Journal: Oncogene

Article Title: Jumonji histone demethylases are therapeutic targets in small cell lung cancer

doi: 10.1038/s41388-024-03125-x

Figure Lengend Snippet: IC 50 values of etoposide, JIB-04, SD70, and GSK-J4 across 31 SCLC cell lines representing all transcription factor subgroups.

Article Snippet: H446, H446 Cas9, and H446 Cas9 KDM4A cell lines were plated into six 175 cm 2 flasks and grown to 75% confluence (Corning, 07-202-000).

Techniques:

Journal: Oncogene

Article Title: Jumonji histone demethylases are therapeutic targets in small cell lung cancer

doi: 10.1038/s41388-024-03125-x

Figure Lengend Snippet: A Range of IC 50 responses to JIB-04, SD70, and GSK-J4 of SCLC cell lines representing all clusters. Each diamond represents the median IC 50 across multiple experiments of a particular cell line, measured by 4-day MTS assay (see also Table ). The IC 50 values are shown on a log scale. B Inhibitors act on target as demonstrated by robust decreases in Jumonji enzymatic activity when cells are treated with their corresponding IC 50 values for 24 h. Bar graphs display the level of H3K9me3 demethylase activity in H446 and H2171 cell lysates and/or nuclear extracts after the indicated treatment. ** = p ≤ 0.01 and * = p ≤ 0.05 by two-tailed t -test, unequal variance. Data are average ± SEM. C Correlation analysis yields negative correlations between the IC 50 of etoposide and the IC 50 of each of the three Jumonji inhibitors tested across 31 SCLC cell lines: JIB-04 (pan-JmjC KDM inhibitor with some selectivity for KDM5s), SD70 (relatively selective KDM4 inhibitor) and GSK-J4 (relatively selective KDM6 inhibitor). Pearson R-values are shown. D Heat map of etoposide and Jumonji inhibitor IC 50 values represented in a blue to red scale for individual drugs in the indicated concentration ranges. Chemoresistant cell lines in the green box are sensitive to Jumonji inhibitors, particularly to JIB-04. E Dose-response curves demonstrate three etoposide most resistant cell lines (H378, H889, and H510) are highly sensitive to JIB-04. H524 and HCC4003 are sensitive to etoposide and are given for reference. Representative curves of n = 4–8 replicates with SEM are shown. See Table for further details.

Article Snippet: H446, H446 Cas9, and H446 Cas9 KDM4A cell lines were plated into six 175 cm 2 flasks and grown to 75% confluence (Corning, 07-202-000).

Techniques: MTS Assay, Activity Assay, Two Tailed Test, Concentration Assay

Journal: Oncogene

Article Title: Jumonji histone demethylases are therapeutic targets in small cell lung cancer

doi: 10.1038/s41388-024-03125-x

Figure Lengend Snippet: A Western blot analysis demonstrates multiple proteins in the ER stress pathway are elevated by treatment with IC 50 doses of JIB-04 or SD70 for 24 h across multiple SCLC lines representing various inhibitor sensitivities and both NEUROD1-high (H446, H2171, H524) and ASCL1-high transcription (H510, H1522, H1417, H2107) factor clusters. Tubulin or GAPDH were used as a loading control. B Activation of phospho-elF2α is also seen by Western analysis in SCLC cell lines treated with inhibitors for 24 h at their respective IC 50 . GAPDH was used as loading control. C Annexin V staining was analyzed by FACS after 48 h (H446, H2171, H510) or 72 h (H524) of inhibitor treatment, and double-positive early or late apoptotic cells quantified. Data are averages across replicates with error bars representing SEM. **** = p ≤ 0.0001, *** = p ≤ 0.001, ** = p ≤ 0.01, * = p < 0.05, by unpaired two-tailed t -test. D Inhibitor treatment with IC 50 for 48 h triggers PARP cleavage in H510 as shown by Western blot, indicating late stage of apoptosis. GAPDH served as a loading control. C shows quantification of early apoptotic H510 cells at this time point.

Article Snippet: H446, H446 Cas9, and H446 Cas9 KDM4A cell lines were plated into six 175 cm 2 flasks and grown to 75% confluence (Corning, 07-202-000).

Techniques: Western Blot, Control, Activation Assay, Staining, Two Tailed Test

Journal: Oncogene

Article Title: Jumonji histone demethylases are therapeutic targets in small cell lung cancer

doi: 10.1038/s41388-024-03125-x

Figure Lengend Snippet: A Immunoblot demonstrating decrease of KDM4A (top) in H446 cells compared to parental and Cas9-only control cells. GAPDH (bottom) was used as the loading control. B Loss of KDM4A in H446 decreases proliferation compared to parental and Cas9-only H446 cells. See the methods section for experimental details. Error bars represent SD from the mean across 4–5 biological replicates. ** = p ≤ 0.01, * = p ≤ 0.05, by two-tailed t -test, unequal variance. C Depletion of KDM4A in H446 decreases colony formation potential compared to parental and Cas9-only H446 cells. Each colony is defined by a cluster of approximately 100 cells. Error bars are SEM across six replicates. **** = p ≤ 0.0001 by two-tailed t -test, unequal variance. D Upregulation of ER stress pathway genes measured by RNA-sequencing in KDM4A knockdown compared to parental and Cas9-only H446 cells. Data are shown as mean ± SD, n = 3. *** = p ≤ 0.001, ** = p ≤ 0.01, * = p ≤ 0.05, by two-tailed t -test, unpaired with Welch’s correction. E ER stress proteins p-elF2α, DDIT3, and CHAC1 are upregulated in H446 KDM4A knockdown cells vs controls, as shown by Western blot. Tubulin is a loading control. F MTS viability curves of H446 Cas9 vs KDM4A knockdown cells in response to mTOR inhibitors. Data are average ± SD for each dose, n = 4. P -values for curve comparisons calculated by GraphPad Prism (top, bottom, IC 50 , Hill coefficient). G Growth of cell line-derived xenografts in nude mice demonstrates suppressed tumor progression in H446 tumors with KDM4A knockdown compared to controls. Data are shown as mean ± SEM. H446 parental ( n = 5), H446 Cas9 ( n = 6), H446 Cas9 KDM4A ( n = 7) and * = p ≤ 0.05, by one-tailed t -test, unequal variance. H Bar graph representing final tumor volumes in H446 xenografts. H446 KDM4A knockdown xenograft volumes were significantly lower compared to parental and Cas9-only controls. Data are shown as means ± SEM. ** = p ≤ 0.01, * = p < 0.05, by one-tailed t -test, unpaired with Welch’s correction.

Article Snippet: H446, H446 Cas9, and H446 Cas9 KDM4A cell lines were plated into six 175 cm 2 flasks and grown to 75% confluence (Corning, 07-202-000).

Techniques: Western Blot, Control, Two Tailed Test, RNA Sequencing, Knockdown, Derivative Assay, One-tailed Test

Journal: Oncogene

Article Title: Jumonji histone demethylases are therapeutic targets in small cell lung cancer

doi: 10.1038/s41388-024-03125-x

Figure Lengend Snippet: A Growth of H446 xenograft tumors grown in nude mice is significantly suppressed after treatment with JIB-04. Tumor weight was likewise significantly decreased in JIB-04-treated mice. JIB-04 was given via oral gavage three times a week (Mon, Wed, Fri) at 75 mg/kg. Data are shown as mean ± SEM ( n = 4 per treatment group). B Growth of H446 xenograft tumors grown in nude mice was suppressed after treatment with SD70. Tumor weight decreased in SD70-treated animals, showing a nearly significant trend. SD70 was given by IP injection Mon-Fri at 10 mg/kg. Data are shown as mean ± SEM ( n = 4 per treatment group). C Growth of H510 xenograft tumors grown in nude mice is significantly suppressed after treatment with SD70. Tumor weight was significantly decreased in SD70-treated animals. Data are shown as mean ± SEM ( n = 3 per treatment group). ** = p ≤ 0.01, * = p ≤ 0.05, by two-tailed t -test, unequal variance. D Venn diagrams depicting the overlap in upregulated genes (≥1.5 over vehicle) between JIB-04 and SD70 in H446 xenografts (left panel, n = 3 per treatment group) and between SD70-treated H446 vs H510 xenografts ( n = 3 per xenograft).

Article Snippet: H446, H446 Cas9, and H446 Cas9 KDM4A cell lines were plated into six 175 cm 2 flasks and grown to 75% confluence (Corning, 07-202-000).

Techniques: Injection, Two Tailed Test

Journal: Oncogene

Article Title: Jumonji histone demethylases are therapeutic targets in small cell lung cancer

doi: 10.1038/s41388-024-03125-x

Figure Lengend Snippet: A The SCLC marker Secretogranin-3 is decreased in KDM4A knockdown cell secreted media compared to Cas9 control H446 cells. A representative immunoblot is shown along with a section of total protein stain for the same gel. Quantification across two independent experiments normalized to total protein lysate is shown in the bar graph. B Western analysis validates the downregulation of INSM1 protein expression in H446 KDM4A KD cells, as well as in HCC4001, H510, and H2107 cells treated with Jumonji inhibitors, as indicated. GAPDH was used as a loading control. C NEUROD1 or ASCL1 Western blots show decreased protein in KDM4A KD or SD70 and JIB-04 treated cells. NEUROD1-high (H446, HCC4001) and ASCL1-high cells (H510, H2107) cells are shown. GAPDH as loading control. Note that in some cases the same gels were used to probe for INSM1 and NEUROD1 or ASCL1 thus sharing the same loading control. Quantifications are given in Fig. . D Western blots showing upregulation of H3K9me3 levels in H510 tumor nuclear extracts ( n = 2 per treatment group) with RNA polymerase II used as a loading control. Quantification is shown on the right with lines connecting the DMSO control and SD70-treated mouse pairs. E Western blots showing decreased INSM1 and ASCL1 protein levels in H510 tumor lysates ( n = 2 per treatment group) with GAPDH as a loading control. Quantifications are shown on the right with lines connecting DMSO control and SD70-treated mouse pairs. D , E dotted lines represent the place in the membrane where an extra control lane between DMSO and drug-treated samples was removed for simplicity of presentation.

Article Snippet: H446, H446 Cas9, and H446 Cas9 KDM4A cell lines were plated into six 175 cm 2 flasks and grown to 75% confluence (Corning, 07-202-000).

Techniques: Marker, Knockdown, Control, Western Blot, Staining, Expressing, Membrane

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) Schematic of Cas9 fused to the HUH endonuclease PCV with a covalently attached ssODN. ( b ) SDS-PAGE of Cas9 variants reacted with an Alexa 488 fluorescently labelled ssDNA containing the PCV recognition sequence. The top panel is the coomassie stained gel, and the bottom panel is the identical fluorescently imaged gel. PCV is fused to either the carboxyl (Cas9-PCV) or amino (PCV-Cas9) terminus of Cas9. Cas9-PCV(Y96F) represents catalytically inactive PCV(Y96F) fused to Cas9. (C) SDS-PAGE gel shift assay of Cas9 reacting with two ssDNA templates containing the PCV recognition sequence of differing lengths in a 1:1 ssDNA:Cas9 molar ratio.

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: SDS Page, Sequencing, Staining, Electrophoretic Mobility Shift Assay

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) Schematic of split luciferase insertion. The C-terminus of NanoLuc nanoluciferase (HiBiT) is encoded on the 200bp ssODN along with the 5’ PCV recognition sequence and targeted to the 3’ end of GAPDH . ( b ) Assaying luminescence using different Cas9 variants when inserting HiBiT into GAPDH in HEK-293T cells. PCV is fused to either the amino (PCV-Cas9) or carboxyl (Cas9-PCV) terminus of Cas9. Transfections were performed with ssODN lacking the PCV recognition sequence (PCV- ssODN) or ssODN containing the PCV recognition sequence (PCV+ ssODN). Units are displayed in relative light units (RLU) normalized to Cas9. ( c ) The calculated fold change from (b) between the PCV-ssODN and PCV+ ssODN is shown for each variant. ( d ) Targeting the GAPDH locus in U2-OS cells. ( e ) Targeting a locus in vinculin in HEK-293T cells using an ssODN containing the PCV recognition sequence. ( f ) Fold change in RLU compared to Cas9 when varying the amount of RNP (equimolar ssODN). All graphs represent data from one of multiple independent experiments exhibiting similar results. Data are shown as mean +/− SD (n=3). Significance calculated using 2-tailed Student’s t-test: ** P < 0.01, *** P < 0.001, ns = no significance (P >0.05).

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: Luciferase, Sequencing, Transfection, Variant Assay

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) HEK-293T cells stably expressing a mutant mCherry-GFP reporter are edited by HDR through a frameshift correction, restoring mCherry activity. ( b ) Representative microscopy images of fluorescent reporter editing. ( c ) The percent of mCherry positive cells determined by flow cytometry at two different RNP concentrations using an ssODN containing the PCV recognition sequence ( d ) RNP transfections at 3 pmol in the presence or absence of ssODN. Data are shown as mean +/− SD (n=3). For (c) and (d), the statistical significance of %mCherry positive cells between PCV-fusions of Cas9 and Cas9 alone was <0.001, calculated using 2-tailed Student’s t-test.

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: Stable Transfection, Expressing, Mutagenesis, Activity Assay, Microscopy, Flow Cytometry, Sequencing, Transfection

Journal: bioRxiv

Article Title: Towards CRISPR/Cas9-based gene drive in the diamondback moth Plutella xylostella

doi: 10.1101/2021.10.05.462963

Figure Lengend Snippet: A: Donor cassettes for piggyBac -mediated transformation of Cas9 lines. B: HDR-based integration of homing elements into endogenous marker genes Pxyellow and Pxkmo .

Article Snippet: The Cas9 Sequencing Kit (Oxford Nanopore Technology) was used to prepare a library targeting the insertion.

Techniques: Transformation Assay, Marker

Journal: bioRxiv

Article Title: Towards CRISPR/Cas9-based gene drive in the diamondback moth Plutella xylostella

doi: 10.1101/2021.10.05.462963

Figure Lengend Snippet: A: Parental Cas9 lines were crossed with the 1619P15 line integrated into and expressing sgRNAs targeting Pxyellow . B: Parental Cas9 lines are crossed with the 1963C line integrated into and expressing sgRNAs targeting Pxkmo . Mosaic phenotypes are indicated using white arrows.

Article Snippet: The Cas9 Sequencing Kit (Oxford Nanopore Technology) was used to prepare a library targeting the insertion.

Techniques: Expressing

Journal: bioRxiv

Article Title: Towards CRISPR/Cas9-based gene drive in the diamondback moth Plutella xylostella

doi: 10.1101/2021.10.05.462963

Figure Lengend Snippet: A: Proportion of F 2 individuals inheriting the relevant homing element. Offspring come from either a male (blue) or female (orange) Cas9-bearing F 1 parent. Due to non-significant effect of ‘grandparental’ (F 0 ) sex on the model, this factor has been collapsed in the data shown. Central tendency and error bars represent estimated mean proportions and associated approximate 95% confidence intervals for that treatment. Shaded areas (violin plots) represent the density distribution of the raw data. B: Germline cleavage efficiency (%) = no. phenotypic mutants (e.g. yellow eyes) / (no. phenotypic mutants + phenotypic wildtypes (e.g. black eyes)). Offspring come from either a male (blue) or female (orange) Cas9-bearing F 1 parent Data are plotted as means overlaid on raw data. For comparison of lines within the same sex, those which share a letter above the plotted data (e.g. ab and bc) are not significantly different from each other. For comparisons between sex, within each line, “*” shows the significant difference between subgroups. *: P < 0.05. **: P < 0.01. ***: P < 0.001.

Article Snippet: The Cas9 Sequencing Kit (Oxford Nanopore Technology) was used to prepare a library targeting the insertion.

Techniques: Comparison