Journal: Advanced Science

Article Title: The Crosstalk Between CRL5 and APC/C E3 Ligases Regulates Metastasis and Chemosensitivity of Cancer Cells

doi: 10.1002/advs.202512652

Figure Lengend Snippet: ITCH stabilizes APC11 by adding K27, K29, and K33‐linked polyubiquitin chains to APC11 at Lys83 in a CUL5‐dependent manner. a) Immunoblot of APC11 and ITCH in Hep3B and PLC/PRF/5 cells upon ITCH knockdown by two distinct siRNA oligos. b) Immunoblot of APC11 and APC/C substrate securin in HEK293 cells after transfection with wild‐type ITCH or its C830S mutant for 48 h. c) The stability of APC11 in Hep3B and PLC/PRF/5 cells upon ITCH knockdown. Hep3B and PLC/PRF/5 cells were transfected with indicated siRNA oligos for 72 h and then treated with CHX (100 µg mL −1 ) for the indicated time periods, followed by IB analysis. Densitometry quantifications were performed with ImageJ, and the decay curves are shown (right). d) K27/K29/K33‐linked ubiquitylation of APC11 promoted by overexpression of ITCH but not its C830S mutant. HEK293 cells were co‐transfected with indicated plasmids, followed by purification with Ni‐NTA. Pull‐downs (top) and WCE (bottom) were subjected to IB with indicated Abs. e) Co‐IP of exogenous FLAG‐tagged APC11 with endogenous ITCH or CUL5 in HEK293 cells. f) The interaction of exogenous FLAG‐tagged CUL5 with endogenous ITCH or APC11 in HEK293 cells. g) Co‐IP of endogenous ITCH with CUL5 or APC11 in Hep3B and PLC/PRF/5 cells. h) Co‐IP of exogenous FLAG‐tagged APC11 in HeLa cells upon CUL5 knockout mediated by CRISPR‐Cas9. HeLa cells with or without CUL5 were infected with lentivirus expressing FLAG‐APC11 for 72 h, and then harvested for IP analysis. Band intensities were quantified using ImageJ, and the relative ITCH‐APC11 binding affinity in sgCUL5 cells was compared to sgCtrl cells. i) Immunoblot of APC11 in sgCtrl or sgCUL5 HeLa cells after transfection with GFP‐ITCH for 48 h. Band intensities were quantified using ImageJ and expressed as relative gray values (normalized to sgCtrl/Vector cells), shown beneath each band. j,k) ITCH facilitates K27/K29/K33‐linked ubiquitylation of wild‐type APC11 but not its W14A (j) or K83R (k) mutant. HEK293 cells were co‐transfected with indicated plasmids, followed by purification with Ni‐NTA. Pull‐downs (top) and WCE (bottom) were subjected to IB with indicated Abs. Data are presented as mean ± SEM, n = 3 (c, h, i; right). For statistical analysis, significances were determined by Student's t ‐test.* p < 0.05, ** p < 0.01, ns, not significant. l A model illustrating that CUL5 stabilizes APC11 through ITCH‐mediated K27/K29/K33‐linked polyubiquitylation at Lys83.

Article Snippet: HeLa cells with CUL5 knockout by CRISPR‐Cas9 were established by puromycin (Selleck, S7417) selection after transfection with a sequence‐verified CRISPR plasmid.

Techniques: Western Blot, Knockdown, Transfection, Mutagenesis, Over Expression, Purification, Co-Immunoprecipitation Assay, Knock-Out, CRISPR, Infection, Expressing, Binding Assay, Plasmid Preparation

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: Mutation of Vsx genes in zebrafish highlights the robustness of the retinal specification network

doi: 10.1101/2022.01.20.477122

Figure Lengend Snippet: a. CRISPR/Cas9 DNA editing tool was used to generate deletions (green box) in the highly conserved DBD from vxs1 (top) and vsx2 (bottom) TFs. Blue boxes represent gene exons, black boxes the location of sgRNAs used to guide Cas9 endonuclease and primers for screening are depicted as opposing arrowheads. b-d and f-h. Histological sections stained with nuclear marker DAPI and phalloidin-Alexa488 for actin filaments from WT (b-d, n≥8) and vsx KO central retinas (f-h, n≥10) at 48hpf (b, f), 72hpf (c, g) and 6dpf (d, h). e, i. Head dorsal view from 6dpf WT (e) and vsx KO (i) larvae with insets showing their pigmentation pattern (white arrowhead). ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, hpf: hours post-fertilization, dpf: days post-fertilization. Scale bar in b-d and f-h: 50μm, scale bar in e and i: 500μm.

Article Snippet: To target individual vsx genes, a solution containing two sgRNAs (40 ng/μL each) and Cas9 protein (250 ng/μL) (Addgene; 47327) ( ) were injected into one-cell-stage zebrafish and medaka embryos.

Techniques: CRISPR, Staining, Marker

Journal: bioRxiv

Article Title: Mutation of Vsx genes in zebrafish highlights the robustness of the retinal specification network

doi: 10.1101/2022.01.20.477122

Figure Lengend Snippet: a. CRISPR/Cas9 was used to eliminate (green box) the DBD from vxs1 (top) and vsx2.1 (bottom) TFs in medaka. Blue boxes represent exons, black boxes the location of sgRNAs used and primers for screening are depicted as opposing arrowheads. b-e. Histological sections from WT (b, d, n=4) and vsx KO central retinas (c, e, n=5) at 12dpf. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, hpf: hours post-fertilization, dpf: days post-fertilization. Scale bar b-c: 50μm, d-e: 20 μm.

Article Snippet: To target individual vsx genes, a solution containing two sgRNAs (40 ng/μL each) and Cas9 protein (250 ng/μL) (Addgene; 47327) ( ) were injected into one-cell-stage zebrafish and medaka embryos.

Techniques: CRISPR