e 800 eclipse nikon fluorescent microscope Search Results


eclipse ti series microscope  (Nikon)
93
Nikon eclipse ti series microscope
Eclipse Ti Series Microscope, supplied by Nikon, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/MM-400%2F800+series/pmc08482497-135-9-8
Average 93 stars, based on 1 article reviews
eclipse ti series microscope - by Bioz Stars, 2026-09
93/100 stars
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inverted fluorescence microscope  (Olympus)
99
Olympus inverted fluorescence microscope
Inverted Fluorescence Microscope, supplied by Olympus, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/CKX53+Cell+Culture+Microscope/pm37529915-308-10-14
Average 99 stars, based on 1 article reviews
inverted fluorescence microscope - by Bioz Stars, 2026-09
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antibody amhr2 neutralizing antibody r d systems  (R&D Systems)
93
R&D Systems antibody amhr2 neutralizing antibody r d systems
Figure 2. In utero pharmacological invalidation of <t>Amhr2</t> disrupts GnRH neuronal migration and the olfactory/terminal nerve targeting. (a) Schematic of in utero injections targeting the olfactory pits. Injections were performed at E12.5 and embryos harvested 48 hr later. (b) Representative coronal section of an embryo head at E14.5 showing that olfactory pit Fluorogold delivery at E12.5 was successful. GnRH immunoreactive neurons are shown in green. (c–f) Representative photomicrographs of sagittal sections of mouse embryos injected at E12.5 with either saline or a neutralizing antibody for Amhr2 (Amhr2-NA) and immunostained for GnRH (green) and Peripherin (magenta) at E14.5. (e, f) Higher magnification confocal photomicrograph of boxed areas in c and d. (g) Quantification of the total number of GnRH immunoreactive neurons in saline-injected (control) and Amhr2-NA injected embryos (n = 4 for both groups, harvested from two independent dams). Data are represented as mean ± s.e.m (n = 4, unpaired two-tailed Student’s t test: mean cell number, t6 = 0.3796, p = 0.7173). (h) Quantitative analysis of GnRH neuronal distribution throughout the migratory pathway in the two experimental groups. Data are represented as mean ± s.e.m (n = 4, two-way ANOVA, F3,24 = 15.09, p<0.0001; followed by Holm-Sˇ ı´da´k multiple Figure 2 continued on next page
Antibody Amhr2 Neutralizing Antibody R D Systems, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/Rat+MIS+RII+Antibody/10__7554_slash_elife__47198-272-60-64
Average 93 stars, based on 1 article reviews
antibody amhr2 neutralizing antibody r d systems - by Bioz Stars, 2026-09
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em-ccd digital camera c9100  (Hamamatsu)
90
Hamamatsu em-ccd digital camera c9100
Figure 2. In utero pharmacological invalidation of <t>Amhr2</t> disrupts GnRH neuronal migration and the olfactory/terminal nerve targeting. (a) Schematic of in utero injections targeting the olfactory pits. Injections were performed at E12.5 and embryos harvested 48 hr later. (b) Representative coronal section of an embryo head at E14.5 showing that olfactory pit Fluorogold delivery at E12.5 was successful. GnRH immunoreactive neurons are shown in green. (c–f) Representative photomicrographs of sagittal sections of mouse embryos injected at E12.5 with either saline or a neutralizing antibody for Amhr2 (Amhr2-NA) and immunostained for GnRH (green) and Peripherin (magenta) at E14.5. (e, f) Higher magnification confocal photomicrograph of boxed areas in c and d. (g) Quantification of the total number of GnRH immunoreactive neurons in saline-injected (control) and Amhr2-NA injected embryos (n = 4 for both groups, harvested from two independent dams). Data are represented as mean ± s.e.m (n = 4, unpaired two-tailed Student’s t test: mean cell number, t6 = 0.3796, p = 0.7173). (h) Quantitative analysis of GnRH neuronal distribution throughout the migratory pathway in the two experimental groups. Data are represented as mean ± s.e.m (n = 4, two-way ANOVA, F3,24 = 15.09, p<0.0001; followed by Holm-Sˇ ı´da´k multiple Figure 2 continued on next page
Em Ccd Digital Camera C9100, supplied by Hamamatsu, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/em+ccd+camera/10__1074_slash_jbc__m802272200-81-14-13
Average 90 stars, based on 1 article reviews
em-ccd digital camera c9100 - by Bioz Stars, 2026-09
90/100 stars
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fluorescence microscope bz-800  (KEYENCE)
90
KEYENCE fluorescence microscope bz-800
Figure 2. In utero pharmacological invalidation of <t>Amhr2</t> disrupts GnRH neuronal migration and the olfactory/terminal nerve targeting. (a) Schematic of in utero injections targeting the olfactory pits. Injections were performed at E12.5 and embryos harvested 48 hr later. (b) Representative coronal section of an embryo head at E14.5 showing that olfactory pit Fluorogold delivery at E12.5 was successful. GnRH immunoreactive neurons are shown in green. (c–f) Representative photomicrographs of sagittal sections of mouse embryos injected at E12.5 with either saline or a neutralizing antibody for Amhr2 (Amhr2-NA) and immunostained for GnRH (green) and Peripherin (magenta) at E14.5. (e, f) Higher magnification confocal photomicrograph of boxed areas in c and d. (g) Quantification of the total number of GnRH immunoreactive neurons in saline-injected (control) and Amhr2-NA injected embryos (n = 4 for both groups, harvested from two independent dams). Data are represented as mean ± s.e.m (n = 4, unpaired two-tailed Student’s t test: mean cell number, t6 = 0.3796, p = 0.7173). (h) Quantitative analysis of GnRH neuronal distribution throughout the migratory pathway in the two experimental groups. Data are represented as mean ± s.e.m (n = 4, two-way ANOVA, F3,24 = 15.09, p<0.0001; followed by Holm-Sˇ ı´da´k multiple Figure 2 continued on next page
Fluorescence Microscope Bz 800, supplied by KEYENCE, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/fluorescence+microscope+bz+9000/pm40619412-174-12-15
Average 90 stars, based on 1 article reviews
fluorescence microscope bz-800 - by Bioz Stars, 2026-09
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pbmc cells  (ATCC)
99
ATCC pbmc cells
Inhibition of GBM cancer cell development by suppressing M2 macrophage infiltration through activation of TAM‐iron death pathway. (A) Flow cytometry was used to determine the proportion of M2 macrophages. (B) BMDMs were transfected with lentivirus for knockdown of MS4A4A or negative control plasmid, then induced into M2 phenotype using IL‐4 (20 ng/mL) and IL‐13 (20 ng/mL). qRT‐PCR was used to measure the interference efficiency of MS4A4A and expression of M2 markers (Mgl2, Arg1, and Tgfb1) and M1 marker iNOS. <t>(C)</t> <t>THP‐1</t> human monocytic cell line was used to establish an overexpression cell line of MS4A4A, followed by differentiation into M0 macrophages using PMA (50 ng/mL). M0 macrophages were then polarized into M2 phenotype using IL‐4 (20 ng/mL) and qRT‐PCR was used to measure expression levels of MS4A4A and M2 markers (CD163, ARG1, and TGFB1) and M1 marker CD86. (D) <t>PBMC</t> cells were used to construct a cell line overexpressing MS4A4A. (E) Procedure for testing macrophage inhibition function: Mouse BMDMs and spleen cells were mixed with CT2A/GL261 cells, treated with MS4A4A antibody, and CD8 + T cells were sorted using flow cytometry and Ki‐67 expression was measured. (F) Analysis of Ki‐67 expression on designated CD8 + T cells using flow cytometry ( n = 3). (G) Apoptosis of CT2A cells was detected using fluorescence‐activated cell sorting (FACS). (H) Iron content in tumor‐associated macrophages (TAMs) of sh‐NC and sh‐MS4A4A groups. (I) Reactive oxygen species (ROS) content in TAM cells of sh‐NC and sh‐MS4A4A groups. (J) Expression of iron death‐related proteins in TAM cells of sh‐NC and sh‐MS4A4A groups. (K) Electron microscopy analysis of TAM cells in sh‐NC and sh‐MS4A4A groups. * p < 0.05, and all cell experiments were repeated three times.
Pbmc Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/Primary+Peripheral+Blood+Mononuclear+Cells+(PBMC)%2C+Normal%2C+Human/pmc11245405-208-65-68
Average 99 stars, based on 1 article reviews
pbmc cells - by Bioz Stars, 2026-09
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fluorescence nikon eclipse e 800 microscope  (Nikon)
99
Nikon fluorescence nikon eclipse e 800 microscope
Inhibition of GBM cancer cell development by suppressing M2 macrophage infiltration through activation of TAM‐iron death pathway. (A) Flow cytometry was used to determine the proportion of M2 macrophages. (B) BMDMs were transfected with lentivirus for knockdown of MS4A4A or negative control plasmid, then induced into M2 phenotype using IL‐4 (20 ng/mL) and IL‐13 (20 ng/mL). qRT‐PCR was used to measure the interference efficiency of MS4A4A and expression of M2 markers (Mgl2, Arg1, and Tgfb1) and M1 marker iNOS. <t>(C)</t> <t>THP‐1</t> human monocytic cell line was used to establish an overexpression cell line of MS4A4A, followed by differentiation into M0 macrophages using PMA (50 ng/mL). M0 macrophages were then polarized into M2 phenotype using IL‐4 (20 ng/mL) and qRT‐PCR was used to measure expression levels of MS4A4A and M2 markers (CD163, ARG1, and TGFB1) and M1 marker CD86. (D) <t>PBMC</t> cells were used to construct a cell line overexpressing MS4A4A. (E) Procedure for testing macrophage inhibition function: Mouse BMDMs and spleen cells were mixed with CT2A/GL261 cells, treated with MS4A4A antibody, and CD8 + T cells were sorted using flow cytometry and Ki‐67 expression was measured. (F) Analysis of Ki‐67 expression on designated CD8 + T cells using flow cytometry ( n = 3). (G) Apoptosis of CT2A cells was detected using fluorescence‐activated cell sorting (FACS). (H) Iron content in tumor‐associated macrophages (TAMs) of sh‐NC and sh‐MS4A4A groups. (I) Reactive oxygen species (ROS) content in TAM cells of sh‐NC and sh‐MS4A4A groups. (J) Expression of iron death‐related proteins in TAM cells of sh‐NC and sh‐MS4A4A groups. (K) Electron microscopy analysis of TAM cells in sh‐NC and sh‐MS4A4A groups. * p < 0.05, and all cell experiments were repeated three times.
Fluorescence Nikon Eclipse E 800 Microscope, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/Objectives/pm38866313-61-19-20
Average 99 stars, based on 1 article reviews
fluorescence nikon eclipse e 800 microscope - by Bioz Stars, 2026-09
99/100 stars
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rabbit mab cd45  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc rabbit mab cd45
Standardization and validation of CTC by IF×3 using breast, ovarian, and lung cancer cell lines: Patients’ blood samples spiked with titrating number (1000 cells, 750 cells, 375 cells, 250 cells/100 cells) of cell lines of different solid tumors using. Pictures were taken at 60× oil objective of an Olympus IX71 Microscope with DAPI/FITC/TRITC/CY5 filter sets. ( A ): MCF7 cells (750 cells/375 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with a CellSieve enumeration kit (Creatv Microtech) with either <t>DAPI/CK-FITC/EpCAM-PE/CD45-Cy5</t> ( Ai ) or DAPI/CK-FITC/CD31 PE/CD45-Cy5 ( Aii ). ( B ): OVCAR3 cells (100 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with cell sieve enumeration kit (Creatv MicroTech) with DAPI/CK-FITC/EpCAM-PE/CD45-Cy5. ( C ): HCC1975 cells (1000 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with cell sieve enumeration kit (Creatv Microtech) with DAPI/CK-FITC/EpCAM-PE/CD45-Cy5. ( D ): NCI-H441 cells (250 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with cell sieve enumeration kit (Creatv Microtech) with DAPI/CK-FITC/EpCAM-PE/CD45-Cy5. The magnification, scale bar, and digital reticle are represented for each photomicrograph. Fluorescence images from DAPI, FITC, TRITC, and Cy5 channels were separated as pictures with a color bar. The fluorescence-photomicrographs presented the diameters (μm) of CTC and a representative WBC and their respective DAPI stained nucleus.
Rabbit Mab Cd45, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/CD45+(Intracellular+Domain)+XP+Rabbit+mAb/pmc09221448-267-5-8
Average 96 stars, based on 1 article reviews
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rabbit polyclonal rab27a  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc rabbit polyclonal rab27a
Characterization and functional analysis of EVs in GCB‐resistant bladder cancer cells . (A) MTT assay was used to evaluate the viability of T24 and 5637 cells co‐cultured with T24‐ConMed and 5637‐ConMed or T24GCB‐ConMed and 5637GCB‐ConMed for 24 h, followed by GCB treatment, as described in panel E ( n = 6 per group). (B) Representative transmission electron microscopy images of EVs derived from bladder cancer cells (5637‐EVs, T24‐EVs and J82‐EVs) and their GCB‐resistant counterparts (5637GCB‐EVs and T24GCB‐EVs). Scale bar: 100 nm. (C) Nanoparticle tracking analysis (NS300) was used to evaluate the size distribution and concentration of EVs from 5637, 5637GCB, T24, T24GCB and J82 cells. (D) Imaging flow cytometry analysis of EVs stained with a lipid bilayer dye and labelled with EV surface markers CD9‐APC, CD63‐FITC and CD81‐PE. Each dot represents a single EV. (E) Representative fluorescence microscopy images of T24 cells following a 6‐h incubation with T24‐EVs, T24GCB‐EVs, or J82‐EVs pre‐labelled with CD9‐APC, CD63‐FITC and CD81‐PE. Scale bar: 10 µm. (F) Western blot analysis of EV markers (CD9, CD63, CD81, Alix and TSG101) and endosomal protein <t>Rab27A</t> in EVs from the indicated cell lines. (G) MTT assay evaluating the viability of T24 and 5637 cells co‐cultured with the indicated EVs for 24 h, followed by GCB treatment (0–3 µM) for 48 h ( n = 6 per group). (H and I) Colony formation assay was used to evaluate the clonogenic potential of T24 and 5637 cells after co‐culture with T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs, or J82‐EVs for 14 days ( n = 3 per group). (J) Western blot analysis of GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) and anti‐apoptotic protein BCL‐2 in T24 and 5637 cells co‐cultured with T24‐ConMed, T24GCB‐ConMed, or 5637‐ConMed, 5637GCB‐ConMed, or T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs for 24 h. (K, M, N) Electron microscopy images of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) in T24 and T24GCB cells. Quantification of MVBs per image (M) and ILVs per MVB (N). (L, O, P) Electron microscopy images of T24 cells incubated with T24‐EVs or T24GCB‐EVs for 24 h showing MVB and ILV structures. Quantification of MVBs per image (O) and ILVs per MVB (P). Data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed independently in triplicate. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; GCB, gemcitabine; EVs, extracellular vesicles; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.
Rabbit Polyclonal Rab27a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/Rab27A+Rabbit+mAb/pmc12570045-70-23-27
Average 95 stars, based on 1 article reviews
rabbit polyclonal rab27a - by Bioz Stars, 2026-09
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fluorescence inverted optical microscope  (Nikon)
99
Nikon fluorescence inverted optical microscope
Characterization and functional analysis of EVs in GCB‐resistant bladder cancer cells . (A) MTT assay was used to evaluate the viability of T24 and 5637 cells co‐cultured with T24‐ConMed and 5637‐ConMed or T24GCB‐ConMed and 5637GCB‐ConMed for 24 h, followed by GCB treatment, as described in panel E ( n = 6 per group). (B) Representative transmission electron microscopy images of EVs derived from bladder cancer cells (5637‐EVs, T24‐EVs and J82‐EVs) and their GCB‐resistant counterparts (5637GCB‐EVs and T24GCB‐EVs). Scale bar: 100 nm. (C) Nanoparticle tracking analysis (NS300) was used to evaluate the size distribution and concentration of EVs from 5637, 5637GCB, T24, T24GCB and J82 cells. (D) Imaging flow cytometry analysis of EVs stained with a lipid bilayer dye and labelled with EV surface markers CD9‐APC, CD63‐FITC and CD81‐PE. Each dot represents a single EV. (E) Representative fluorescence microscopy images of T24 cells following a 6‐h incubation with T24‐EVs, T24GCB‐EVs, or J82‐EVs pre‐labelled with CD9‐APC, CD63‐FITC and CD81‐PE. Scale bar: 10 µm. (F) Western blot analysis of EV markers (CD9, CD63, CD81, Alix and TSG101) and endosomal protein <t>Rab27A</t> in EVs from the indicated cell lines. (G) MTT assay evaluating the viability of T24 and 5637 cells co‐cultured with the indicated EVs for 24 h, followed by GCB treatment (0–3 µM) for 48 h ( n = 6 per group). (H and I) Colony formation assay was used to evaluate the clonogenic potential of T24 and 5637 cells after co‐culture with T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs, or J82‐EVs for 14 days ( n = 3 per group). (J) Western blot analysis of GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) and anti‐apoptotic protein BCL‐2 in T24 and 5637 cells co‐cultured with T24‐ConMed, T24GCB‐ConMed, or 5637‐ConMed, 5637GCB‐ConMed, or T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs for 24 h. (K, M, N) Electron microscopy images of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) in T24 and T24GCB cells. Quantification of MVBs per image (M) and ILVs per MVB (N). (L, O, P) Electron microscopy images of T24 cells incubated with T24‐EVs or T24GCB‐EVs for 24 h showing MVB and ILV structures. Quantification of MVBs per image (O) and ILVs per MVB (P). Data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed independently in triplicate. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; GCB, gemcitabine; EVs, extracellular vesicles; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.
Fluorescence Inverted Optical Microscope, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/Inverted+Microscopes/10__1590_slash_0104___1428__2372-46-6-10
Average 99 stars, based on 1 article reviews
fluorescence inverted optical microscope - by Bioz Stars, 2026-09
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immunofluorescence microscope  (Nikon)
96
Nikon immunofluorescence microscope
Characterization and functional analysis of EVs in GCB‐resistant bladder cancer cells . (A) MTT assay was used to evaluate the viability of T24 and 5637 cells co‐cultured with T24‐ConMed and 5637‐ConMed or T24GCB‐ConMed and 5637GCB‐ConMed for 24 h, followed by GCB treatment, as described in panel E ( n = 6 per group). (B) Representative transmission electron microscopy images of EVs derived from bladder cancer cells (5637‐EVs, T24‐EVs and J82‐EVs) and their GCB‐resistant counterparts (5637GCB‐EVs and T24GCB‐EVs). Scale bar: 100 nm. (C) Nanoparticle tracking analysis (NS300) was used to evaluate the size distribution and concentration of EVs from 5637, 5637GCB, T24, T24GCB and J82 cells. (D) Imaging flow cytometry analysis of EVs stained with a lipid bilayer dye and labelled with EV surface markers CD9‐APC, CD63‐FITC and CD81‐PE. Each dot represents a single EV. (E) Representative fluorescence microscopy images of T24 cells following a 6‐h incubation with T24‐EVs, T24GCB‐EVs, or J82‐EVs pre‐labelled with CD9‐APC, CD63‐FITC and CD81‐PE. Scale bar: 10 µm. (F) Western blot analysis of EV markers (CD9, CD63, CD81, Alix and TSG101) and endosomal protein <t>Rab27A</t> in EVs from the indicated cell lines. (G) MTT assay evaluating the viability of T24 and 5637 cells co‐cultured with the indicated EVs for 24 h, followed by GCB treatment (0–3 µM) for 48 h ( n = 6 per group). (H and I) Colony formation assay was used to evaluate the clonogenic potential of T24 and 5637 cells after co‐culture with T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs, or J82‐EVs for 14 days ( n = 3 per group). (J) Western blot analysis of GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) and anti‐apoptotic protein BCL‐2 in T24 and 5637 cells co‐cultured with T24‐ConMed, T24GCB‐ConMed, or 5637‐ConMed, 5637GCB‐ConMed, or T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs for 24 h. (K, M, N) Electron microscopy images of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) in T24 and T24GCB cells. Quantification of MVBs per image (M) and ILVs per MVB (N). (L, O, P) Electron microscopy images of T24 cells incubated with T24‐EVs or T24GCB‐EVs for 24 h showing MVB and ILV structures. Quantification of MVBs per image (O) and ILVs per MVB (P). Data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed independently in triplicate. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; GCB, gemcitabine; EVs, extracellular vesicles; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.
Immunofluorescence Microscope, supplied by Nikon, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/Fluorescence+Filter+Cubes/pmc01266095-95-10-12
Average 96 stars, based on 1 article reviews
immunofluorescence microscope - by Bioz Stars, 2026-09
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rat anti mouse cd68  (Bio-Rad)
96
Bio-Rad rat anti mouse cd68
Characterization and functional analysis of EVs in GCB‐resistant bladder cancer cells . (A) MTT assay was used to evaluate the viability of T24 and 5637 cells co‐cultured with T24‐ConMed and 5637‐ConMed or T24GCB‐ConMed and 5637GCB‐ConMed for 24 h, followed by GCB treatment, as described in panel E ( n = 6 per group). (B) Representative transmission electron microscopy images of EVs derived from bladder cancer cells (5637‐EVs, T24‐EVs and J82‐EVs) and their GCB‐resistant counterparts (5637GCB‐EVs and T24GCB‐EVs). Scale bar: 100 nm. (C) Nanoparticle tracking analysis (NS300) was used to evaluate the size distribution and concentration of EVs from 5637, 5637GCB, T24, T24GCB and J82 cells. (D) Imaging flow cytometry analysis of EVs stained with a lipid bilayer dye and labelled with EV surface markers CD9‐APC, CD63‐FITC and CD81‐PE. Each dot represents a single EV. (E) Representative fluorescence microscopy images of T24 cells following a 6‐h incubation with T24‐EVs, T24GCB‐EVs, or J82‐EVs pre‐labelled with CD9‐APC, CD63‐FITC and CD81‐PE. Scale bar: 10 µm. (F) Western blot analysis of EV markers (CD9, CD63, CD81, Alix and TSG101) and endosomal protein <t>Rab27A</t> in EVs from the indicated cell lines. (G) MTT assay evaluating the viability of T24 and 5637 cells co‐cultured with the indicated EVs for 24 h, followed by GCB treatment (0–3 µM) for 48 h ( n = 6 per group). (H and I) Colony formation assay was used to evaluate the clonogenic potential of T24 and 5637 cells after co‐culture with T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs, or J82‐EVs for 14 days ( n = 3 per group). (J) Western blot analysis of GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) and anti‐apoptotic protein BCL‐2 in T24 and 5637 cells co‐cultured with T24‐ConMed, T24GCB‐ConMed, or 5637‐ConMed, 5637GCB‐ConMed, or T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs for 24 h. (K, M, N) Electron microscopy images of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) in T24 and T24GCB cells. Quantification of MVBs per image (M) and ILVs per MVB (N). (L, O, P) Electron microscopy images of T24 cells incubated with T24‐EVs or T24GCB‐EVs for 24 h showing MVB and ILV structures. Quantification of MVBs per image (O) and ILVs per MVB (P). Data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed independently in triplicate. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; GCB, gemcitabine; EVs, extracellular vesicles; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.
Rat Anti Mouse Cd68, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/e+800+eclipse+nikon+fluorescent+microscope/Rat+anti+Mouse+CD68/pm22555796-15-36-39
Average 96 stars, based on 1 article reviews
rat anti mouse cd68 - by Bioz Stars, 2026-09
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Figure 2. In utero pharmacological invalidation of Amhr2 disrupts GnRH neuronal migration and the olfactory/terminal nerve targeting. (a) Schematic of in utero injections targeting the olfactory pits. Injections were performed at E12.5 and embryos harvested 48 hr later. (b) Representative coronal section of an embryo head at E14.5 showing that olfactory pit Fluorogold delivery at E12.5 was successful. GnRH immunoreactive neurons are shown in green. (c–f) Representative photomicrographs of sagittal sections of mouse embryos injected at E12.5 with either saline or a neutralizing antibody for Amhr2 (Amhr2-NA) and immunostained for GnRH (green) and Peripherin (magenta) at E14.5. (e, f) Higher magnification confocal photomicrograph of boxed areas in c and d. (g) Quantification of the total number of GnRH immunoreactive neurons in saline-injected (control) and Amhr2-NA injected embryos (n = 4 for both groups, harvested from two independent dams). Data are represented as mean ± s.e.m (n = 4, unpaired two-tailed Student’s t test: mean cell number, t6 = 0.3796, p = 0.7173). (h) Quantitative analysis of GnRH neuronal distribution throughout the migratory pathway in the two experimental groups. Data are represented as mean ± s.e.m (n = 4, two-way ANOVA, F3,24 = 15.09, p<0.0001; followed by Holm-Sˇ ı´da´k multiple Figure 2 continued on next page

Journal: eLife

Article Title: Defective AMH signaling disrupts GnRH neuron development and function and contributes to hypogonadotropic hypogonadism

doi: 10.7554/elife.47198

Figure Lengend Snippet: Figure 2. In utero pharmacological invalidation of Amhr2 disrupts GnRH neuronal migration and the olfactory/terminal nerve targeting. (a) Schematic of in utero injections targeting the olfactory pits. Injections were performed at E12.5 and embryos harvested 48 hr later. (b) Representative coronal section of an embryo head at E14.5 showing that olfactory pit Fluorogold delivery at E12.5 was successful. GnRH immunoreactive neurons are shown in green. (c–f) Representative photomicrographs of sagittal sections of mouse embryos injected at E12.5 with either saline or a neutralizing antibody for Amhr2 (Amhr2-NA) and immunostained for GnRH (green) and Peripherin (magenta) at E14.5. (e, f) Higher magnification confocal photomicrograph of boxed areas in c and d. (g) Quantification of the total number of GnRH immunoreactive neurons in saline-injected (control) and Amhr2-NA injected embryos (n = 4 for both groups, harvested from two independent dams). Data are represented as mean ± s.e.m (n = 4, unpaired two-tailed Student’s t test: mean cell number, t6 = 0.3796, p = 0.7173). (h) Quantitative analysis of GnRH neuronal distribution throughout the migratory pathway in the two experimental groups. Data are represented as mean ± s.e.m (n = 4, two-way ANOVA, F3,24 = 15.09, p<0.0001; followed by Holm-Sˇ ı´da´k multiple Figure 2 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.47198 19 of 36 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody GnRH (guinea pig) Dr. Erik Hrabovszky, Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary Lab Stock 1:3000; https://doi.org/ 10.3389/fendo.2011.00080 Antibody Peripherin (Contactin1) (rabbit) Millipore #AB1530; RRID:AB_90725 1:1000 Antibody b-III tubulin (TUJ-1) (mouse) Sigma Aldrich #T8660; RRID:AB_477590 1:800 Antibody AMHR2 Neutralizing Antibody R&D systems #AF1618 ; RRID: AB_2226485 1:200 Antibody TAG-1 (goat) R&D systems AF2215 Antibody Actin (mouse) Sigma Aldrich #A5316; RRID:AB_476743 1:1000 Antibody Donkey anti-rabbit IgG AlexaFluor 488 (H + L) Molecular Probes #A-21026; RRID:AB_141708 1:500 Antibody Donkey anti-rabbit IgG AlexaFluor 555 (H + L) Molecular Probes #A-31572; RRID:AB_162543 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 488 (H + L) Molecular Probes #A-21202; RRID:AB_141607 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 555 (H + L) Molecular Probes #A-31570; RRID:AB_2536180 1:500 Antibody Donkey anti-goat IgG AlexaFluor 488 (H + L) Molecular Probes #A-11055; RRID:AB_142672 1:500 Antibody Donkey anti-goat IgG AlexaFluor 555 (H + L) Molecular Probes #A-21432; RRID:AB_141788 1:500 Antibody Donkey anti-goat IgG AlexaFluor 647 (H + L) Molecular Probes #A-21447; RRID:AB_141844 1:500 Antibody Donkey anti-guinea pig IgG AlexaFluor 488 (H + L) Jackson ImmunoResearch #706-545-148; RRID:AB_2340472 1:500 Antibody Horse anti-mouse IgG peroxidase labelled Vector #PI-2000; RRID:AB_2336177 1:5000 Sequencebased reagent Amh Taqman gene expression assay Thermofisher Scientific Mm00431795_g1 Sequencebased reagent GnRH Taqman gene expression assay Thermofisher Scientific Mm01315605 Sequencebased reagent Amhr2 Taqman gene expression assay Thermofisher Scientific Mm00513847_m1 Sequencebased reagent Acvr1 Taqman gene expression assay Thermofisher Scientific Mm01331069_m1 Continued on next page Malone et al. eLife 2019;8:e47198.

Techniques: In Utero, Migration, Injection, Saline, Control, Two Tailed Test

Figure 3. GnRH migration and olfactory innervation are perturbed in Amhr2-/- mice. (a) Schematic representation depicting whole-body iDISCO experiments in E13.5 Amhr2+/+ and Amhr2-/- embryos. E13.5 embryos (n = 2 per genotype) were immunolabelled for Peripherin and GnRH, rendered optically transparent using iDISCO and imaged with a light-sheet microscope (LSM). (b, c) Frontal projection of the embryo heads, arrowheads indicate noticeable differences in Peripherin-positive fibers innervating the olfactory bulb (OB). Lateral projection views (d, e) showing defective GnRH migration and terminal nerve projections to the ventral forebrain (vFB, arrows). (f, g) Higher magnification photomicrographs depicting olfactory axon innervations of the right OB shown in b and c. Dotted circles define the anatomical border of the OB. (h, i) 3D rendering of figures in f and g. Arrowheads indicate observed differences in olfactory axon innervation between Amhr2+/+ and Amhr2-/- embryos. (j, k) 3D rendering of peripherin and GnRH staining observed from a lateral projection in a representative Amhr2+/+ and Amhr2-/- embryo. Cx: cortex; VNO: vomeronasal organ. Scale bars: (b) 400 mm; (d) 300 mm; (f) 130 mm. DOI: https://doi.org/10.7554/eLife.47198.006 The following video is available for figure 3: Figure 3—video 1. Light-sheet fluorescence microscopy video of solvent-cleared E13.5 Amhr2+/+ and Amhr2-/- embryos immunostained in toto for GnRH (green) and peripherin (red). DOI: https://doi.org/10.7554/eLife.47198.007

Journal: eLife

Article Title: Defective AMH signaling disrupts GnRH neuron development and function and contributes to hypogonadotropic hypogonadism

doi: 10.7554/elife.47198

Figure Lengend Snippet: Figure 3. GnRH migration and olfactory innervation are perturbed in Amhr2-/- mice. (a) Schematic representation depicting whole-body iDISCO experiments in E13.5 Amhr2+/+ and Amhr2-/- embryos. E13.5 embryos (n = 2 per genotype) were immunolabelled for Peripherin and GnRH, rendered optically transparent using iDISCO and imaged with a light-sheet microscope (LSM). (b, c) Frontal projection of the embryo heads, arrowheads indicate noticeable differences in Peripherin-positive fibers innervating the olfactory bulb (OB). Lateral projection views (d, e) showing defective GnRH migration and terminal nerve projections to the ventral forebrain (vFB, arrows). (f, g) Higher magnification photomicrographs depicting olfactory axon innervations of the right OB shown in b and c. Dotted circles define the anatomical border of the OB. (h, i) 3D rendering of figures in f and g. Arrowheads indicate observed differences in olfactory axon innervation between Amhr2+/+ and Amhr2-/- embryos. (j, k) 3D rendering of peripherin and GnRH staining observed from a lateral projection in a representative Amhr2+/+ and Amhr2-/- embryo. Cx: cortex; VNO: vomeronasal organ. Scale bars: (b) 400 mm; (d) 300 mm; (f) 130 mm. DOI: https://doi.org/10.7554/eLife.47198.006 The following video is available for figure 3: Figure 3—video 1. Light-sheet fluorescence microscopy video of solvent-cleared E13.5 Amhr2+/+ and Amhr2-/- embryos immunostained in toto for GnRH (green) and peripherin (red). DOI: https://doi.org/10.7554/eLife.47198.007

Article Snippet: DOI: https://doi.org/10.7554/eLife.47198 19 of 36 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody GnRH (guinea pig) Dr. Erik Hrabovszky, Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary Lab Stock 1:3000; https://doi.org/ 10.3389/fendo.2011.00080 Antibody Peripherin (Contactin1) (rabbit) Millipore #AB1530; RRID:AB_90725 1:1000 Antibody b-III tubulin (TUJ-1) (mouse) Sigma Aldrich #T8660; RRID:AB_477590 1:800 Antibody AMHR2 Neutralizing Antibody R&D systems #AF1618 ; RRID: AB_2226485 1:200 Antibody TAG-1 (goat) R&D systems AF2215 Antibody Actin (mouse) Sigma Aldrich #A5316; RRID:AB_476743 1:1000 Antibody Donkey anti-rabbit IgG AlexaFluor 488 (H + L) Molecular Probes #A-21026; RRID:AB_141708 1:500 Antibody Donkey anti-rabbit IgG AlexaFluor 555 (H + L) Molecular Probes #A-31572; RRID:AB_162543 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 488 (H + L) Molecular Probes #A-21202; RRID:AB_141607 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 555 (H + L) Molecular Probes #A-31570; RRID:AB_2536180 1:500 Antibody Donkey anti-goat IgG AlexaFluor 488 (H + L) Molecular Probes #A-11055; RRID:AB_142672 1:500 Antibody Donkey anti-goat IgG AlexaFluor 555 (H + L) Molecular Probes #A-21432; RRID:AB_141788 1:500 Antibody Donkey anti-goat IgG AlexaFluor 647 (H + L) Molecular Probes #A-21447; RRID:AB_141844 1:500 Antibody Donkey anti-guinea pig IgG AlexaFluor 488 (H + L) Jackson ImmunoResearch #706-545-148; RRID:AB_2340472 1:500 Antibody Horse anti-mouse IgG peroxidase labelled Vector #PI-2000; RRID:AB_2336177 1:5000 Sequencebased reagent Amh Taqman gene expression assay Thermofisher Scientific Mm00431795_g1 Sequencebased reagent GnRH Taqman gene expression assay Thermofisher Scientific Mm01315605 Sequencebased reagent Amhr2 Taqman gene expression assay Thermofisher Scientific Mm00513847_m1 Sequencebased reagent Acvr1 Taqman gene expression assay Thermofisher Scientific Mm01331069_m1 Continued on next page Malone et al. eLife 2019;8:e47198.

Techniques: Migration, Microscopy, Staining, Fluorescence, Solvent

Figure 4. Amhr2 mutant mice show reduced GnRH cell number and impaired LH secretion and fertility. (a–h) Immunolabelling of GnRH (red staining) in adult wild type and Amhr2-/- adult female mice (P90–P120). The majority of GnRH cell bodies are located at the level of the organum vasculosum laminae terminalis (OVLT) in both Amhr2 +/+ and Amhr2 -/- mice, (arrows (c, d). (e–h) GnRH fiber projections at the level of the median eminence. (i) Total mean GnRH population in Amhr2+/+, Amhr2+/- and Amhr2-/- adult female mice brains (3–4 months old). Comparisons between groups were Figure 4 continued on next page

Journal: eLife

Article Title: Defective AMH signaling disrupts GnRH neuron development and function and contributes to hypogonadotropic hypogonadism

doi: 10.7554/elife.47198

Figure Lengend Snippet: Figure 4. Amhr2 mutant mice show reduced GnRH cell number and impaired LH secretion and fertility. (a–h) Immunolabelling of GnRH (red staining) in adult wild type and Amhr2-/- adult female mice (P90–P120). The majority of GnRH cell bodies are located at the level of the organum vasculosum laminae terminalis (OVLT) in both Amhr2 +/+ and Amhr2 -/- mice, (arrows (c, d). (e–h) GnRH fiber projections at the level of the median eminence. (i) Total mean GnRH population in Amhr2+/+, Amhr2+/- and Amhr2-/- adult female mice brains (3–4 months old). Comparisons between groups were Figure 4 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.47198 19 of 36 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody GnRH (guinea pig) Dr. Erik Hrabovszky, Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary Lab Stock 1:3000; https://doi.org/ 10.3389/fendo.2011.00080 Antibody Peripherin (Contactin1) (rabbit) Millipore #AB1530; RRID:AB_90725 1:1000 Antibody b-III tubulin (TUJ-1) (mouse) Sigma Aldrich #T8660; RRID:AB_477590 1:800 Antibody AMHR2 Neutralizing Antibody R&D systems #AF1618 ; RRID: AB_2226485 1:200 Antibody TAG-1 (goat) R&D systems AF2215 Antibody Actin (mouse) Sigma Aldrich #A5316; RRID:AB_476743 1:1000 Antibody Donkey anti-rabbit IgG AlexaFluor 488 (H + L) Molecular Probes #A-21026; RRID:AB_141708 1:500 Antibody Donkey anti-rabbit IgG AlexaFluor 555 (H + L) Molecular Probes #A-31572; RRID:AB_162543 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 488 (H + L) Molecular Probes #A-21202; RRID:AB_141607 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 555 (H + L) Molecular Probes #A-31570; RRID:AB_2536180 1:500 Antibody Donkey anti-goat IgG AlexaFluor 488 (H + L) Molecular Probes #A-11055; RRID:AB_142672 1:500 Antibody Donkey anti-goat IgG AlexaFluor 555 (H + L) Molecular Probes #A-21432; RRID:AB_141788 1:500 Antibody Donkey anti-goat IgG AlexaFluor 647 (H + L) Molecular Probes #A-21447; RRID:AB_141844 1:500 Antibody Donkey anti-guinea pig IgG AlexaFluor 488 (H + L) Jackson ImmunoResearch #706-545-148; RRID:AB_2340472 1:500 Antibody Horse anti-mouse IgG peroxidase labelled Vector #PI-2000; RRID:AB_2336177 1:5000 Sequencebased reagent Amh Taqman gene expression assay Thermofisher Scientific Mm00431795_g1 Sequencebased reagent GnRH Taqman gene expression assay Thermofisher Scientific Mm01315605 Sequencebased reagent Amhr2 Taqman gene expression assay Thermofisher Scientific Mm00513847_m1 Sequencebased reagent Acvr1 Taqman gene expression assay Thermofisher Scientific Mm01331069_m1 Continued on next page Malone et al. eLife 2019;8:e47198.

Techniques: Mutagenesis, Staining

Figure 5. AMH promotes GnRH cell motility via Amhr2 and Bmpr1b signaling. (a) Quantitative RT-PCR analysis for Amh, Amhr2, Acvr1 (Activin Receptor1; ALK2), Bmpr1a (Bone Morphogenetic Protein Receptor1a; ALK3) and Bmpr1b (Bone Morphogenetic Protein Receptor1b; ALK6) mRNA in GN11 (n = 4) and GT1-7 (n = 3) cells. Comparisons between treatment groups were performed using unpaired two-tailed Student’s t test (Amh t5 = 1.139, p = 0.0004; Amhr2 t5 = 1.6, p<0.0001; Acvr1 t5 = 5.044, p<0.0001); Bmpr1a t5 = 2.374, p<0.0044. (b) Representative western blot showing P-ERK1/ Figure 5 continued on next page

Journal: eLife

Article Title: Defective AMH signaling disrupts GnRH neuron development and function and contributes to hypogonadotropic hypogonadism

doi: 10.7554/elife.47198

Figure Lengend Snippet: Figure 5. AMH promotes GnRH cell motility via Amhr2 and Bmpr1b signaling. (a) Quantitative RT-PCR analysis for Amh, Amhr2, Acvr1 (Activin Receptor1; ALK2), Bmpr1a (Bone Morphogenetic Protein Receptor1a; ALK3) and Bmpr1b (Bone Morphogenetic Protein Receptor1b; ALK6) mRNA in GN11 (n = 4) and GT1-7 (n = 3) cells. Comparisons between treatment groups were performed using unpaired two-tailed Student’s t test (Amh t5 = 1.139, p = 0.0004; Amhr2 t5 = 1.6, p<0.0001; Acvr1 t5 = 5.044, p<0.0001); Bmpr1a t5 = 2.374, p<0.0044. (b) Representative western blot showing P-ERK1/ Figure 5 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.47198 19 of 36 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody GnRH (guinea pig) Dr. Erik Hrabovszky, Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary Lab Stock 1:3000; https://doi.org/ 10.3389/fendo.2011.00080 Antibody Peripherin (Contactin1) (rabbit) Millipore #AB1530; RRID:AB_90725 1:1000 Antibody b-III tubulin (TUJ-1) (mouse) Sigma Aldrich #T8660; RRID:AB_477590 1:800 Antibody AMHR2 Neutralizing Antibody R&D systems #AF1618 ; RRID: AB_2226485 1:200 Antibody TAG-1 (goat) R&D systems AF2215 Antibody Actin (mouse) Sigma Aldrich #A5316; RRID:AB_476743 1:1000 Antibody Donkey anti-rabbit IgG AlexaFluor 488 (H + L) Molecular Probes #A-21026; RRID:AB_141708 1:500 Antibody Donkey anti-rabbit IgG AlexaFluor 555 (H + L) Molecular Probes #A-31572; RRID:AB_162543 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 488 (H + L) Molecular Probes #A-21202; RRID:AB_141607 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 555 (H + L) Molecular Probes #A-31570; RRID:AB_2536180 1:500 Antibody Donkey anti-goat IgG AlexaFluor 488 (H + L) Molecular Probes #A-11055; RRID:AB_142672 1:500 Antibody Donkey anti-goat IgG AlexaFluor 555 (H + L) Molecular Probes #A-21432; RRID:AB_141788 1:500 Antibody Donkey anti-goat IgG AlexaFluor 647 (H + L) Molecular Probes #A-21447; RRID:AB_141844 1:500 Antibody Donkey anti-guinea pig IgG AlexaFluor 488 (H + L) Jackson ImmunoResearch #706-545-148; RRID:AB_2340472 1:500 Antibody Horse anti-mouse IgG peroxidase labelled Vector #PI-2000; RRID:AB_2336177 1:5000 Sequencebased reagent Amh Taqman gene expression assay Thermofisher Scientific Mm00431795_g1 Sequencebased reagent GnRH Taqman gene expression assay Thermofisher Scientific Mm01315605 Sequencebased reagent Amhr2 Taqman gene expression assay Thermofisher Scientific Mm00513847_m1 Sequencebased reagent Acvr1 Taqman gene expression assay Thermofisher Scientific Mm01331069_m1 Continued on next page Malone et al. eLife 2019;8:e47198.

Techniques: Quantitative RT-PCR, Two Tailed Test, Western Blot

Figure 6. AMH and AMHR2 heterozygous mutations in CHH probands. (a) Schematic illustration of AMH mutations in nCHH and KS probands. (b) Pedigrees of patients harboring AMH mutations. Circles denote females, squares denote males. The phenotype interpretation is explained in the square legend on the top of the figure. (c) The AMH mutations affect evolutionarily conserved amino acid residues. Alignment of partial protein sequences of AMH orthologs showing in red text the amino acid residues evolutionarily conserved. Purple highlights correspond to variants identified Figure 6 continued on next page

Journal: eLife

Article Title: Defective AMH signaling disrupts GnRH neuron development and function and contributes to hypogonadotropic hypogonadism

doi: 10.7554/elife.47198

Figure Lengend Snippet: Figure 6. AMH and AMHR2 heterozygous mutations in CHH probands. (a) Schematic illustration of AMH mutations in nCHH and KS probands. (b) Pedigrees of patients harboring AMH mutations. Circles denote females, squares denote males. The phenotype interpretation is explained in the square legend on the top of the figure. (c) The AMH mutations affect evolutionarily conserved amino acid residues. Alignment of partial protein sequences of AMH orthologs showing in red text the amino acid residues evolutionarily conserved. Purple highlights correspond to variants identified Figure 6 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.47198 19 of 36 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody GnRH (guinea pig) Dr. Erik Hrabovszky, Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary Lab Stock 1:3000; https://doi.org/ 10.3389/fendo.2011.00080 Antibody Peripherin (Contactin1) (rabbit) Millipore #AB1530; RRID:AB_90725 1:1000 Antibody b-III tubulin (TUJ-1) (mouse) Sigma Aldrich #T8660; RRID:AB_477590 1:800 Antibody AMHR2 Neutralizing Antibody R&D systems #AF1618 ; RRID: AB_2226485 1:200 Antibody TAG-1 (goat) R&D systems AF2215 Antibody Actin (mouse) Sigma Aldrich #A5316; RRID:AB_476743 1:1000 Antibody Donkey anti-rabbit IgG AlexaFluor 488 (H + L) Molecular Probes #A-21026; RRID:AB_141708 1:500 Antibody Donkey anti-rabbit IgG AlexaFluor 555 (H + L) Molecular Probes #A-31572; RRID:AB_162543 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 488 (H + L) Molecular Probes #A-21202; RRID:AB_141607 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 555 (H + L) Molecular Probes #A-31570; RRID:AB_2536180 1:500 Antibody Donkey anti-goat IgG AlexaFluor 488 (H + L) Molecular Probes #A-11055; RRID:AB_142672 1:500 Antibody Donkey anti-goat IgG AlexaFluor 555 (H + L) Molecular Probes #A-21432; RRID:AB_141788 1:500 Antibody Donkey anti-goat IgG AlexaFluor 647 (H + L) Molecular Probes #A-21447; RRID:AB_141844 1:500 Antibody Donkey anti-guinea pig IgG AlexaFluor 488 (H + L) Jackson ImmunoResearch #706-545-148; RRID:AB_2340472 1:500 Antibody Horse anti-mouse IgG peroxidase labelled Vector #PI-2000; RRID:AB_2336177 1:5000 Sequencebased reagent Amh Taqman gene expression assay Thermofisher Scientific Mm00431795_g1 Sequencebased reagent GnRH Taqman gene expression assay Thermofisher Scientific Mm01315605 Sequencebased reagent Amhr2 Taqman gene expression assay Thermofisher Scientific Mm00513847_m1 Sequencebased reagent Acvr1 Taqman gene expression assay Thermofisher Scientific Mm01331069_m1 Continued on next page Malone et al. eLife 2019;8:e47198.

Techniques:

Figure 7. Functional validation of AMH variants. (a) AMH released in the medium of COS-7 cells transiently transfected either with lipofectamine alone (mock), or with a WT AMH or a variant AMH identified in CHH and KS probands. Bar graph illustrates the mean amount of AMH secreted in the conditioned medium of transfected COS-7 cells (n = 3 independent experiments per condition). Comparisons between treatment groups were performed using a one-way ANOVA followed by Tukey’s post hoc comparison test (F4,10 = 1193, Mock vs AMH WT p<0.0001, AMH WT vs p.Pro151Ser p<0.0001, AMH WT vs p.Asp238Glu p<0.0001, AMH WT vs p.Thr99Ser p<0.0001). No significant motility difference was detected between Mock, p. Thr99Ser and p.Pro151Ser mutated forms of AMH treatment, p>0.9 for all. (b) Transwell assay was performed on GN11 cells transiently transfected either with lipofectamine alone (mock), or with a WT AMH or a variant AMH identified in CHH and KS probands. Comparisons between treatment groups were performed using a one-way ANOVA followed by Tukey’s post hoc comparison test (F4,50 = 13.94, Mock vs AMH WT p<0.0001, AMH WT vs p.Pro151Ser p<0.0001, AMH WT vs p.Asp238Glu p<0.0014, AMH WT vs p.Thr99Ser p = 0.0218. No significant motility difference was detected between Mock and mutated forms of AMH treatment, p>0.9 for all. (c) Quantification of GnRH secretion from GT1-7 cells transfected with lipofectamine alone (mock), or with a WT AMH or the p.Pro151Ser AMH variant identified in a nCHH proband. GnRH mean concentration measured in the medium (n = 3, one-way ANOVA: F 2,6 = 43.84, p = 0.0003; followed by Tukey’s multiple comparison post hoc test, mock vs. AMH WT p = 0.0003, mock vs p.Thr99Ser p = 0.5220, AMH WT vs p.Thr99Ser p = 0.0007. (d) Transwell assay was performed on GN11 cells transiently transfected with the AMHR2 plasmid or with the AMHR2 variant and stimulated with either serum-free medium (SFM) or with recombinant AMH (50 ng/ml). Bar graph illustrates the mean number of migrated GN11 cells under different treatment conditions (SFM n = 10 for both WT and mutant AMHR2, AMH 50 ng/ml n = 12 for both WT and mutant AMHR2). Comparisons between treatment groups were performed using two-way ANOVA (F1,43 = 16.5 P = 0.0002; followed by Sidak’s multiple comparison post hoc test, AMHR2 WT SFM vs AMHR2 WT + AMH 50 ng/ml p<0.0001, p.Gly445_Leu453del SFM vs p. Gly445_Leu453del + AMH 50 ng/ml P = 0.1036). (e) Quantification of GnRH secretion from GT1-7 cells transfected with the same plasmids as in d (n = 3 independent experiments per condition). Experiments were replicated three times with comparable results. Two-way ANOVA, F1,8 = 1.927, p<0.02025; followed by Holm-Sˇ ı´da´k multiple comparison post hoc test, AMHR2 WT SFM vs AMHR2 WT + AMH 50 ng/ml P = 0.0269, p.Gly445_Leu453del SFM vs p.Gly445_Leu453del + AMH 50 ng/ml P = 0.4652. (f) Initial three-dimensional models of WT and p.Gly445_Leu453del catalytic intracellular serine/ threonine domains of AMHR2. The backbone of the WT and deleted proteins are shown in tan or white cartoon representations, respectively, with the deleted 445–453 residues colored in red. The activation loop is depicted in blue. (g–i) Root-mean-square fluctuations (RMSF) of the Ca atoms along the simulations for the AMHR2 WT and the p.Gly445_Leu453del models. (g) RMSF (in A˚ ) for the WT (black line) and the p.Gly445_Leu453del models (red Figure 7 continued on next page

Journal: eLife

Article Title: Defective AMH signaling disrupts GnRH neuron development and function and contributes to hypogonadotropic hypogonadism

doi: 10.7554/elife.47198

Figure Lengend Snippet: Figure 7. Functional validation of AMH variants. (a) AMH released in the medium of COS-7 cells transiently transfected either with lipofectamine alone (mock), or with a WT AMH or a variant AMH identified in CHH and KS probands. Bar graph illustrates the mean amount of AMH secreted in the conditioned medium of transfected COS-7 cells (n = 3 independent experiments per condition). Comparisons between treatment groups were performed using a one-way ANOVA followed by Tukey’s post hoc comparison test (F4,10 = 1193, Mock vs AMH WT p<0.0001, AMH WT vs p.Pro151Ser p<0.0001, AMH WT vs p.Asp238Glu p<0.0001, AMH WT vs p.Thr99Ser p<0.0001). No significant motility difference was detected between Mock, p. Thr99Ser and p.Pro151Ser mutated forms of AMH treatment, p>0.9 for all. (b) Transwell assay was performed on GN11 cells transiently transfected either with lipofectamine alone (mock), or with a WT AMH or a variant AMH identified in CHH and KS probands. Comparisons between treatment groups were performed using a one-way ANOVA followed by Tukey’s post hoc comparison test (F4,50 = 13.94, Mock vs AMH WT p<0.0001, AMH WT vs p.Pro151Ser p<0.0001, AMH WT vs p.Asp238Glu p<0.0014, AMH WT vs p.Thr99Ser p = 0.0218. No significant motility difference was detected between Mock and mutated forms of AMH treatment, p>0.9 for all. (c) Quantification of GnRH secretion from GT1-7 cells transfected with lipofectamine alone (mock), or with a WT AMH or the p.Pro151Ser AMH variant identified in a nCHH proband. GnRH mean concentration measured in the medium (n = 3, one-way ANOVA: F 2,6 = 43.84, p = 0.0003; followed by Tukey’s multiple comparison post hoc test, mock vs. AMH WT p = 0.0003, mock vs p.Thr99Ser p = 0.5220, AMH WT vs p.Thr99Ser p = 0.0007. (d) Transwell assay was performed on GN11 cells transiently transfected with the AMHR2 plasmid or with the AMHR2 variant and stimulated with either serum-free medium (SFM) or with recombinant AMH (50 ng/ml). Bar graph illustrates the mean number of migrated GN11 cells under different treatment conditions (SFM n = 10 for both WT and mutant AMHR2, AMH 50 ng/ml n = 12 for both WT and mutant AMHR2). Comparisons between treatment groups were performed using two-way ANOVA (F1,43 = 16.5 P = 0.0002; followed by Sidak’s multiple comparison post hoc test, AMHR2 WT SFM vs AMHR2 WT + AMH 50 ng/ml p<0.0001, p.Gly445_Leu453del SFM vs p. Gly445_Leu453del + AMH 50 ng/ml P = 0.1036). (e) Quantification of GnRH secretion from GT1-7 cells transfected with the same plasmids as in d (n = 3 independent experiments per condition). Experiments were replicated three times with comparable results. Two-way ANOVA, F1,8 = 1.927, p<0.02025; followed by Holm-Sˇ ı´da´k multiple comparison post hoc test, AMHR2 WT SFM vs AMHR2 WT + AMH 50 ng/ml P = 0.0269, p.Gly445_Leu453del SFM vs p.Gly445_Leu453del + AMH 50 ng/ml P = 0.4652. (f) Initial three-dimensional models of WT and p.Gly445_Leu453del catalytic intracellular serine/ threonine domains of AMHR2. The backbone of the WT and deleted proteins are shown in tan or white cartoon representations, respectively, with the deleted 445–453 residues colored in red. The activation loop is depicted in blue. (g–i) Root-mean-square fluctuations (RMSF) of the Ca atoms along the simulations for the AMHR2 WT and the p.Gly445_Leu453del models. (g) RMSF (in A˚ ) for the WT (black line) and the p.Gly445_Leu453del models (red Figure 7 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.47198 19 of 36 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody GnRH (guinea pig) Dr. Erik Hrabovszky, Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary Lab Stock 1:3000; https://doi.org/ 10.3389/fendo.2011.00080 Antibody Peripherin (Contactin1) (rabbit) Millipore #AB1530; RRID:AB_90725 1:1000 Antibody b-III tubulin (TUJ-1) (mouse) Sigma Aldrich #T8660; RRID:AB_477590 1:800 Antibody AMHR2 Neutralizing Antibody R&D systems #AF1618 ; RRID: AB_2226485 1:200 Antibody TAG-1 (goat) R&D systems AF2215 Antibody Actin (mouse) Sigma Aldrich #A5316; RRID:AB_476743 1:1000 Antibody Donkey anti-rabbit IgG AlexaFluor 488 (H + L) Molecular Probes #A-21026; RRID:AB_141708 1:500 Antibody Donkey anti-rabbit IgG AlexaFluor 555 (H + L) Molecular Probes #A-31572; RRID:AB_162543 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 488 (H + L) Molecular Probes #A-21202; RRID:AB_141607 1:500 Antibody Donkey anti-mouse IgG AlexaFluor 555 (H + L) Molecular Probes #A-31570; RRID:AB_2536180 1:500 Antibody Donkey anti-goat IgG AlexaFluor 488 (H + L) Molecular Probes #A-11055; RRID:AB_142672 1:500 Antibody Donkey anti-goat IgG AlexaFluor 555 (H + L) Molecular Probes #A-21432; RRID:AB_141788 1:500 Antibody Donkey anti-goat IgG AlexaFluor 647 (H + L) Molecular Probes #A-21447; RRID:AB_141844 1:500 Antibody Donkey anti-guinea pig IgG AlexaFluor 488 (H + L) Jackson ImmunoResearch #706-545-148; RRID:AB_2340472 1:500 Antibody Horse anti-mouse IgG peroxidase labelled Vector #PI-2000; RRID:AB_2336177 1:5000 Sequencebased reagent Amh Taqman gene expression assay Thermofisher Scientific Mm00431795_g1 Sequencebased reagent GnRH Taqman gene expression assay Thermofisher Scientific Mm01315605 Sequencebased reagent Amhr2 Taqman gene expression assay Thermofisher Scientific Mm00513847_m1 Sequencebased reagent Acvr1 Taqman gene expression assay Thermofisher Scientific Mm01331069_m1 Continued on next page Malone et al. eLife 2019;8:e47198.

Techniques: Functional Assay, Biomarker Discovery, Transfection, Variant Assay, Comparison, Transwell Assay, Concentration Assay, Plasmid Preparation, Recombinant, Mutagenesis, Activation Assay

Inhibition of GBM cancer cell development by suppressing M2 macrophage infiltration through activation of TAM‐iron death pathway. (A) Flow cytometry was used to determine the proportion of M2 macrophages. (B) BMDMs were transfected with lentivirus for knockdown of MS4A4A or negative control plasmid, then induced into M2 phenotype using IL‐4 (20 ng/mL) and IL‐13 (20 ng/mL). qRT‐PCR was used to measure the interference efficiency of MS4A4A and expression of M2 markers (Mgl2, Arg1, and Tgfb1) and M1 marker iNOS. (C) THP‐1 human monocytic cell line was used to establish an overexpression cell line of MS4A4A, followed by differentiation into M0 macrophages using PMA (50 ng/mL). M0 macrophages were then polarized into M2 phenotype using IL‐4 (20 ng/mL) and qRT‐PCR was used to measure expression levels of MS4A4A and M2 markers (CD163, ARG1, and TGFB1) and M1 marker CD86. (D) PBMC cells were used to construct a cell line overexpressing MS4A4A. (E) Procedure for testing macrophage inhibition function: Mouse BMDMs and spleen cells were mixed with CT2A/GL261 cells, treated with MS4A4A antibody, and CD8 + T cells were sorted using flow cytometry and Ki‐67 expression was measured. (F) Analysis of Ki‐67 expression on designated CD8 + T cells using flow cytometry ( n = 3). (G) Apoptosis of CT2A cells was detected using fluorescence‐activated cell sorting (FACS). (H) Iron content in tumor‐associated macrophages (TAMs) of sh‐NC and sh‐MS4A4A groups. (I) Reactive oxygen species (ROS) content in TAM cells of sh‐NC and sh‐MS4A4A groups. (J) Expression of iron death‐related proteins in TAM cells of sh‐NC and sh‐MS4A4A groups. (K) Electron microscopy analysis of TAM cells in sh‐NC and sh‐MS4A4A groups. * p < 0.05, and all cell experiments were repeated three times.

Journal: CNS Neuroscience & Therapeutics

Article Title: Targeting MS4A4A: A novel pathway to improve immunotherapy responses in glioblastoma

doi: 10.1111/cns.14791

Figure Lengend Snippet: Inhibition of GBM cancer cell development by suppressing M2 macrophage infiltration through activation of TAM‐iron death pathway. (A) Flow cytometry was used to determine the proportion of M2 macrophages. (B) BMDMs were transfected with lentivirus for knockdown of MS4A4A or negative control plasmid, then induced into M2 phenotype using IL‐4 (20 ng/mL) and IL‐13 (20 ng/mL). qRT‐PCR was used to measure the interference efficiency of MS4A4A and expression of M2 markers (Mgl2, Arg1, and Tgfb1) and M1 marker iNOS. (C) THP‐1 human monocytic cell line was used to establish an overexpression cell line of MS4A4A, followed by differentiation into M0 macrophages using PMA (50 ng/mL). M0 macrophages were then polarized into M2 phenotype using IL‐4 (20 ng/mL) and qRT‐PCR was used to measure expression levels of MS4A4A and M2 markers (CD163, ARG1, and TGFB1) and M1 marker CD86. (D) PBMC cells were used to construct a cell line overexpressing MS4A4A. (E) Procedure for testing macrophage inhibition function: Mouse BMDMs and spleen cells were mixed with CT2A/GL261 cells, treated with MS4A4A antibody, and CD8 + T cells were sorted using flow cytometry and Ki‐67 expression was measured. (F) Analysis of Ki‐67 expression on designated CD8 + T cells using flow cytometry ( n = 3). (G) Apoptosis of CT2A cells was detected using fluorescence‐activated cell sorting (FACS). (H) Iron content in tumor‐associated macrophages (TAMs) of sh‐NC and sh‐MS4A4A groups. (I) Reactive oxygen species (ROS) content in TAM cells of sh‐NC and sh‐MS4A4A groups. (J) Expression of iron death‐related proteins in TAM cells of sh‐NC and sh‐MS4A4A groups. (K) Electron microscopy analysis of TAM cells in sh‐NC and sh‐MS4A4A groups. * p < 0.05, and all cell experiments were repeated three times.

Article Snippet: To induce differentiation into M0 macrophages, THP‐1 cells were incubated with 50 ng/mL of phorbol 12‐myristate 13‐acetate (PMA) from InvivoGen (USA) for 48 h. To further polarize M1, cells were incubated with 100 ng/mL lipopolysaccharide (LPS, L2630, Sigma‐Aldrich, USA) and 20 ng/mL recombinant human interferon‐γ (Recombinant Human IFN‐γ, AF‐300‐02, PeproTech, USA) for 48 h. As a control experiment, we conducted the same incubation treatment in PBMC cells (PCS‐800‐011, ATCC, USA) as in THP‐1 cells.

Techniques: Inhibition, Activation Assay, Flow Cytometry, Transfection, Knockdown, Negative Control, Plasmid Preparation, Quantitative RT-PCR, Expressing, Marker, Over Expression, Construct, Fluorescence, FACS, Electron Microscopy

Standardization and validation of CTC by IF×3 using breast, ovarian, and lung cancer cell lines: Patients’ blood samples spiked with titrating number (1000 cells, 750 cells, 375 cells, 250 cells/100 cells) of cell lines of different solid tumors using. Pictures were taken at 60× oil objective of an Olympus IX71 Microscope with DAPI/FITC/TRITC/CY5 filter sets. ( A ): MCF7 cells (750 cells/375 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with a CellSieve enumeration kit (Creatv Microtech) with either DAPI/CK-FITC/EpCAM-PE/CD45-Cy5 ( Ai ) or DAPI/CK-FITC/CD31 PE/CD45-Cy5 ( Aii ). ( B ): OVCAR3 cells (100 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with cell sieve enumeration kit (Creatv MicroTech) with DAPI/CK-FITC/EpCAM-PE/CD45-Cy5. ( C ): HCC1975 cells (1000 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with cell sieve enumeration kit (Creatv Microtech) with DAPI/CK-FITC/EpCAM-PE/CD45-Cy5. ( D ): NCI-H441 cells (250 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with cell sieve enumeration kit (Creatv Microtech) with DAPI/CK-FITC/EpCAM-PE/CD45-Cy5. The magnification, scale bar, and digital reticle are represented for each photomicrograph. Fluorescence images from DAPI, FITC, TRITC, and Cy5 channels were separated as pictures with a color bar. The fluorescence-photomicrographs presented the diameters (μm) of CTC and a representative WBC and their respective DAPI stained nucleus.

Journal: Cancers

Article Title: A Laboratory-Friendly CTC Identification: Comparable Double-Immunocytochemistry with Triple-Immunofluorescence

doi: 10.3390/cancers14122871

Figure Lengend Snippet: Standardization and validation of CTC by IF×3 using breast, ovarian, and lung cancer cell lines: Patients’ blood samples spiked with titrating number (1000 cells, 750 cells, 375 cells, 250 cells/100 cells) of cell lines of different solid tumors using. Pictures were taken at 60× oil objective of an Olympus IX71 Microscope with DAPI/FITC/TRITC/CY5 filter sets. ( A ): MCF7 cells (750 cells/375 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with a CellSieve enumeration kit (Creatv Microtech) with either DAPI/CK-FITC/EpCAM-PE/CD45-Cy5 ( Ai ) or DAPI/CK-FITC/CD31 PE/CD45-Cy5 ( Aii ). ( B ): OVCAR3 cells (100 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with cell sieve enumeration kit (Creatv MicroTech) with DAPI/CK-FITC/EpCAM-PE/CD45-Cy5. ( C ): HCC1975 cells (1000 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with cell sieve enumeration kit (Creatv Microtech) with DAPI/CK-FITC/EpCAM-PE/CD45-Cy5. ( D ): NCI-H441 cells (250 cells per 7.5 mL of patient’s blood) were used for spiking blood samples, and cells were captured on a microfilter and stained with cell sieve enumeration kit (Creatv Microtech) with DAPI/CK-FITC/EpCAM-PE/CD45-Cy5. The magnification, scale bar, and digital reticle are represented for each photomicrograph. Fluorescence images from DAPI, FITC, TRITC, and Cy5 channels were separated as pictures with a color bar. The fluorescence-photomicrographs presented the diameters (μm) of CTC and a representative WBC and their respective DAPI stained nucleus.

Article Snippet: Number: 818M-90) and 1:800 diluted rabbit mAb CD45 (Cell Signaling Technology; D9M8I XP; Catalog # 13917) primary antibodies.

Techniques: Biomarker Discovery, Microscopy, Staining, Fluorescence

Characterization and functional analysis of EVs in GCB‐resistant bladder cancer cells . (A) MTT assay was used to evaluate the viability of T24 and 5637 cells co‐cultured with T24‐ConMed and 5637‐ConMed or T24GCB‐ConMed and 5637GCB‐ConMed for 24 h, followed by GCB treatment, as described in panel E ( n = 6 per group). (B) Representative transmission electron microscopy images of EVs derived from bladder cancer cells (5637‐EVs, T24‐EVs and J82‐EVs) and their GCB‐resistant counterparts (5637GCB‐EVs and T24GCB‐EVs). Scale bar: 100 nm. (C) Nanoparticle tracking analysis (NS300) was used to evaluate the size distribution and concentration of EVs from 5637, 5637GCB, T24, T24GCB and J82 cells. (D) Imaging flow cytometry analysis of EVs stained with a lipid bilayer dye and labelled with EV surface markers CD9‐APC, CD63‐FITC and CD81‐PE. Each dot represents a single EV. (E) Representative fluorescence microscopy images of T24 cells following a 6‐h incubation with T24‐EVs, T24GCB‐EVs, or J82‐EVs pre‐labelled with CD9‐APC, CD63‐FITC and CD81‐PE. Scale bar: 10 µm. (F) Western blot analysis of EV markers (CD9, CD63, CD81, Alix and TSG101) and endosomal protein Rab27A in EVs from the indicated cell lines. (G) MTT assay evaluating the viability of T24 and 5637 cells co‐cultured with the indicated EVs for 24 h, followed by GCB treatment (0–3 µM) for 48 h ( n = 6 per group). (H and I) Colony formation assay was used to evaluate the clonogenic potential of T24 and 5637 cells after co‐culture with T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs, or J82‐EVs for 14 days ( n = 3 per group). (J) Western blot analysis of GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) and anti‐apoptotic protein BCL‐2 in T24 and 5637 cells co‐cultured with T24‐ConMed, T24GCB‐ConMed, or 5637‐ConMed, 5637GCB‐ConMed, or T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs for 24 h. (K, M, N) Electron microscopy images of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) in T24 and T24GCB cells. Quantification of MVBs per image (M) and ILVs per MVB (N). (L, O, P) Electron microscopy images of T24 cells incubated with T24‐EVs or T24GCB‐EVs for 24 h showing MVB and ILV structures. Quantification of MVBs per image (O) and ILVs per MVB (P). Data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed independently in triplicate. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; GCB, gemcitabine; EVs, extracellular vesicles; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Characterization and functional analysis of EVs in GCB‐resistant bladder cancer cells . (A) MTT assay was used to evaluate the viability of T24 and 5637 cells co‐cultured with T24‐ConMed and 5637‐ConMed or T24GCB‐ConMed and 5637GCB‐ConMed for 24 h, followed by GCB treatment, as described in panel E ( n = 6 per group). (B) Representative transmission electron microscopy images of EVs derived from bladder cancer cells (5637‐EVs, T24‐EVs and J82‐EVs) and their GCB‐resistant counterparts (5637GCB‐EVs and T24GCB‐EVs). Scale bar: 100 nm. (C) Nanoparticle tracking analysis (NS300) was used to evaluate the size distribution and concentration of EVs from 5637, 5637GCB, T24, T24GCB and J82 cells. (D) Imaging flow cytometry analysis of EVs stained with a lipid bilayer dye and labelled with EV surface markers CD9‐APC, CD63‐FITC and CD81‐PE. Each dot represents a single EV. (E) Representative fluorescence microscopy images of T24 cells following a 6‐h incubation with T24‐EVs, T24GCB‐EVs, or J82‐EVs pre‐labelled with CD9‐APC, CD63‐FITC and CD81‐PE. Scale bar: 10 µm. (F) Western blot analysis of EV markers (CD9, CD63, CD81, Alix and TSG101) and endosomal protein Rab27A in EVs from the indicated cell lines. (G) MTT assay evaluating the viability of T24 and 5637 cells co‐cultured with the indicated EVs for 24 h, followed by GCB treatment (0–3 µM) for 48 h ( n = 6 per group). (H and I) Colony formation assay was used to evaluate the clonogenic potential of T24 and 5637 cells after co‐culture with T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs, or J82‐EVs for 14 days ( n = 3 per group). (J) Western blot analysis of GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) and anti‐apoptotic protein BCL‐2 in T24 and 5637 cells co‐cultured with T24‐ConMed, T24GCB‐ConMed, or 5637‐ConMed, 5637GCB‐ConMed, or T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs for 24 h. (K, M, N) Electron microscopy images of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) in T24 and T24GCB cells. Quantification of MVBs per image (M) and ILVs per MVB (N). (L, O, P) Electron microscopy images of T24 cells incubated with T24‐EVs or T24GCB‐EVs for 24 h showing MVB and ILV structures. Quantification of MVBs per image (O) and ILVs per MVB (P). Data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed independently in triplicate. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; GCB, gemcitabine; EVs, extracellular vesicles; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.

Article Snippet: After centrifugation, the cells were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal H3C14 monoclonal (1:500, LSBio, Beijing, China) and rabbit polyclonal Rab27A (1:800, Cell Signalling Technology, Danvers, MA, USA).

Techniques: Functional Assay, MTT Assay, Cell Culture, Transmission Assay, Electron Microscopy, Derivative Assay, Concentration Assay, Imaging, Flow Cytometry, Staining, Fluorescence, Microscopy, Incubation, Western Blot, Colony Assay, Co-Culture Assay, Two Tailed Test

Rab27A regulates EV release and histone H3.2 (H3C14) protein excretion in GCB‐resistant bladder cancer cells . (A) RT‐qPCR analysis was used to evaluate Rab27A knockdown efficiency in T24GCB and 5637GCB cells following siRab27A transfection. (B) Representative fluorescence microscopy images of T24GCB‐Vector and T24GCB‐siRab27A cells stained with H3C14‐FITC, Rab27A‐APC and DAPI. Scale bar: 20 µm. (C) Nanoparticle tracking analysis (NS300) of EVs released from T24GCB‐Vector, T24GCB‐siRab27A and 5637GCB‐Vector, 5637GCB‐siRab27A cells revealed particle size distribution and concentration. (D) MTT assay was used to evaluate cell viability of T24GCB‐Vector, T24GCB‐siRab27A and 5637GCB‐Vector, 5637GCB‐siRab27A cells after 48 h treatment with GCB concentrations ranging from 0 to 3 µM ( n = 6 per group). (E) Representative electron microscopy images show MVBs and ILVs in T24GCB‐Vector, T24GCB‐siRab27A and 5637‐Vector, 5637GCB‐siRab27A cells. (F, G) Quantification of MVBs per image (F) and ILVs per MVB (G) from panel L. (H, I) Colony formation assays were used to evaluate clonogenicity of T24GCB and 5637GCB cells transfected with Vector or siRab27A over 10 days ( n = 3 per group). (J) Western blot analysis of Rab27A, H3C14, CNT3 and TK1, as well as EV‐associated proteins (CD9, CD63, CD81, TSG101 and Alix) in T24GCB and 5637GCB cells transfected with Vector or siRab27A. (K) Nanoparticle tracking analysis (NS300) of EVs derived from T24GCB and 5637GCB cells treated with DMSO, neticonazole (1 µM), or ketoconazole (1 µM) for 24 h. (L, M) Colony formation assays in T24GCB and 5637GCB cells treated with DMSO, neticonazole, or ketoconazole (1 µM each) ± GCB (0.001 µM) for 7 days ( n = 3 per group). (N) Western blot analysis of Rab27A, H3C14, Alix, p‐ERK1/2, total ERK1/2 and BCL‐2 in T24GCB and 5637GCB cells after 24 h treatment with DMSO, neticonazole, or ketoconazole. (O) RT‐qPCR analysis was used to evaluate CNT3 knockdown efficiency in T24GCB and 5637GCB cells after siCNT3 transfection. (P) Nanoparticle tracking analysis (NS300) of EVs derived from T24GCB‐Vector, T24GCB‐siCNT3, and 5637GCB‐Vector, 5637GCB‐siCNT3 cells. (Q) MTT assay was used to evaluate viability of T24GCB‐Vector, T24GCB‐siCNT3 and 5637GCB‐Vector, 5637GCB‐siCNT3 cells treated with GCB (0–3 µM) for 48 h ( n = 6 per group). (R) Western blot analysis of CNT3, H3C14, Rab27A, EV markers (CD9, CD63, CD81, Alix and TSG101) and anti‐apoptotic proteins (BCL‐2 and BCL‐XL) in T24GCB and 5637GCB cells transfected with Vector or siCNT3. All data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were repeated independently at least three times. GCB, gemcitabine; EVs, extracellular vesicles; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Rab27A regulates EV release and histone H3.2 (H3C14) protein excretion in GCB‐resistant bladder cancer cells . (A) RT‐qPCR analysis was used to evaluate Rab27A knockdown efficiency in T24GCB and 5637GCB cells following siRab27A transfection. (B) Representative fluorescence microscopy images of T24GCB‐Vector and T24GCB‐siRab27A cells stained with H3C14‐FITC, Rab27A‐APC and DAPI. Scale bar: 20 µm. (C) Nanoparticle tracking analysis (NS300) of EVs released from T24GCB‐Vector, T24GCB‐siRab27A and 5637GCB‐Vector, 5637GCB‐siRab27A cells revealed particle size distribution and concentration. (D) MTT assay was used to evaluate cell viability of T24GCB‐Vector, T24GCB‐siRab27A and 5637GCB‐Vector, 5637GCB‐siRab27A cells after 48 h treatment with GCB concentrations ranging from 0 to 3 µM ( n = 6 per group). (E) Representative electron microscopy images show MVBs and ILVs in T24GCB‐Vector, T24GCB‐siRab27A and 5637‐Vector, 5637GCB‐siRab27A cells. (F, G) Quantification of MVBs per image (F) and ILVs per MVB (G) from panel L. (H, I) Colony formation assays were used to evaluate clonogenicity of T24GCB and 5637GCB cells transfected with Vector or siRab27A over 10 days ( n = 3 per group). (J) Western blot analysis of Rab27A, H3C14, CNT3 and TK1, as well as EV‐associated proteins (CD9, CD63, CD81, TSG101 and Alix) in T24GCB and 5637GCB cells transfected with Vector or siRab27A. (K) Nanoparticle tracking analysis (NS300) of EVs derived from T24GCB and 5637GCB cells treated with DMSO, neticonazole (1 µM), or ketoconazole (1 µM) for 24 h. (L, M) Colony formation assays in T24GCB and 5637GCB cells treated with DMSO, neticonazole, or ketoconazole (1 µM each) ± GCB (0.001 µM) for 7 days ( n = 3 per group). (N) Western blot analysis of Rab27A, H3C14, Alix, p‐ERK1/2, total ERK1/2 and BCL‐2 in T24GCB and 5637GCB cells after 24 h treatment with DMSO, neticonazole, or ketoconazole. (O) RT‐qPCR analysis was used to evaluate CNT3 knockdown efficiency in T24GCB and 5637GCB cells after siCNT3 transfection. (P) Nanoparticle tracking analysis (NS300) of EVs derived from T24GCB‐Vector, T24GCB‐siCNT3, and 5637GCB‐Vector, 5637GCB‐siCNT3 cells. (Q) MTT assay was used to evaluate viability of T24GCB‐Vector, T24GCB‐siCNT3 and 5637GCB‐Vector, 5637GCB‐siCNT3 cells treated with GCB (0–3 µM) for 48 h ( n = 6 per group). (R) Western blot analysis of CNT3, H3C14, Rab27A, EV markers (CD9, CD63, CD81, Alix and TSG101) and anti‐apoptotic proteins (BCL‐2 and BCL‐XL) in T24GCB and 5637GCB cells transfected with Vector or siCNT3. All data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were repeated independently at least three times. GCB, gemcitabine; EVs, extracellular vesicles; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.

Article Snippet: After centrifugation, the cells were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal H3C14 monoclonal (1:500, LSBio, Beijing, China) and rabbit polyclonal Rab27A (1:800, Cell Signalling Technology, Danvers, MA, USA).

Techniques: Quantitative RT-PCR, Knockdown, Transfection, Fluorescence, Microscopy, Plasmid Preparation, Staining, Concentration Assay, MTT Assay, Electron Microscopy, Western Blot, Derivative Assay, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction