mabe191 nono rabbit polyclonal proteintech Search Results


mabe191  (Proteintech)
95
Proteintech mabe191
Mabe191, supplied by Proteintech, 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/mabe191+nono+rabbit+polyclonal+proteintech/bio_rxiv__2025__01__21__634125-169-87-89?v=Proteintech
Average 95 stars, based on 1 article reviews
mabe191 - by Bioz Stars, 2026-08
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nsd1 rabbit polyclonal abx135901 antibody  (Abbexa Ltd)
90
Abbexa Ltd nsd1 rabbit polyclonal abx135901 antibody
Nsd1 Rabbit Polyclonal Abx135901 Antibody, supplied by Abbexa Ltd, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
nsd1 rabbit polyclonal abx135901 antibody - by Bioz Stars, 2026-08
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nsd1 mouse monoclonal n312/10 antibody  (NeuroMab)
90
NeuroMab nsd1 mouse monoclonal n312/10 antibody
Nsd1 Mouse Monoclonal N312/10 Antibody, supplied by NeuroMab, 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/mabe191+nono+rabbit+polyclonal+proteintech/bio_rxiv__2025__01__21__634125-169-22-18?v=NeuroMab
Average 90 stars, based on 1 article reviews
nsd1 mouse monoclonal n312/10 antibody - by Bioz Stars, 2026-08
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f1804  (Proteintech)
94
Proteintech f1804
F1804, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mabe191+nono+rabbit+polyclonal+proteintech/bio_rxiv__2025__01__21__634125-169-121-125?v=Proteintech
Average 94 stars, based on 1 article reviews
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h2av (active motif) rabbit polyclonal  (Active Motif)
90
Active Motif h2av (active motif) rabbit polyclonal
H2av (Active Motif) Rabbit Polyclonal, supplied by Active Motif, 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/mabe191+nono+rabbit+polyclonal+proteintech/bio_rxiv__2021__01__06__425580-13-118-116?v=Active+Motif
Average 90 stars, based on 1 article reviews
h2av (active motif) rabbit polyclonal - by Bioz Stars, 2026-08
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mouse monoclonal antibody catalog no  (Proteintech)
94
Proteintech mouse monoclonal antibody catalog no
Mouse Monoclonal Antibody Catalog No, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mabe191+nono+rabbit+polyclonal+proteintech/bio_rxiv__2025__01__21__634125-169-126-125?v=Proteintech
Average 94 stars, based on 1 article reviews
mouse monoclonal antibody catalog no - by Bioz Stars, 2026-08
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hdgf2  (Proteintech)
92
Proteintech hdgf2
( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including NSD1 and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, <t>HDGF2,</t> GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.
Hdgf2, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mabe191+nono+rabbit+polyclonal+proteintech/pmc08279504-105-16-17?v=Proteintech
Average 92 stars, based on 1 article reviews
hdgf2 - by Bioz Stars, 2026-08
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nsd1  (Bethyl)
92
Bethyl nsd1
( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including <t>NSD1</t> and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.
Nsd1, supplied by Bethyl, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mabe191+nono+rabbit+polyclonal+proteintech/pmc08279504-105-53-54?v=Bethyl
Average 92 stars, based on 1 article reviews
nsd1 - by Bioz Stars, 2026-08
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a301 748a  (Bethyl)
93
Bethyl a301 748a
( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including <t>NSD1</t> and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.
A301 748a, supplied by Bethyl, 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/mabe191+nono+rabbit+polyclonal+proteintech/pmc08279504-105-100-94?v=Bethyl
Average 93 stars, based on 1 article reviews
a301 748a - by Bioz Stars, 2026-08
93/100 stars
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nsd1  (NeuroMab)
93
NeuroMab nsd1
( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including <t>NSD1</t> and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.
Nsd1, supplied by NeuroMab, 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/mabe191+nono+rabbit+polyclonal+proteintech/pmc08279504-105-62-70?v=NeuroMab
Average 93 stars, based on 1 article reviews
nsd1 - by Bioz Stars, 2026-08
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cell lines sf9 insect cell line fisher scientific  (Fisher Scientific)
86
Fisher Scientific cell lines sf9 insect cell line fisher scientific
( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including <t>NSD1</t> and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.
Cell Lines Sf9 Insect Cell Line Fisher Scientific, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mabe191+nono+rabbit+polyclonal+proteintech/pm40913772-273-188-194?v=Fisher+Scientific
Average 86 stars, based on 1 article reviews
cell lines sf9 insect cell line fisher scientific - by Bioz Stars, 2026-08
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Image Search Results


( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including NSD1 and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.

Journal: Science Advances

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1126/sciadv.abg7444

Figure Lengend Snippet: ( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including NSD1 and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Proteintech) rabbit polyclonal, catalog no. 25504-1-AP; HDGF2 (Proteintech) rabbit polyclonal, catalog no. 15134-1-AP; H3K27me3 (Cell Signaling Technology) rabbit monoclonal C36B11, catalog no. 9733; H3K36me2 (Cell Signaling Technology) rabbit monoclonal C75H12, catalog no. 2901; H3K36me3 (Cell Signaling Technology) rabbit monoclonal D5A7, catalog no. 4909; NSD1 (Bethyl Laboratories) rabbit monoclonal, catalog no. A300-BL715 (discontinued); NSD1 (University of California, Davis/National Institutes of Health NeuroMab Facility) mouse monoclonal, N312/10; NSD2 (Millipore) mouse monoclonal 29D1, catalog no. MABE191; NSD3 (Cell Signaling Technology) rabbit monoclonal D4N9N, catalog no. 92056; ASH1L (Bethyl Laboratories) rabbit polyclonal, catalog no. A301-748A; SETD2 (Bio-Rad) mouse monoclonal OTI1E1, catalog no. VMA00449; glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology) rabbit monoclonal D16H11, catalog no. 5174; histone H3 (Abcam) rabbit monoclonal EPR16987, catalog no. ab176842; anti-Flag (Sigma-Aldrich) mouse monoclonal M2, catalog no. F1804; and H2Av (Active Motif) rabbit polyclonal, catalog no. 39715.

Techniques: Western Blot, Expressing, Construct, MTT Assay, Transfection, Control, Stable Transfection, Plasmid Preparation

( A ) Left: A Kaplan-Meier survival curve plot of mice bearing xenograft tumors with endpoints defined by a sign of distress. DIPG13 cells stably expressing a firefly luciferase and control or shRNAs against NSD1 and/or NSD2 were implanted in the cortex of NSG mice by intracranial injection. A total of 250,000 cells were implanted in each mouse ( n = 10 mice for each condition). Right: A quantification of firefly luciferase signals in mice described in the left panel. Bioluminescent imaging (BLI) data were presented at day 35 after injection before the first mouse exhibited a sign of distress. Bottom: Representative BLI images from indicated conditions. ( B ) Left: A Kaplan-Meier survival curve of mice implanted with DIPG13 cells stably expressing control or shRNAs against LEDGF and/or HDGF2 in the same experimental conditions described in (A) ( n = 10 mice for control; n = 5 for shLEDGF; n = 4 for shHDGF2; and n = 9 for shLEDGF + shHDGF2). Right: A quantification of BLI signals in mice describe in the left panel. Bottom: Representative BLI images from indicated conditions. * P < 0.05 and *** P < 0.001 by log-rank test; n.s., not significant ( P > 0.05) for Kaplan-Meier survival analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001 by a nonparametric Mann-Whitney test for BLI quantifications.

Journal: Science Advances

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1126/sciadv.abg7444

Figure Lengend Snippet: ( A ) Left: A Kaplan-Meier survival curve plot of mice bearing xenograft tumors with endpoints defined by a sign of distress. DIPG13 cells stably expressing a firefly luciferase and control or shRNAs against NSD1 and/or NSD2 were implanted in the cortex of NSG mice by intracranial injection. A total of 250,000 cells were implanted in each mouse ( n = 10 mice for each condition). Right: A quantification of firefly luciferase signals in mice described in the left panel. Bioluminescent imaging (BLI) data were presented at day 35 after injection before the first mouse exhibited a sign of distress. Bottom: Representative BLI images from indicated conditions. ( B ) Left: A Kaplan-Meier survival curve of mice implanted with DIPG13 cells stably expressing control or shRNAs against LEDGF and/or HDGF2 in the same experimental conditions described in (A) ( n = 10 mice for control; n = 5 for shLEDGF; n = 4 for shHDGF2; and n = 9 for shLEDGF + shHDGF2). Right: A quantification of BLI signals in mice describe in the left panel. Bottom: Representative BLI images from indicated conditions. * P < 0.05 and *** P < 0.001 by log-rank test; n.s., not significant ( P > 0.05) for Kaplan-Meier survival analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001 by a nonparametric Mann-Whitney test for BLI quantifications.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Proteintech) rabbit polyclonal, catalog no. 25504-1-AP; HDGF2 (Proteintech) rabbit polyclonal, catalog no. 15134-1-AP; H3K27me3 (Cell Signaling Technology) rabbit monoclonal C36B11, catalog no. 9733; H3K36me2 (Cell Signaling Technology) rabbit monoclonal C75H12, catalog no. 2901; H3K36me3 (Cell Signaling Technology) rabbit monoclonal D5A7, catalog no. 4909; NSD1 (Bethyl Laboratories) rabbit monoclonal, catalog no. A300-BL715 (discontinued); NSD1 (University of California, Davis/National Institutes of Health NeuroMab Facility) mouse monoclonal, N312/10; NSD2 (Millipore) mouse monoclonal 29D1, catalog no. MABE191; NSD3 (Cell Signaling Technology) rabbit monoclonal D4N9N, catalog no. 92056; ASH1L (Bethyl Laboratories) rabbit polyclonal, catalog no. A301-748A; SETD2 (Bio-Rad) mouse monoclonal OTI1E1, catalog no. VMA00449; glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology) rabbit monoclonal D16H11, catalog no. 5174; histone H3 (Abcam) rabbit monoclonal EPR16987, catalog no. ab176842; anti-Flag (Sigma-Aldrich) mouse monoclonal M2, catalog no. F1804; and H2Av (Active Motif) rabbit polyclonal, catalog no. 39715.

Techniques: Stable Transfection, Expressing, Luciferase, Control, Injection, Imaging, MANN-WHITNEY

( A ) Left: Representative ChIP-seq tracks of H3K36me2, LEDGF, and HDGF2 chromatin co-occupancy in H3-WT (DIPG10 and pcGBM2) and H3K27M (DIPG4, DIPG6, and DIPG13) cells. Right: Representative heatmaps of ChIP-seq for DIPG4 cells centered to H3K36me2- or H3K27me3-enriched genes. ( B ) Violin plots showing enrichment of H3K36me2, LEDGF, or HDGF2, respectively, for genes categorized as low, mid, or high based on their mRNA expression. The central thick dash line indicates the mean value of each plot; the upper thin dash line indicates the top 25% percentile and the lower one for the bottom 25% percentile. ( C ) Metaprofile plots of H3K27me3, H3K36me2, LEDGF, and HDGF2 ChIP-seq data in HEK293T cells ectopically expressing H3-WT or H3K27M for 24 hours. Data were presented within a 500-kb window upstream or downstream from TSS. ( D ) Top: Metaprofile plots of LEDGF and HDGF2 occupancy in WT or NSD2-KO DIPG13 cells (clone #7 and clone #10). Bottom: Representative ChIP-seq tracks for the top panel. Overlaid panels were presented at the bottom to better illustrate the differences. TSS, transcription start site; TES, transcription end site. **** P < 0.0001 by Student’s t test.

Journal: Science Advances

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1126/sciadv.abg7444

Figure Lengend Snippet: ( A ) Left: Representative ChIP-seq tracks of H3K36me2, LEDGF, and HDGF2 chromatin co-occupancy in H3-WT (DIPG10 and pcGBM2) and H3K27M (DIPG4, DIPG6, and DIPG13) cells. Right: Representative heatmaps of ChIP-seq for DIPG4 cells centered to H3K36me2- or H3K27me3-enriched genes. ( B ) Violin plots showing enrichment of H3K36me2, LEDGF, or HDGF2, respectively, for genes categorized as low, mid, or high based on their mRNA expression. The central thick dash line indicates the mean value of each plot; the upper thin dash line indicates the top 25% percentile and the lower one for the bottom 25% percentile. ( C ) Metaprofile plots of H3K27me3, H3K36me2, LEDGF, and HDGF2 ChIP-seq data in HEK293T cells ectopically expressing H3-WT or H3K27M for 24 hours. Data were presented within a 500-kb window upstream or downstream from TSS. ( D ) Top: Metaprofile plots of LEDGF and HDGF2 occupancy in WT or NSD2-KO DIPG13 cells (clone #7 and clone #10). Bottom: Representative ChIP-seq tracks for the top panel. Overlaid panels were presented at the bottom to better illustrate the differences. TSS, transcription start site; TES, transcription end site. **** P < 0.0001 by Student’s t test.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Proteintech) rabbit polyclonal, catalog no. 25504-1-AP; HDGF2 (Proteintech) rabbit polyclonal, catalog no. 15134-1-AP; H3K27me3 (Cell Signaling Technology) rabbit monoclonal C36B11, catalog no. 9733; H3K36me2 (Cell Signaling Technology) rabbit monoclonal C75H12, catalog no. 2901; H3K36me3 (Cell Signaling Technology) rabbit monoclonal D5A7, catalog no. 4909; NSD1 (Bethyl Laboratories) rabbit monoclonal, catalog no. A300-BL715 (discontinued); NSD1 (University of California, Davis/National Institutes of Health NeuroMab Facility) mouse monoclonal, N312/10; NSD2 (Millipore) mouse monoclonal 29D1, catalog no. MABE191; NSD3 (Cell Signaling Technology) rabbit monoclonal D4N9N, catalog no. 92056; ASH1L (Bethyl Laboratories) rabbit polyclonal, catalog no. A301-748A; SETD2 (Bio-Rad) mouse monoclonal OTI1E1, catalog no. VMA00449; glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology) rabbit monoclonal D16H11, catalog no. 5174; histone H3 (Abcam) rabbit monoclonal EPR16987, catalog no. ab176842; anti-Flag (Sigma-Aldrich) mouse monoclonal M2, catalog no. F1804; and H2Av (Active Motif) rabbit polyclonal, catalog no. 39715.

Techniques: ChIP-sequencing, Expressing

( A ) Venn diagrams showing overlaps of gene signatures/pathways down-regulated in LEDGF/HDGF2 dKD DIPG13 cells and NSD2-KO#7 + siNSD1 or NSD2-KO#10 + siNSD1 DIPG13 cells ( P = 4.2 × 10 −9 and 1.8 × 10 −4 , respectively). The alteration of gene signatures/pathways was detected by GSEA. P values were obtained by the hypergeometric distribution to compute the significance of the overlap of two pathway sets. ( B ) Representative images of highly ranked GSEA signatures/pathways detected in (A), including a CHEK2 pathway, an embryonic stem cell (ESC) steamness signature, and a set of EGFR signaling target genes. ( C ) Top: A schematic illustration of the design of CPP. A HIV-based cell entry peptide was linked to a H3K36me2 peptide (histone H3 21 to 43 amino acids, cargo peptide) by a disulfide bond. Bottom: Amino acid sequence of H3K36me2-linked CPP. ( D ) A CellTiter-Glo cell survival assay for H3-WT (DIPG10 and pcGBM2) and H3K27M (DIPG-N and DIPG13) cells treated with control (vehicle only) or H3K36me2-CPP. Cells were assayed at 72 hours after dosing with a titration of control or H3K36me2-CPP and data were presented by ratios of CellTiter-Glo signals in control versus H3K36me2-CPP treated cells. PBS, phosphate-buffered saline. ( E ) Metaprofiles of ChIP-seq analysis for changes in LEDGF and HDGF2 occupancy of genes enriched with H3K36me2 or H3K36me3 and the rest of genes in DIPG13 cells treated with a control vehicle (red) or a H3K36me2-CPP (blue).

Journal: Science Advances

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1126/sciadv.abg7444

Figure Lengend Snippet: ( A ) Venn diagrams showing overlaps of gene signatures/pathways down-regulated in LEDGF/HDGF2 dKD DIPG13 cells and NSD2-KO#7 + siNSD1 or NSD2-KO#10 + siNSD1 DIPG13 cells ( P = 4.2 × 10 −9 and 1.8 × 10 −4 , respectively). The alteration of gene signatures/pathways was detected by GSEA. P values were obtained by the hypergeometric distribution to compute the significance of the overlap of two pathway sets. ( B ) Representative images of highly ranked GSEA signatures/pathways detected in (A), including a CHEK2 pathway, an embryonic stem cell (ESC) steamness signature, and a set of EGFR signaling target genes. ( C ) Top: A schematic illustration of the design of CPP. A HIV-based cell entry peptide was linked to a H3K36me2 peptide (histone H3 21 to 43 amino acids, cargo peptide) by a disulfide bond. Bottom: Amino acid sequence of H3K36me2-linked CPP. ( D ) A CellTiter-Glo cell survival assay for H3-WT (DIPG10 and pcGBM2) and H3K27M (DIPG-N and DIPG13) cells treated with control (vehicle only) or H3K36me2-CPP. Cells were assayed at 72 hours after dosing with a titration of control or H3K36me2-CPP and data were presented by ratios of CellTiter-Glo signals in control versus H3K36me2-CPP treated cells. PBS, phosphate-buffered saline. ( E ) Metaprofiles of ChIP-seq analysis for changes in LEDGF and HDGF2 occupancy of genes enriched with H3K36me2 or H3K36me3 and the rest of genes in DIPG13 cells treated with a control vehicle (red) or a H3K36me2-CPP (blue).

Article Snippet: The antibodies used in this study are as follows: LEDGF (Proteintech) rabbit polyclonal, catalog no. 25504-1-AP; HDGF2 (Proteintech) rabbit polyclonal, catalog no. 15134-1-AP; H3K27me3 (Cell Signaling Technology) rabbit monoclonal C36B11, catalog no. 9733; H3K36me2 (Cell Signaling Technology) rabbit monoclonal C75H12, catalog no. 2901; H3K36me3 (Cell Signaling Technology) rabbit monoclonal D5A7, catalog no. 4909; NSD1 (Bethyl Laboratories) rabbit monoclonal, catalog no. A300-BL715 (discontinued); NSD1 (University of California, Davis/National Institutes of Health NeuroMab Facility) mouse monoclonal, N312/10; NSD2 (Millipore) mouse monoclonal 29D1, catalog no. MABE191; NSD3 (Cell Signaling Technology) rabbit monoclonal D4N9N, catalog no. 92056; ASH1L (Bethyl Laboratories) rabbit polyclonal, catalog no. A301-748A; SETD2 (Bio-Rad) mouse monoclonal OTI1E1, catalog no. VMA00449; glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology) rabbit monoclonal D16H11, catalog no. 5174; histone H3 (Abcam) rabbit monoclonal EPR16987, catalog no. ab176842; anti-Flag (Sigma-Aldrich) mouse monoclonal M2, catalog no. F1804; and H2Av (Active Motif) rabbit polyclonal, catalog no. 39715.

Techniques: Sequencing, Clonogenic Cell Survival Assay, Control, Titration, Saline, ChIP-sequencing

( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including NSD1 and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.

Journal: Science Advances

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1126/sciadv.abg7444

Figure Lengend Snippet: ( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including NSD1 and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Proteintech) rabbit polyclonal, catalog no. 25504-1-AP; HDGF2 (Proteintech) rabbit polyclonal, catalog no. 15134-1-AP; H3K27me3 (Cell Signaling Technology) rabbit monoclonal C36B11, catalog no. 9733; H3K36me2 (Cell Signaling Technology) rabbit monoclonal C75H12, catalog no. 2901; H3K36me3 (Cell Signaling Technology) rabbit monoclonal D5A7, catalog no. 4909; NSD1 (Bethyl Laboratories) rabbit monoclonal, catalog no. A300-BL715 (discontinued); NSD1 (University of California, Davis/National Institutes of Health NeuroMab Facility) mouse monoclonal, N312/10; NSD2 (Millipore) mouse monoclonal 29D1, catalog no. MABE191; NSD3 (Cell Signaling Technology) rabbit monoclonal D4N9N, catalog no. 92056; ASH1L (Bethyl Laboratories) rabbit polyclonal, catalog no. A301-748A; SETD2 (Bio-Rad) mouse monoclonal OTI1E1, catalog no. VMA00449; glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology) rabbit monoclonal D16H11, catalog no. 5174; histone H3 (Abcam) rabbit monoclonal EPR16987, catalog no. ab176842; anti-Flag (Sigma-Aldrich) mouse monoclonal M2, catalog no. F1804; and H2Av (Active Motif) rabbit polyclonal, catalog no. 39715.

Techniques: Western Blot, Expressing, Construct, MTT Assay, Transfection, Control, Stable Transfection, Plasmid Preparation

( A ) Left: A Kaplan-Meier survival curve plot of mice bearing xenograft tumors with endpoints defined by a sign of distress. DIPG13 cells stably expressing a firefly luciferase and control or shRNAs against NSD1 and/or NSD2 were implanted in the cortex of NSG mice by intracranial injection. A total of 250,000 cells were implanted in each mouse ( n = 10 mice for each condition). Right: A quantification of firefly luciferase signals in mice described in the left panel. Bioluminescent imaging (BLI) data were presented at day 35 after injection before the first mouse exhibited a sign of distress. Bottom: Representative BLI images from indicated conditions. ( B ) Left: A Kaplan-Meier survival curve of mice implanted with DIPG13 cells stably expressing control or shRNAs against LEDGF and/or HDGF2 in the same experimental conditions described in (A) ( n = 10 mice for control; n = 5 for shLEDGF; n = 4 for shHDGF2; and n = 9 for shLEDGF + shHDGF2). Right: A quantification of BLI signals in mice describe in the left panel. Bottom: Representative BLI images from indicated conditions. * P < 0.05 and *** P < 0.001 by log-rank test; n.s., not significant ( P > 0.05) for Kaplan-Meier survival analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001 by a nonparametric Mann-Whitney test for BLI quantifications.

Journal: Science Advances

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1126/sciadv.abg7444

Figure Lengend Snippet: ( A ) Left: A Kaplan-Meier survival curve plot of mice bearing xenograft tumors with endpoints defined by a sign of distress. DIPG13 cells stably expressing a firefly luciferase and control or shRNAs against NSD1 and/or NSD2 were implanted in the cortex of NSG mice by intracranial injection. A total of 250,000 cells were implanted in each mouse ( n = 10 mice for each condition). Right: A quantification of firefly luciferase signals in mice described in the left panel. Bioluminescent imaging (BLI) data were presented at day 35 after injection before the first mouse exhibited a sign of distress. Bottom: Representative BLI images from indicated conditions. ( B ) Left: A Kaplan-Meier survival curve of mice implanted with DIPG13 cells stably expressing control or shRNAs against LEDGF and/or HDGF2 in the same experimental conditions described in (A) ( n = 10 mice for control; n = 5 for shLEDGF; n = 4 for shHDGF2; and n = 9 for shLEDGF + shHDGF2). Right: A quantification of BLI signals in mice describe in the left panel. Bottom: Representative BLI images from indicated conditions. * P < 0.05 and *** P < 0.001 by log-rank test; n.s., not significant ( P > 0.05) for Kaplan-Meier survival analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001 by a nonparametric Mann-Whitney test for BLI quantifications.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Proteintech) rabbit polyclonal, catalog no. 25504-1-AP; HDGF2 (Proteintech) rabbit polyclonal, catalog no. 15134-1-AP; H3K27me3 (Cell Signaling Technology) rabbit monoclonal C36B11, catalog no. 9733; H3K36me2 (Cell Signaling Technology) rabbit monoclonal C75H12, catalog no. 2901; H3K36me3 (Cell Signaling Technology) rabbit monoclonal D5A7, catalog no. 4909; NSD1 (Bethyl Laboratories) rabbit monoclonal, catalog no. A300-BL715 (discontinued); NSD1 (University of California, Davis/National Institutes of Health NeuroMab Facility) mouse monoclonal, N312/10; NSD2 (Millipore) mouse monoclonal 29D1, catalog no. MABE191; NSD3 (Cell Signaling Technology) rabbit monoclonal D4N9N, catalog no. 92056; ASH1L (Bethyl Laboratories) rabbit polyclonal, catalog no. A301-748A; SETD2 (Bio-Rad) mouse monoclonal OTI1E1, catalog no. VMA00449; glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology) rabbit monoclonal D16H11, catalog no. 5174; histone H3 (Abcam) rabbit monoclonal EPR16987, catalog no. ab176842; anti-Flag (Sigma-Aldrich) mouse monoclonal M2, catalog no. F1804; and H2Av (Active Motif) rabbit polyclonal, catalog no. 39715.

Techniques: Stable Transfection, Expressing, Luciferase, Control, Injection, Imaging, MANN-WHITNEY

( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including NSD1 and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.

Journal: Science Advances

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1126/sciadv.abg7444

Figure Lengend Snippet: ( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including NSD1 and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Proteintech) rabbit polyclonal, catalog no. 25504-1-AP; HDGF2 (Proteintech) rabbit polyclonal, catalog no. 15134-1-AP; H3K27me3 (Cell Signaling Technology) rabbit monoclonal C36B11, catalog no. 9733; H3K36me2 (Cell Signaling Technology) rabbit monoclonal C75H12, catalog no. 2901; H3K36me3 (Cell Signaling Technology) rabbit monoclonal D5A7, catalog no. 4909; NSD1 (Bethyl Laboratories) rabbit monoclonal, catalog no. A300-BL715 (discontinued); NSD1 (University of California, Davis/National Institutes of Health NeuroMab Facility) mouse monoclonal, N312/10; NSD2 (Millipore) mouse monoclonal 29D1, catalog no. MABE191; NSD3 (Cell Signaling Technology) rabbit monoclonal D4N9N, catalog no. 92056; ASH1L (Bethyl Laboratories) rabbit polyclonal, catalog no. A301-748A; SETD2 (Bio-Rad) mouse monoclonal OTI1E1, catalog no. VMA00449; glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology) rabbit monoclonal D16H11, catalog no. 5174; histone H3 (Abcam) rabbit monoclonal EPR16987, catalog no. ab176842; anti-Flag (Sigma-Aldrich) mouse monoclonal M2, catalog no. F1804; and H2Av (Active Motif) rabbit polyclonal, catalog no. 39715.

Techniques: Western Blot, Expressing, Construct, MTT Assay, Transfection, Control, Stable Transfection, Plasmid Preparation

( A ) Left: A Kaplan-Meier survival curve plot of mice bearing xenograft tumors with endpoints defined by a sign of distress. DIPG13 cells stably expressing a firefly luciferase and control or shRNAs against NSD1 and/or NSD2 were implanted in the cortex of NSG mice by intracranial injection. A total of 250,000 cells were implanted in each mouse ( n = 10 mice for each condition). Right: A quantification of firefly luciferase signals in mice described in the left panel. Bioluminescent imaging (BLI) data were presented at day 35 after injection before the first mouse exhibited a sign of distress. Bottom: Representative BLI images from indicated conditions. ( B ) Left: A Kaplan-Meier survival curve of mice implanted with DIPG13 cells stably expressing control or shRNAs against LEDGF and/or HDGF2 in the same experimental conditions described in (A) ( n = 10 mice for control; n = 5 for shLEDGF; n = 4 for shHDGF2; and n = 9 for shLEDGF + shHDGF2). Right: A quantification of BLI signals in mice describe in the left panel. Bottom: Representative BLI images from indicated conditions. * P < 0.05 and *** P < 0.001 by log-rank test; n.s., not significant ( P > 0.05) for Kaplan-Meier survival analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001 by a nonparametric Mann-Whitney test for BLI quantifications.

Journal: Science Advances

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1126/sciadv.abg7444

Figure Lengend Snippet: ( A ) Left: A Kaplan-Meier survival curve plot of mice bearing xenograft tumors with endpoints defined by a sign of distress. DIPG13 cells stably expressing a firefly luciferase and control or shRNAs against NSD1 and/or NSD2 were implanted in the cortex of NSG mice by intracranial injection. A total of 250,000 cells were implanted in each mouse ( n = 10 mice for each condition). Right: A quantification of firefly luciferase signals in mice described in the left panel. Bioluminescent imaging (BLI) data were presented at day 35 after injection before the first mouse exhibited a sign of distress. Bottom: Representative BLI images from indicated conditions. ( B ) Left: A Kaplan-Meier survival curve of mice implanted with DIPG13 cells stably expressing control or shRNAs against LEDGF and/or HDGF2 in the same experimental conditions described in (A) ( n = 10 mice for control; n = 5 for shLEDGF; n = 4 for shHDGF2; and n = 9 for shLEDGF + shHDGF2). Right: A quantification of BLI signals in mice describe in the left panel. Bottom: Representative BLI images from indicated conditions. * P < 0.05 and *** P < 0.001 by log-rank test; n.s., not significant ( P > 0.05) for Kaplan-Meier survival analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001 by a nonparametric Mann-Whitney test for BLI quantifications.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Proteintech) rabbit polyclonal, catalog no. 25504-1-AP; HDGF2 (Proteintech) rabbit polyclonal, catalog no. 15134-1-AP; H3K27me3 (Cell Signaling Technology) rabbit monoclonal C36B11, catalog no. 9733; H3K36me2 (Cell Signaling Technology) rabbit monoclonal C75H12, catalog no. 2901; H3K36me3 (Cell Signaling Technology) rabbit monoclonal D5A7, catalog no. 4909; NSD1 (Bethyl Laboratories) rabbit monoclonal, catalog no. A300-BL715 (discontinued); NSD1 (University of California, Davis/National Institutes of Health NeuroMab Facility) mouse monoclonal, N312/10; NSD2 (Millipore) mouse monoclonal 29D1, catalog no. MABE191; NSD3 (Cell Signaling Technology) rabbit monoclonal D4N9N, catalog no. 92056; ASH1L (Bethyl Laboratories) rabbit polyclonal, catalog no. A301-748A; SETD2 (Bio-Rad) mouse monoclonal OTI1E1, catalog no. VMA00449; glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology) rabbit monoclonal D16H11, catalog no. 5174; histone H3 (Abcam) rabbit monoclonal EPR16987, catalog no. ab176842; anti-Flag (Sigma-Aldrich) mouse monoclonal M2, catalog no. F1804; and H2Av (Active Motif) rabbit polyclonal, catalog no. 39715.

Techniques: Stable Transfection, Expressing, Luciferase, Control, Injection, Imaging, MANN-WHITNEY