or without maraviroc. E,F) The ratio of F4/80 positive cells and CD31 + vessel density was quantified and shown as a bar graph ( n = 6).

Article Snippet: Mice were treated with clodronate liposomes (Yeasen, Shanghai, China; i.p., 200 μL, every 3 days), cisplatin (Yeasen, Shanghai, China; i.v., 4 mg kg −1 , every 3 days), maraviroc (MCE, Shanghai, China; i.p., 10 mg kg −1 , every other day), or corresponding controls.

Techniques: Blocking Assay, Plasmid Preparation, Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Scaffolding Protein ENH Promotes Tumor Angiogenesis and Growth Through Macrophage Recruitment and Polarization

doi: 10.1002/advs.202416476

Figure Lengend Snippet: ENH activates macrophage STAT3 signaling through CCL5 to promote M2 polarization. A) Schematic model of the co‐culture system of THP1 and human primary monocytes‐drived macrophages with LUAD cells. B) Representative images of THP‐1 cells before and after PMA treatment. C) Flow cytometric analysis of CD68 expression in PMA‐treated or untreated THP‐1 cells. D) Flow cytometric analysis of CD206 expression in THP1‐drived macrophages co‐cultured with ENH knockdown LUAD cells. The mean fluorescence intensities (MFI) value of each group was represented in the histogram ( n = 3). E,F) The mRNA levels of M2 markers in THP1‐drived macrophages co‐cultured with ENH knockdown LUAD cells were measured by qPCR (n = 3). G) The correlation of ENH with M2 macrophage infiltration in LUAD was analyzed using the TIMER 2.0 database. H, I) Flow cytometric analysis of CD206 expression in THP1‐drived macrophages co‐cultured with ENH overexpression and CCL5 knockdown LUAD cells. The MFI value of each group was represented in histogram ( n = 3). J) Western blot analysis of STAT3/p‐STAT3 levels in THP1‐drived macrophages co‐cultured with ENH knockdown LUAD cells. K) Western blot analysis of STAT3/p‐STAT3 levels in THP1‐drived macrophages co‐cultured with ENH overexpression and CCL5 knockdown LUAD cells. L,M) Flow cytometric analysis of CD206 expression in THP1‐drived macrophages co‐cultured with ENH overexpression LUAD cells treated with or without STAT3 inhibitor (STAT3‐IN). The MFI value of each group was represented in the histogram ( n = 3). N,O) Flow cytometric analysis of CD206 expression in THP1‐ and human primary monocytes‐derived macrophages co‐cultured with ENH overexpression LUAD cells treated with or without maraviroc (MRC). The MFI value of each group was represented in histogram ( n = 3).

Article Snippet: Mice were treated with clodronate liposomes (Yeasen, Shanghai, China; i.p., 200 μL, every 3 days), cisplatin (Yeasen, Shanghai, China; i.v., 4 mg kg −1 , every 3 days), maraviroc (MCE, Shanghai, China; i.p., 10 mg kg −1 , every other day), or corresponding controls.

Techniques: Co-Culture Assay, Expressing, Cell Culture, Knockdown, Fluorescence, Over Expression, Western Blot, Derivative Assay

Journal: Molecular Oncology

Article Title: Exosomes secreted by ST3GAL5 high cancer cells promote peritoneal dissemination by establishing a premetastatic microenvironment

doi: 10.1002/1878-0261.13524

Figure Lengend Snippet: Incorporation of ST3G5 high ‐cExo into macrophages accelerated mesothelial–mesenchymal transition (MMT). (A, B) Macrophages (MΦs) were treated by exosomes of 44As3 cells (wt or ST3G5 KO clones; 5 μg·mL −1 ) for 2 days, and the cells were subjected to immunoblot analysis (A, representative results, n = 2), or quantitative real‐time PCR (qRT‐PCR; B, n = 3). (B) Mean ± SD, * P < 0.05, ** P < 0.01, Student's t ‐test. (C) Rat peritoneal mesothelial cells (PMCs) were treated by 44As3‐cExos as above for 5 days. Cell lysates were prepared for immunoblot analysis ( n = 2). (D) Cell surface total proteins of PMCs were labeled by membrane‐impermeable biotin. MΦs were incubated with exosomes as above for 2 days, and mixed with the biotin‐labeled PMCs (1 : 1 Ratio) and co‐cultured for 3 days. Biotin + cells were collected using streptavidin mag‐beads column, and then, the contaminated MΦs were sequentially removed by CD11b and F4/80 mag‐beads columns. Biotin + /CD11b − /F4/80 − cells represent PMCs and subjected to immunoblot analysis ( n = 2). (A, C, D) The intensities of each band were quantified and normalized by β‐Actin or α‐tubulin. The relative ratios were described below the panel. (E) Representative optical microscope images of PMCs cultured alone or with MΦs‐incorporated wt or ST3G5 KO 44As3‐cExo (5 μg·mL −1 ). Bar, 50 μm. n = 3. (F) Co‐culture was performed as above after the cExo‐uptake MΦs were labeled by DiI (red; n = 3). The cells were fixed and immunostained with phalloidin and DAPI. Bar, 50 μm. (G, H) Expression of αSMA in PMC was analyzed by qRT‐PCR (G, n = 3) or immunofluorescence staining (H, Bar: 20 μm). In G, PMCs and MΦs were derived from rat and mouse, respectively; all primers used for PCR were designed from the rat sequence (i.e., they do not amplify the corresponding mouse gene). MVC: Maraviroc. In H, the percentage of the area of αSMA + cells in the total cell area was calculated in three independent images and summarized (right panel). (G, H) Mean ± SD, * P < 0.01, Student's t ‐test.

Article Snippet: ExoSparkler exosome membrane labeling kit (Dojindo, Red: 340‐09671, Green: 343‐09661), EZ‐Link Sulfo‐NHS‐LC‐biotin (membrane‐impermeable, Thermo Fisher Scientific, Waltham, MA, USA, 21335), GM3, and NGcGM3 (Cayman, Ann Arbor, MI, USA, GM3: 15587, NGcGM3: 33263), Alexa546‐streptavidin (Life Science, Waltham, MA, USA, S11225), Maraviroc (TargetMol, Boston, MA, USA, T6016).

Techniques: Exosomes, Clone Assay, Western Blot, Analysis, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Labeling, Membrane, Incubation, Cell Culture, Microscopy, Co-culture Assay, Expressing, Immunofluorescence, Staining, Derivative Assay, Sequencing

Journal: Molecular Oncology

Article Title: Exosomes secreted by ST3GAL5 high cancer cells promote peritoneal dissemination by establishing a premetastatic microenvironment

doi: 10.1002/1878-0261.13524

Figure Lengend Snippet: ST3G5 high ‐cExo caused immunosuppression in milky spots (MS) and accelerated omentum metastasis. (A) Exosomes of wt (a) or ST3G5 KO ‐B16 cells (b) (30 μg, each) were i.p injected into C57BL/6 mice ( n = 5, each group), and wt B16 cells (1 × 10 6 ) were i.p injected on Day 5. Mice were sacrificed on Day 10. (Control mice without preinjection of exosomes were shown in Fig. D.) Upper panels: Representative images of the omentum and mesentery. Arrows indicate the omentum. Tumors were detected by melanin in cancer cells (black). Asterisk: stomach. Bar, 2.5 mm. Lower panels: Omentums were fixed, paraffin‐embedded, and sectioned for H&E staining (left) and immunostaining with anti‐CD8 (green), anti‐CD25 (red), and DAPI. Merged image (middle) and CD25 alone (right). Bar, 200 μm. (c) Tumor area in omentum was quantified by imagej software (examples were shown in right) and expressed as percentage of tumor area in total area of omentum ( n = 5, mean ± SD, * P < 0.05, ** P < 0.01, Student's t ‐test). (d) The number of tumors in mesentery was counted and analyzed the significance of the difference by Student's t ‐test. Results are shown as means ± SD ( n = 5, * P < 0.01, Student's t ‐test). (e) The area of CD8 + CD25 + T cells in omentum tumors was measured and expressed as the percentage of the tumor area in each specimen. Five tumors were analyzed individually (mean ± SD, * P < 0.01, ** P < 0.006, Student's t ‐test). (B) Omentums were excised from C57BL/6 mice and cultured in medium containing Wt or ST3G5 KO ‐B16‐cExo (10 μg·mL −1 ), or left untreated for 3 days. Omentums were dispersed and subjected to FACS as indicated. Mean ± SD, * P < 0.05, ** P < 0.01, Student's t ‐test. (C) Omentum explants were treated by ExoSparkler‐labeled cExo (red) as above. In some experiments, Maraviroc (10 μg·mL −1 ) was added together with the exosomes. EGFP + B16 cells were added to these explants (5 × 10 5 cells per well) and further incubated for 3 days. Representative appearance of the omentum. Left: Pseudo‐color image of the co‐localization of B16 cells and cExo on the explant. Tumor area was measured by quantification of green fluorescence (EGFP) and normalized to the cExo area (red) in the omentum. Bar, 1.5 mm. The results are shown by the relative ratio of the explant treated by wt B16‐cExo without inhibitor. Mean ± SD, * P < 0.01, Student's t ‐test. (B, C) Five explants were analyzed in each group.

Article Snippet: ExoSparkler exosome membrane labeling kit (Dojindo, Red: 340‐09671, Green: 343‐09661), EZ‐Link Sulfo‐NHS‐LC‐biotin (membrane‐impermeable, Thermo Fisher Scientific, Waltham, MA, USA, 21335), GM3, and NGcGM3 (Cayman, Ann Arbor, MI, USA, GM3: 15587, NGcGM3: 33263), Alexa546‐streptavidin (Life Science, Waltham, MA, USA, S11225), Maraviroc (TargetMol, Boston, MA, USA, T6016).

Techniques: Exosomes, Injection, Control, Staining, Immunostaining, Software, Cell Culture, Labeling, Incubation, Fluorescence, FACS, Immunofluorescence

Journal: Cancer research

Article Title: Inducible nitric oxide synthase (iNOS) drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2

doi: 10.1158/0008-5472.CAN-13-0588

Figure Lengend Snippet: A375 cells were transfected with (A) iNOS-targeting or control siRNA or (B) the indicated concentrations of L-NIL and CAM assay performed as described in Figure 1. Whole cell lysates were subjected to SDS-PAGE and western blot analysis for mTOR phosphorylated at serine 2448 (Ser2448), p-P70S6K phosphorylated at threonine 389 (Thr389), p-4EBP1 phosphorylated at threonine 37/46 (Thr37/46), p-S6RP phospho at serine 235/236 (Ser235/236), and actin as a loading control. (C) MeWo cells were transfected with iNOS-targeting or control siRNA before CAM assay and western blot for mTOR, p-P70S6K, p-4EBP1, p-S6RP, and actin. (D) Quantitation of phospho-4EBP1 versus total protein and phospho-S6RP versus actin were by densitometric analysis. All graphs represent data derived from 3 independent experiments.

Article Snippet: Western blots Following SDS-PAGE and transfer to nitrocellulose, membranes were incubated overnight at 4°C with primary antibodies against iNOS (sc-651, Santa Cruz Biotech), mTOR (sc-2983 Santa Cruz), p-mTOR Ser 2448 (sc-2971 Santa Cruz), Phospho-4E-BP1 (Thr37/46) (sc-9459, Santa Cruz), 4E-BP1 (sc-9977, Santa Cruz), Tuberin/TSC2 (D57A9) (sc-3990 , Santa Cruz), Hamartin/TSC1 (sc-4906), Actin (sc-1616, Santa Cruz) followed by appropriate secondary antibody conjugated to horseradish peroxidase.

Techniques: Transfection, Control, Chick Chorioallantoic Membrane Assay, SDS Page, Western Blot, Quantitation Assay, Derivative Assay

Journal: Cancer research

Article Title: Inducible nitric oxide synthase (iNOS) drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2

doi: 10.1158/0008-5472.CAN-13-0588

Figure Lengend Snippet: (A) A375 Cells were transfected with GFP or murine GFP-iNOS constructs and imaged 48 hours later by fluorescence microscopy. Note that only iNOS-GFP-transfected cells display aggresomes (arrowhead). (B) To rescue the effects of iNOS knockdown, cells were transfected with iNOS siRNA, Scramble siRNA, murine GFP-iNOS, or GFP constructs alone or in combination before inoculation on the CAM as described above. Western blot analysis was performed to confirm the knockdown of endogenous iNOS and overexpression of murine GFP-iNOS; rescue of mTOR pathway activation by mouse GFP-iNOS was assayed by Western blot for the downstream pathway member S6RP. Actin was used as the loading control. One representative blot of 3 is shown. The bar graphs show the relative expression level (normalized to actin).

Article Snippet: Western blots Following SDS-PAGE and transfer to nitrocellulose, membranes were incubated overnight at 4°C with primary antibodies against iNOS (sc-651, Santa Cruz Biotech), mTOR (sc-2983 Santa Cruz), p-mTOR Ser 2448 (sc-2971 Santa Cruz), Phospho-4E-BP1 (Thr37/46) (sc-9459, Santa Cruz), 4E-BP1 (sc-9977, Santa Cruz), Tuberin/TSC2 (D57A9) (sc-3990 , Santa Cruz), Hamartin/TSC1 (sc-4906), Actin (sc-1616, Santa Cruz) followed by appropriate secondary antibody conjugated to horseradish peroxidase.

Techniques: Transfection, Construct, Fluorescence, Microscopy, Knockdown, Western Blot, Over Expression, Activation Assay, Control, Expressing

Journal: Cancer research

Article Title: Inducible nitric oxide synthase (iNOS) drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2

doi: 10.1158/0008-5472.CAN-13-0588

Figure Lengend Snippet: A375 cells were cultured with the indicated concentrations of L-NIL, the NO donor S-nitroso-acetyl-penicillamine (SNAP), or the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (PTIO) for 16 Hours prior to harvest and preparation of protein lysates. As a negative control, parallel lysates were exposed to 10 mM of SH-reducing agent dithiothreitol (DTT) for 1 hour in vitro. (A) Total S-nitrosylated protein levels were determined by biotin switch assay and anti-biotin immunoblot; “unbiotinylated” shows endogenous biotin levels. (B) S-Nitrosylation of TSC2 in biotin switch assay followed by immunoprecipitation with streptavidine agarose and immunoblot analysis with anti-TSC2 polyclonal antibody. “SNO” (S-nitrosothiol) indicates biotinylated (nitrosylated) TSC2 levels following biotin switch. “Total” indicates total TSC2 levels prior to immunoprecipitation. Actin was used as a loading control. (C) Effect of iNOS/NO on TSC2/TSC1 dimerization. A375 cells were treated with NO donors DEANONOate or SNAP, or the selective iNOS inhibitor L-NIL, and cell lysates were immunoprecipitated (IP) with anti-TSC2 antibody. Immunocomplexes (upper panel) and crude lysates (lower panel) were immunoblotted with anti-TSC1, -TSC2, or –actin antibody. The ratio of TSC1 to TSC2 was calculated and normalized against the amount of TSC2 present after the immnunoprecipitation (D) Effect of iNOS/NO on Rheb activation. A375 cells were transfected with iNOS-targeting siRNA or scrambled control and cultured for 48 hours. Western blots for activated Rheb (Rheb/GTP) total Rheb, including the inactive GDP-Bound form (GDP), and actin loading control were performed. Actived Rheb was quantified by densitometry and normalized to total Rheb levels. All graphs represent data from 3 independent experiments.

Article Snippet: Western blots Following SDS-PAGE and transfer to nitrocellulose, membranes were incubated overnight at 4°C with primary antibodies against iNOS (sc-651, Santa Cruz Biotech), mTOR (sc-2983 Santa Cruz), p-mTOR Ser 2448 (sc-2971 Santa Cruz), Phospho-4E-BP1 (Thr37/46) (sc-9459, Santa Cruz), 4E-BP1 (sc-9977, Santa Cruz), Tuberin/TSC2 (D57A9) (sc-3990 , Santa Cruz), Hamartin/TSC1 (sc-4906), Actin (sc-1616, Santa Cruz) followed by appropriate secondary antibody conjugated to horseradish peroxidase.

Techniques: Cell Culture, Negative Control, In Vitro, Biotin Switch Assay, Western Blot, Immunoprecipitation, Control, Activation Assay, Transfection

Journal: Cancer research

Article Title: Inducible nitric oxide synthase (iNOS) drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2

doi: 10.1158/0008-5472.CAN-13-0588

Figure Lengend Snippet: (A) Formalin-fixed paraffin-embedded (FFPE) tissue sections of tumor samples were stained for iNOS, nitrotyrosine (NT), phospho-4EBP1, or p70S6K expression by IHC. Pre-immune normal mouse IgG and anti-vimentin antibody were used as negative and positive controls, respectively. Hematoxylin was applied as a counter stain. 20× magnification. (B) Immunolabeling was scored separately for 2 variables: first, for number of positive melanoma cells; second, for the overall staining intensity of positive cells as described in the materials and methods, with 0 being the lowest composite score, and 3 the highest. The slides were independently interpreted by 2 readers without knowledge of the clinical data. (C) Effects of the BRAF inhibitor Vemurafenib (VEMU) and NO donor DEANONOATE (DEA) alone and in combination on mTOR pathway activation. A375 melanoma cells were exposed for 3 hours to 3 µM Vemurafenib, 100 µM of the donor DEANONOate, or the combination. mTOR pathway activation was analyzed by 4EBP1 and p-S6RP western blot. Signals were quantified by densitometry, and the ratio of activated p-4EBP1 and p-S6RP was calculated, normalized against total protein (right panel). All graphs represent data from 3 independent experiments.

Article Snippet: Western blots Following SDS-PAGE and transfer to nitrocellulose, membranes were incubated overnight at 4°C with primary antibodies against iNOS (sc-651, Santa Cruz Biotech), mTOR (sc-2983 Santa Cruz), p-mTOR Ser 2448 (sc-2971 Santa Cruz), Phospho-4E-BP1 (Thr37/46) (sc-9459, Santa Cruz), 4E-BP1 (sc-9977, Santa Cruz), Tuberin/TSC2 (D57A9) (sc-3990 , Santa Cruz), Hamartin/TSC1 (sc-4906), Actin (sc-1616, Santa Cruz) followed by appropriate secondary antibody conjugated to horseradish peroxidase.

Techniques: Formalin-fixed Paraffin-Embedded, Staining, Expressing, Immunolabeling, Activation Assay, Western Blot

Journal:

Article Title: Structural Basis of Focal Adhesion Localization of LIM-only Adaptor PINCH by Integrin-linked Kinase * S⃞

doi: 10.1074/jbc.M805319200

Figure Lengend Snippet: The effect of structure-based interface mutations on PINCH/ILK interaction and the PINCH localization to FAs. A, the expression levels of GFP vector alone, WT GFP-PINCH-1, F42A, and R56A. Lysates of C2C12 cells expressing GFP-tagged wild type or mutant (Phe-42 → Ala and Arg-56 → Ala) forms of PINCH were mixed with rabbit anti-GFP antibodies and measured by Western blotting. B, complex formation of WT PINCH and PINCH mutants with ILK. The GFP-PINCH (lane 2), GFP-F42A (lane 3), and GFP-R56A (lane 4) immunoprecipitates were analyzed by Western blotting with mouse monoclonal anti-ILK antibody 65.1 and horseradish peroxidase-conjugated anti-mouse IgG antibodies. C-H, subcellular localization. C2C12 cells transfected with expression vectors encoding GFP-WT PINCH (C and D), F42A (E and F), or R56A (G and H) were plated on fibronectin-coated coverslips and stained with a mouse monoclonal anti-Mig-2 antibody (as a marker of focal adhesions) and a Rhodamine Red™-conjugated anti-mouse IgG antibody. GFP-F42A, GFP-R56A, GFP-PINCH, and Mig-2 were visualized under a fluorescence microscope equipped with GFP (C, E, and G) and rhodamine (D, F, and H) filters. Bar, 10 μm.

Article Snippet: After washing, the precipitated proteins were released from the beads by boiling in 30 μl of SDS-PAGE sample buffer for 5 min and analyzed by Western blotting with a polyclonal anti-GFP antibody (Santa Cruz Biotechnology) or a monoclonal anti-ILK antibody (clone 65.1).

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Transfection, Staining, Marker, Fluorescence, Microscopy

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Phytosomal curcumin causes natural killer cell-dependent repolarization of glioblastoma (GBM) tumor-associated microglia/macrophages and elimination of GBM and GBM stem cells

doi: 10.1186/s13046-018-0792-5

Figure Lengend Snippet: Peripheral injection of the NK cell-neutralizing NK1.1 antibody partially reverses the CCP-mediated M2➔M1 repolarization, IL10 suppression, and IL12 induction in TAM. a The dispersed Iba1(+) cells in the CCP-treated GBM displayed an 80% decrease in integrated ARG1 fluorescence with respect to the Vehicle-treated (* p = 0.01, Vehicle versus CCP) and this decrease was only 60% in the CCP + NK1.1 samples (Δ p = 0.03, CCP versus CCP + NK1.1; ** p = 0.02, Vehicle versus CCP + NK1.1) ( a i - v ). Compared to the iNOS IF values in the Vehicle-treated, the Iba1(+) cells in the other two groups displayed a 293% increase in the CCP-treated (* p = 0.004, CCP versus Vehicle) and an 89% increase in the CCP + NK1.1-treated (Δ p = 0.014 CCP + NK1.1 versus CCP; ** p = 0.04, Vehicle versus CCP + NK1.1) ( b i-v ). The graphs represent mean ± S.D. for iNOS/ARG1 obtained from Vehicle ( n = 4), CCP (n = 4), and CCP + NK1.1 ( n = 3) mice. c - f The Iba1(+) cells displayed a 92% decrease IL10 IF in the CCP-treated (* p = 8.7 × 10 − 6 , CCP versus Vehicle) and only a 65% decrease in the CCP + NK1.1-treated samples (Δ p = 0.03, CCP + NK1.1 versus CCP; ** p = 5.0 × 10 − 3 , Vehicle versus CCP + NK1.1) ( c i - iv and e ). The Iba1(+) cells displayed a 445% increase in IL12 IF in the CCP-treated mice (* p = 0.01, CCP versus Vehicle) and a 69% increase in the CCP + NK1.1 mice (Δ p = 0.01, CCP + NK1.1 versus CCP; ** p = 0.04, Vehicle versus CCP + NK1.1) ( d i - iv and f ). The graphs represent mean ± S.D. obtained from Vehicle ( n = 4), CCP ( n = 4), and CCP + NK1.1 ( n = 3) mice

Article Snippet: Antibodies against Iba1 (C20) (1:50), iNOS (rabbit IgG) (NOS2 sc-651) (1:100), ARG1 (rabbit IgG) (sc-20,150) (1:100), NKp46 (1:100), IL12p40 (1:50), IL10 (1:50), CD68 (H-255) (Rabbit IgG) (sc-9139) (1:50), Active-Caspase3 (Asp175) (Rabbit IgG) (CST #9661) (1:100), and CD133 (1:50) were used for staining.

Techniques: Injection, Fluorescence

Journal: Discover Oncology

Article Title: Identification of the disulfidptosis-related gene G6PD as a potential biomarker and therapeutic target in pancreatic ductal adenocarcinoma by integrative bioinformatics analysis and experimental validation

doi: 10.1007/s12672-026-04753-3

Figure Lengend Snippet: Variations in G6PD Expression Across Pan-Cancer Types. A Expression levels of G6PD mRNA in normal tissues and pan-cancer tissues were determined using the TCGA and GTEx datasets. B G6PD protein expression levels provided by the HPA database. C, D Relationships between G6PD expression and tumor clinical stage ( C ) and tumor histological grade ( D ). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The G6PD inhibitor RRx-001 was obtained from Med Chem Express (MCE) and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution.

Techniques: Expressing

Journal: Discover Oncology

Article Title: Identification of the disulfidptosis-related gene G6PD as a potential biomarker and therapeutic target in pancreatic ductal adenocarcinoma by integrative bioinformatics analysis and experimental validation

doi: 10.1007/s12672-026-04753-3

Figure Lengend Snippet: Expression of G6PD in PAAD. A Boxplot showing differential G6PD expression between PAAD tumor tissues and normal tissues. B-D G6PD expression levels in three GEO cohorts. E RT-qPCR detection of G6PD mRNA expression levels. F Immunohistochemical sections of G6PD from the HPA database. G Analysis of G6PD protein expression levels using the CPTAC database. H Western blot analysis of G6PD protein levels. I Quantification of the Western blot results. J Immunohistochemical staining of G6PD protein expression in pancreatic cancer tissues. K Quantification of the IHC staining data. L RT-qPCR assessment of G6PD mRNA levels in pancreatic cancer tissues. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The G6PD inhibitor RRx-001 was obtained from Med Chem Express (MCE) and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Western Blot, Staining, Immunohistochemistry

Journal: Discover Oncology

Article Title: Identification of the disulfidptosis-related gene G6PD as a potential biomarker and therapeutic target in pancreatic ductal adenocarcinoma by integrative bioinformatics analysis and experimental validation

doi: 10.1007/s12672-026-04753-3

Figure Lengend Snippet: Prognostic Value of G6PD Expression in PAAD. A Kaplan-Meier survival curves comparing DFS in patients with different G6PD expression levels. B ROC curve analysis of the diagnostic potential of G6PD in PAAD. ( C ) Time-dependent ROC analysis. D-J Graphical representation of the association between G6PD mRNA expression and various clinical variables in PAAD patients, including clinical stage ( D ), histological grade ( E ), T stage ( F ), N stage ( G ), M stage ( H ), age ( I ), and gender ( J ). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The G6PD inhibitor RRx-001 was obtained from Med Chem Express (MCE) and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution.

Techniques: Expressing, Diagnostic Assay

Journal: Discover Oncology

Article Title: Identification of the disulfidptosis-related gene G6PD as a potential biomarker and therapeutic target in pancreatic ductal adenocarcinoma by integrative bioinformatics analysis and experimental validation

doi: 10.1007/s12672-026-04753-3

Figure Lengend Snippet: Correlation Analysis of G6PD Expression with Immune Cell Infiltration in PAAD. A Lollipop plot showing the correlation between G6PD expression and infiltration of 24 immune cell types. B Scatter plot displaying the correlation between G6PD expression and various immune cells. C Dot plot of immune infiltration scores for high and low G6PD expression groups. D-F Scatter plots showing the correlation between G6PD and ImmuneScore ( D ), StromalScore ( E ), and ESTIMATEScore ( F ). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The G6PD inhibitor RRx-001 was obtained from Med Chem Express (MCE) and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution.

Techniques: Expressing

Journal: Discover Oncology

Article Title: Identification of the disulfidptosis-related gene G6PD as a potential biomarker and therapeutic target in pancreatic ductal adenocarcinoma by integrative bioinformatics analysis and experimental validation

doi: 10.1007/s12672-026-04753-3

Figure Lengend Snippet: Correlation of G6PD Expression with Immune Regulatory Genes. A-C Correlation of G6PD expression with three types of immune regulators (including immunosuppressive agents, immunostimulatory agents, and MHC-related molecules). D, E Correlation of G6PD expression with chemokines and their receptors. On the right side of each panel, the two cells/molecules with the highest positive or negative correlation with G6PD expression are shown. F, G The linked medications that target G6PD were predicted using the CTRP ( F ) and GDSC ( G ) databases. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The G6PD inhibitor RRx-001 was obtained from Med Chem Express (MCE) and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution.

Techniques: Expressing, Medications

Journal: Discover Oncology

Article Title: Identification of the disulfidptosis-related gene G6PD as a potential biomarker and therapeutic target in pancreatic ductal adenocarcinoma by integrative bioinformatics analysis and experimental validation

doi: 10.1007/s12672-026-04753-3

Figure Lengend Snippet: Functional Enrichment Analysis of G6PD-Interacting and Co-Expressed Genes. A Co-expression heatmap of the top 20 genes most strongly correlated with G6PD. B-E GO and KEGG enrichment analysis of genes correlated with G6PD expression. F-G Gene Set Enrichment Analysis (GSEA) of PAAD patient cohorts stratified by G6PD expression levels. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The G6PD inhibitor RRx-001 was obtained from Med Chem Express (MCE) and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution.

Techniques: Functional Assay, Expressing

Journal: Discover Oncology

Article Title: Identification of the disulfidptosis-related gene G6PD as a potential biomarker and therapeutic target in pancreatic ductal adenocarcinoma by integrative bioinformatics analysis and experimental validation

doi: 10.1007/s12672-026-04753-3

Figure Lengend Snippet: G6PD Inhibitor RRx-001 Activates Disulfide-Mediated Cell Death in SW1990 Cells. A Molecular docking and optimization of RRx-001 with G6PD. B CCK-8 assay to detect the proliferation capacity of SW1990 cells. C-E Flow cytometry analysis of SW1990 cell death rates. F WST-8 colorimetric assay to measure NADPH levels in SW1990 cells. G Fluorescence microplate assay to determine cystine uptake capacity in SW1990 cells. H Representative immunofluorescence images of F-actin (phalloidin staining) and nuclei (DAPI) in SW1990 cells treated with RRx-001 or vehicle control. I Quantification of immunofluorescence staining. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The G6PD inhibitor RRx-001 was obtained from Med Chem Express (MCE) and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution.

Techniques: CCK-8 Assay, Flow Cytometry, Colorimetric Assay, Fluorescence, Immunofluorescence, Staining, Control

Journal: ACS Omega

Article Title: Using Bibliometric Analysis and Machine Learning to Identify Compounds Binding to Sialidase-1

doi: 10.1021/acsomega.0c05591

Figure Lengend Snippet: In vitro data was generated using Microscale Thermophoresis for sulfameter and mexenone. The binding analysis for the interaction between sialidase-1 and these compounds is presented. The concentration of labeled sialidase-1 is kept constant at 5 nM, while the ligand concentration varies from 250 μM and 7.629 nM. The serial titrations result in measurable changes in the fluorescence signal within a temperature gradient that can be used to calculate the dissociation constant K d = 2.15 ( K d confidence ± 1.02 μM) for sulfameter and K d = 8.88 μM ( K d confidence ± 4.02 μM) for mexenone. The curve is shown as Fraction Bound [-] against compound concentration on a log scale.

Article Snippet: Mexenone and sulfameter were purchased from MedChem Express (Monmouth Junction, NJ).

Techniques: In Vitro, Generated, Microscale Thermophoresis, Binding Assay, Concentration Assay, Labeling, Fluorescence

Journal: Biomedical Optics Express

Article Title: Tomographic STED microscopy

doi: 10.1364/BOE.391787

Figure Lengend Snippet: Bleaching experiments for Abberior STAR 635P-stained vimentin network in fixed Vero cells. (a) Ten consecutive frames displayed for 2D STED and for tomoSTED (MFV reconstruction with N=Nopt=6). Total pixel dwell time (30 μs) and pixel size (15 nm) are equal for both methods compared (scale bar: 1 μm). (b) Normalized fluorescence intensity vs. number of frames. The data for tomoSTED with N=Nopt/2=3 depletion pattern orientations is derived by allocating the N=Nopt=6 data into two separate data sets. Error bars denote the standard error of the mean.

Article Snippet: Several washing steps are followed by an incubation time of 1 hour for the secondary antibody (Abberior Star 635P goat anti-mouse, red fluorescent (635/651), 1:50 in BS; Abberior , Germany) at room temperature.

Techniques: Staining, Fluorescence, Derivative Assay