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a. IHC for dsRNA in K-Ctrl , K-Setd2 KO , KH3 K36M -Ctrl , and KH3 K36M -Setd2 KO mice. Scale bars are 119 μm; insets are magnified x3. b. Quantification of dsRNA IHC in K-Ctrl , K-Setd2 KO , KH3 K36M -Ctrl , and KH3 K36M -Setd2 KO tumors. Significance was determined by unpaired Student’s t-test. Error bars represent mean±SEM. c. Immunocytochemistry for dsRNA in KP-H3 WT -Ctrl , KP-H3 WT -Setd2 KO , KP-H3 K36M -Ctrl , and KP-H3 K36M -Setd2 KO cells. Sparallel using 3 dsRNA antibodies: K1, J2, and 9D5. Scale bars are 25 μm. d. IFN-β ELISA analysis of cell culture supernatant collected from KP-H3 WT -Ctrl , KP-H3 WT -Setd2 KO , KP-H3 K36M -Ctrl , KP-H3 K36M -Setd2 KO , and cells. Significance was determined by unpaired Student’s t-test. Error bars represent mean±SEM. e. Schematic of plasmid used for CRISPR enrichment screen targeting dsRNA sensing pathway genes. f. Brightfield and fluorescent micrographs of lungs from athymic nude mice injected with KP-H3 WT -Ctrl or KP-H3 K36M -Ctrl cells infected with <t>lentiCRISPRv2mCherry</t> + sgRNA library. GFP marks all tumor cells while mCherry marks infected cells. Scale bars are 4.4 mm. g. Top-ranked genes enriched in CRISPR screen determined by comparing KP-H3 K36M -Ctrl cells with KP-H3 WT -Ctrl cells. Enriched genes are marked in red. Positive control Setd2 is marked in blue. Non-targeting controls are marked in black.
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a. IHC for dsRNA in K-Ctrl , K-Setd2 KO , KH3 K36M -Ctrl , and KH3 K36M -Setd2 KO mice. Scale bars are 119 μm; insets are magnified x3. b. Quantification of dsRNA IHC in K-Ctrl , K-Setd2 KO , KH3 K36M -Ctrl , and KH3 K36M -Setd2 KO tumors. Significance was determined by unpaired Student’s t-test. Error bars represent mean±SEM. c. Immunocytochemistry for dsRNA in KP-H3 WT -Ctrl , KP-H3 WT -Setd2 KO , KP-H3 K36M -Ctrl , and KP-H3 K36M -Setd2 KO cells. Sparallel using 3 dsRNA antibodies: K1, J2, and 9D5. Scale bars are 25 μm. d. IFN-β ELISA analysis of cell culture supernatant collected from KP-H3 WT -Ctrl , KP-H3 WT -Setd2 KO , KP-H3 K36M -Ctrl , KP-H3 K36M -Setd2 KO , and cells. Significance was determined by unpaired Student’s t-test. Error bars represent mean±SEM. e. Schematic of plasmid used for CRISPR enrichment screen targeting dsRNA sensing pathway genes. f. Brightfield and fluorescent micrographs of lungs from athymic nude mice injected with KP-H3 WT -Ctrl or KP-H3 K36M -Ctrl cells infected with <t>lentiCRISPRv2mCherry</t> + sgRNA library. GFP marks all tumor cells while mCherry marks infected cells. Scale bars are 4.4 mm. g. Top-ranked genes enriched in CRISPR screen determined by comparing KP-H3 K36M -Ctrl cells with KP-H3 WT -Ctrl cells. Enriched genes are marked in red. Positive control Setd2 is marked in blue. Non-targeting controls are marked in black.
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a. IHC for dsRNA in K-Ctrl , K-Setd2 KO , KH3 K36M -Ctrl , and KH3 K36M -Setd2 KO mice. Scale bars are 119 μm; insets are magnified x3. b. Quantification of dsRNA IHC in K-Ctrl , K-Setd2 KO , KH3 K36M -Ctrl , and KH3 K36M -Setd2 KO tumors. Significance was determined by unpaired Student’s t-test. Error bars represent mean±SEM. c. Immunocytochemistry for dsRNA in KP-H3 WT -Ctrl , KP-H3 WT -Setd2 KO , KP-H3 K36M -Ctrl , and KP-H3 K36M -Setd2 KO cells. Sparallel using 3 dsRNA antibodies: K1, J2, and 9D5. Scale bars are 25 μm. d. IFN-β ELISA analysis of cell culture supernatant collected from KP-H3 WT -Ctrl , KP-H3 WT -Setd2 KO , KP-H3 K36M -Ctrl , KP-H3 K36M -Setd2 KO , and cells. Significance was determined by unpaired Student’s t-test. Error bars represent mean±SEM. e. Schematic of plasmid used for CRISPR enrichment screen targeting dsRNA sensing pathway genes. f. Brightfield and fluorescent micrographs of lungs from athymic nude mice injected with KP-H3 WT -Ctrl or KP-H3 K36M -Ctrl cells infected with <t>lentiCRISPRv2mCherry</t> + sgRNA library. GFP marks all tumor cells while mCherry marks infected cells. Scale bars are 4.4 mm. g. Top-ranked genes enriched in CRISPR screen determined by comparing KP-H3 K36M -Ctrl cells with KP-H3 WT -Ctrl cells. Enriched genes are marked in red. Positive control Setd2 is marked in blue. Non-targeting controls are marked in black.
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a. IHC for dsRNA in K-Ctrl , K-Setd2 KO , KH3 K36M -Ctrl , and KH3 K36M -Setd2 KO mice. Scale bars are 119 μm; insets are magnified x3. b. Quantification of dsRNA IHC in K-Ctrl , K-Setd2 KO , KH3 K36M -Ctrl , and KH3 K36M -Setd2 KO tumors. Significance was determined by unpaired Student’s t-test. Error bars represent mean±SEM. c. Immunocytochemistry for dsRNA in KP-H3 WT -Ctrl , KP-H3 WT -Setd2 KO , KP-H3 K36M -Ctrl , and KP-H3 K36M -Setd2 KO cells. Sparallel using 3 dsRNA antibodies: K1, J2, and 9D5. Scale bars are 25 μm. d. IFN-β ELISA analysis of cell culture supernatant collected from KP-H3 WT -Ctrl , KP-H3 WT -Setd2 KO , KP-H3 K36M -Ctrl , KP-H3 K36M -Setd2 KO , and cells. Significance was determined by unpaired Student’s t-test. Error bars represent mean±SEM. e. Schematic of plasmid used for CRISPR enrichment screen targeting dsRNA sensing pathway genes. f. Brightfield and fluorescent micrographs of lungs from athymic nude mice injected with KP-H3 WT -Ctrl or KP-H3 K36M -Ctrl cells infected with lentiCRISPRv2mCherry + sgRNA library. GFP marks all tumor cells while mCherry marks infected cells. Scale bars are 4.4 mm. g. Top-ranked genes enriched in CRISPR screen determined by comparing KP-H3 K36M -Ctrl cells with KP-H3 WT -Ctrl cells. Enriched genes are marked in red. Positive control Setd2 is marked in blue. Non-targeting controls are marked in black.

Journal: bioRxiv

Article Title: The H3 K36M oncohistone inhibits NSD2 to activate a SETD2-dependent antiviral-like immune response in KRAS-driven lung cancer

doi: 10.1101/2025.05.25.655971

Figure Lengend Snippet: a. IHC for dsRNA in K-Ctrl , K-Setd2 KO , KH3 K36M -Ctrl , and KH3 K36M -Setd2 KO mice. Scale bars are 119 μm; insets are magnified x3. b. Quantification of dsRNA IHC in K-Ctrl , K-Setd2 KO , KH3 K36M -Ctrl , and KH3 K36M -Setd2 KO tumors. Significance was determined by unpaired Student’s t-test. Error bars represent mean±SEM. c. Immunocytochemistry for dsRNA in KP-H3 WT -Ctrl , KP-H3 WT -Setd2 KO , KP-H3 K36M -Ctrl , and KP-H3 K36M -Setd2 KO cells. Sparallel using 3 dsRNA antibodies: K1, J2, and 9D5. Scale bars are 25 μm. d. IFN-β ELISA analysis of cell culture supernatant collected from KP-H3 WT -Ctrl , KP-H3 WT -Setd2 KO , KP-H3 K36M -Ctrl , KP-H3 K36M -Setd2 KO , and cells. Significance was determined by unpaired Student’s t-test. Error bars represent mean±SEM. e. Schematic of plasmid used for CRISPR enrichment screen targeting dsRNA sensing pathway genes. f. Brightfield and fluorescent micrographs of lungs from athymic nude mice injected with KP-H3 WT -Ctrl or KP-H3 K36M -Ctrl cells infected with lentiCRISPRv2mCherry + sgRNA library. GFP marks all tumor cells while mCherry marks infected cells. Scale bars are 4.4 mm. g. Top-ranked genes enriched in CRISPR screen determined by comparing KP-H3 K36M -Ctrl cells with KP-H3 WT -Ctrl cells. Enriched genes are marked in red. Positive control Setd2 is marked in blue. Non-targeting controls are marked in black.

Article Snippet: The sgRNA pool was cloned by annealing two DNA oligos for each guide and ligating into a BsmB1-digested lentiCRISPRv2mCherry vector (Addgene 99154), as described [ ].

Techniques: Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Cell Culture, Plasmid Preparation, CRISPR, Injection, Infection, Positive Control