EMBO/SourceData huggingface dataset:
Figure 4A Tandem affinity purification of MAGE-A3 from HEK293 cells identified DDB1, DCAF5, and DCAF12 as binding partners. Mascot score, number of peptides identified, and percent coverage is shown.B MAGE-A3 interacts with DCAF12, but not DCAF5. HeLa cells were transfected with the indicated constructs (Myc-MAGE-A3 and HA-DCAF5 or HA-DCAF12). Two days later cells were treated with complete media or EBSS for 4 hrs. Cells were treated in the presence of 1 µM MLN4924 to stabilize MAGE-A3. Cell extracts were subjected to anti-Myc IP.C Nutrient deprivation increases endogenous DCAF12 binding to MAGE-A3. HeLa cells were transfect with Myc-vector or Myc-MAGE-A3 for 48 hrs. Cells were then treated with 10 µM MG132 in complete media, EBSS, or EBSS containing 20 µg/mL insulin for 6 hrs. Cell extracts were subjected to anti-Myc IP.Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control, DCAF5 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM).E Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control or DCAF12 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM).F Knockdown of DCAF12 with individual siRNAs blocks MAGE-A3/6 degradation upon nutrient deprivation. HeLa cells were treated with the indicated siRNAs for 72 hrs before incubation in EBSS for the indicated times. Note, time 0 samples between siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation.G Ubiquitination of MAGE-A3/6 upon nutrient deprivation is dependent on DCAF12. A375 parental or DCAF12 KO cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.
Figure 5A Identification of DCAF12 substrates. Control (WT) or DCAF12 knockout (KO) A375 cells were subjected to quantitative TMT proteomics to identify potential DCAF12 targets. MAGE-A proteins (shown in blue) are stabilized in DCAF12 knockout cells.B Knockout of DCAF12 rescues degradation of MAGE-A proteins in A375 cells. DCAF12 KO A375 cells were treated with complete media or EBSS before quantitative TMT proteomics. Note MAGE-A genes are not significantly altered by EBSS in DCAF12 KO cells.C DCAF12 target proteins are differentially affected by EBSS compared to remainder of the proteome. DCAF12 targets (n=33) identified show significantly greater decrease upon EBSS treatment compared to non-DCAF12 altered genes (n=8243). Average with standard deviation is shown with statistical analysis by student t-test. Asterisks *** indicate p<0.001 (student t-test).E Overexpression of DCAF12 decreases wild-type MAGE-A3, but not MAGE-A3 degron mutant. The indicated constructs were expressed for 48 hrs in HeLa cells before immunoblotting. The MAGE-A3 degron mutant was created ], in which DNYNEPKANQ* were added to the extreme C-term to obscure the DCAF12 degron motif.F Analysis of proteomics indicates that MAGE-A1, MAGE-A4, MAGE-A10, and MAGE-A11 are not regulated by nutrient deprivation or DCAF12. Average (n=3) with standard deviation is shown with statistical analysis by student t-test. Asterisks *** indicate p<0.001 (student t-test).G MAGE-A10 and MAGE-A11 are not altered upon nutrient deprivation or DCAF12 deletion. A375 parental or DCAF12 KO cells were treated with EBSS for the indicated times. Cell lysates were probed for the indicated proteins.
Figure 6A-B U2OS GFP-LC3 cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, imaged, and the number of GFP-LC3 puncta were quantitated per cell. Representative images (A) and quantitation of >50 cells from three independent experiments (B) are shown. Scale bar indicates 50 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test.C-D A375 parental or DCAF12 KO cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, endogenous LC3 was immunostained, and the number of LC3 puncta were quantitated per cell. Representative images (C) and quantitation of >50 cells from three independent experiments (D) are shown. Scale bar indicates 20 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test.E A375 parental or DCAF12 KO cells were treated Cell lysates were collected and immunoblotted for the indicated antibodies to confirm MAGE-A3/6 depletion and monitor the autophagy adaptor/substrate p62/SQSTM1.
Figure 1a, Expression levels of ISX, TWIST1, Snail1, Fibronectin (FN1), VEGF, and E-cadherin (CDH1) mRNA were examined in a ISX-GFP inducible Tet-ON transformants at 8 h after addition of DOX. Data are presented as mean ± SD in graph (p< 0.001 compared to time point 0h; Student's t‐test) of 3 independent experiments, each performed in triplicate.b, Western blotting analysis of the protein levels of ISX, HIF1α, TWIST1, Snail1, Slug, ZEB1, Bmi1, E-cadherin (E-Cad.), Fibronectin, N-cadherin (N-Cad.), Vimentin, and VEGF in A549 and H1299 cells with DOX-inducible ISX expression system 8hs after DOX induction.ISX transcriptionally activates luciferase activity driven by TWIST1 (c) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.d, ISX transcriptionally activates luciferase activity driven by Snail1 (d) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.e, ChIP analysis of ISX binding to the TWIST1 promoter region in A549 and H1299 cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.f, ISX transactivation activity analyzed by luciferase activity driven by the TWIST1 promoter. Data are presented as mean ± SD in graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.g, ChIP analysis of ISX binding to the endogenous promoters of TWIST1 (-180/+35) and Snail1(-160/+40) in A549 and cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.h, Effect of forced expression and knockdown of ISX on cell migration (wound healing) measured in A549 cells. ISXi, ISX-specific shRNAi Data are presented as mean ± SD in graph (**, p < 0.01, ***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.i, Effect of forced expression and knockdown of ISX on cell invasion (transwell) activity measured in A549 cells. Data are presented as mean ± SD in bar graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.
Figure 2a, BRD4 peptide (291-314 a.a) obtained from anti-ISX immunoprecipitates of A549 cell lysates identified through liquid chromatography‒tandem mass spectrometry (LC-tandem -MS). Data information: Each experiment was repeated at least three times.b, ISX association proteins (PCAF, BRD4, CBP, CREB, RNA Pol II, and Histone H3) determined by Western blot in immunoprecipitation of A549 cells. Data information: Each experiment was repeated at least three times.c, ISX association proteins (PCAF, BRD4, CBP, CREB, and RNA Pol II) determined by Western blot in anti-ISX immunoprecipitates of tumor tissues from patients with lung cancer. Data information: Each experiment was repeated at least three times.d, Proximity ligation assay (PLA) of ISX and BRD4(PCAF) interaction in A549 cells. Red foci indicate close proximity of the two proteins. NC: negative control. Blank arrows indicate interactions of ISX with BRD4 (PCAF) in the cells. Data information: Each experiment was repeated at least three times.e, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in DOX-inducible ISX expression system A549 cells treated with TH1834, Garcinol, C646, MB-3 for 8 h after DOX induction. Data information: Each experiment was repeated at least three times.f, Proximity ligation assay of ISX and BRD4 interaction in A549 cells treated with Garcinol. Red foci indicate close proximity of the two proteins. Blank arrows indicate interactions of ISX with BRD4 in the cells. NC: negative control. Data information: Each experiment was repeated at least three times.g, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in A549 cells with PCAF knockdown and DOX-inducible ISX expression system at 8 h time point after DOX induction. Data information: Each experiment was repeated at least three times.Cell migration (wound healing, h) ssay of A549 lung cancer cells treated with Garcinol. V, vehicle. Data are presented as mean ± SD in graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times. i, Cell invasion (transwell, i) assay of A549 lung cancer cells treated with Garcinol. V, vehicle. Data are presented as mean ± SD in graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.
Figure 3c, Recombinant PCAF acetylates His6-ISX at lysine residue 69 by in vitro acetylation assay. Acetylated ISX was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.d and e, The protein levels of GFP-tagged WT or mutant ISX, PCAF and BRD4 were determined in cytosol, nuclei, and anti-GFP immunoprecipites of A549 cells by Western blotting. Acetylated ISX was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.f, The protein levels of total and acetylated histone H3 were determined in anti-histone H3 immunoprecipites of A549 cells by Western blotting. Data information: Each experiment was repeated at least three times.The cell migration (wound healing, g) activity were determined in A549 cells with GFP-tagged wild or ISX mutants. Data are presented as mean ± SD in graph (***, p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.h, The cell invasion (transwell, h) activity were determined in A549 cells with GFP-tagged wild or ISX mutants. Data are presented as mean ± SD in graph (***, p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.j. Tumor xenografts metastasis activity of constitutively expressing RFP A549 cells transfected with wild type or ISX AC3 mutant cDNA were imaged by IVIS imaging system at fifth weeks Data information: Each experiment was repeated at least three times.k, Kaplan-Meier survival curve analysis of nude mice xenograft injected with A549 cells carrying GFP, ISX-GFP and ISX Ac-GFP (AC3) (n = 10). p = 0.0003. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves. Data information: Each experiment was repeated at least three times.
Figure 4c, Recombinant PCAF acetylates His6-BRD4 at lysine residue 332. Acetylated BRD4 was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.d, The mCherry-tagged WT and BRD4 mutants were detected in anti-GFP immunoprecipitates by Western blotting in A549 cells. Data information: Each experiment was repeated at least three times.e and f, The mRNA levels of TWIST1(e) and Snail1(f) were verified in A549 cells co-expressing mCherry-tagged WT or mutant BRD4 and GFP-tagged ISX by RT-PCR. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.g and h, The DNA binding activity of TWIST1 and Snail1 were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190/+40 bp, negative control) (10% input of each groups were pulled down and used for qPCR analysis). Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.i, The cell invasion (transwell) activity were determined in A549 cells co-transfected with cDNA coding for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.
Figure 5a, Schematic depiction of ISX and deletion constructs used. b GFP-tagged truncated ISX mutant proteins or HA-tagged BRD4 were detected in anti-HA or GFP immunoprecipitates by Western blotting analysis. Data information: Each experiment was repeated at least three times.
Figure 6a mCherry-tagged truncated BRD4 mutant proteins and GFP-tagged ISX were detected in anti-GFP immunoprecipitates by Western blot. Data information: Each experiment was repeated at least three times.The DNA binding activity of Snail (b) were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190-+40 bp, negative control). (10% input of each groups were pull down and applied to qPCR) Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.c, The DNA binding activity of TWIST1 (c) were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190-+40 bp, negative control). (10% input of each groups were pull down and applied to qPCR) Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.The cell migration (wound healing, d) activity were determined in A549 cells co-transfected with cDNAs for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.e, The cell invasion (transwell, e) activity were determined in A549 cells co-transfected with cDNAs for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.
Figure 7a, IHC staining with ISX (brown) or PCAF (brown) antibody in lung tumors from patients with lung cancer. N, normal tissue; T, tumor mass; Nuclei, hematoxylin (blue); Blue and red arrow, cytosol and nuclear ISX (PCAF). Scale bar, 500μm (400X); Scale bar, 2mm (100X).b, Confocal immunofluorescence detection of ISX (green) and BRD4 (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass.c, Confocal immunofluorescence detection of ISX (green) and PCAF (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass.Correlation analysis of mRNA expression for, (d) ISX and BRD4, in 157 lung cancer samples.e, Correlation analysis of mRNA expression for, (e) ISX and PCAF in 157 lung cancer samples.f, Protein expression of ISX, ISX(69Kac) and Vimentin in tumor and normal parts from patients with NSCLC by western blotting. N, normal tissue; T, tumor mass; Arrow, target protein; *, unspecific band.The Kaplan-Meier survival curve was used to analyze survival correlation between patients (n=157) with NSCLC and ISX (g) levels. On the basis of the cut-off values of fold differences, the study population was dichotomized into the "high" and "low" expression group. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves.g and h, The Kaplan-Meier survival curve was used to analyze survival correlation between patients (n=157) with NSCLC and PCAF (h) levels. On the basis of the cut-off values of fold differences, the study population was dichotomized into the "high" and "low" expression group. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves.
Figure 2A, RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection of A549-ACE2 cells with indicated SARS-CoV-2 strains at 24 hpi. Cells were pretreated with BB94 (10 µM) or DMSO for 6 h. Data are normalized to DMSO (N = 3-7). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI from at least three (A, biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.B. RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection of A549-ACE2 cells with indicated SARS-CoV-2 strains at 24 hpi. Cells were pretreated with BB94 (10 µM), DPC-333 (10 µM) or DMSO for 6 h. Data are normalized to DMSO (N = 3-7). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI from at least three , B) biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.C. Normal Human Bronchial Epithelial cells (NHBE) from ten donors were infected with SARS-CoV-2. NHBE cells were pre-treated with camostat mesylate (10µM), BB94 (1 µM or 10 µM) or DMSO for 6 hours. RNA was isolated at 24 hpi and levels of SARS-CoV-2 N relative to RPLP0 from total cellular RNA was measured by RT-qPCR. Data are normalized to the DMSO control (N = 5-10). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.D, NHBE cells from five donors were infected with GFP-expressing SARS-CoV-2. Infection of cells was monitored microscopically as green fluorescence through live-cell imaging at 72 hours post-infection (hpi) and is shown as the mean of the GFP-positive area relative to the whole area covered by cells in the same well. In (D) NHBE cells were pre-treated with the indicated drugs for 6 hours before infection Data are normalized to the DMSO control (N = 5). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.E. NHBE cells from five donors were infected with GFP-expressing SARS-CoV-2. Infection of cells was monitored microscopically as green fluorescence through live-cell imaging at 72 hours post-infection (hpi) and is shown as the mean of the GFP-positive area relative to the whole area covered by cells in the same well. NHBE cells were were treated in (E) with the indicated drugs 4 hours after infection. Data are normalized to the DMSO control (N = 5). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3A. Western Blot (WB) analysis of A549-ACE2 knock-out (KO) lines. Two different KO lines were generated using distinct CRISPR guide RNAs for both ADAM10 and ADAM17. A non-targeting control (NTC) guide RNA served as control. Shown are two replicates for each cell line. Mature (m) and immature (im) forms of ADAM10 and ADAM17 are indicated. ACE2 levels remained unaffected. α-tubulin served as loading control. *: remaining, low amount of immature ADAM10 and ADAM17. The double KO (dKO) was generated from guide 1 of each ADAM10 and ADAM17. B. Quantification of the abundance of mature ADAM10 and ADAM17 protein forms from (A). Data are normalized to loading control tubulin and to NTC (N = 3-4). The band of mADAM17 runs very close to three unspecific bands (marked with arrowheads) - one closely above and two closely below mADAM17 (A). Thus, quantification of remaining mADAM17 in the ADAM10 KO includes also these three unspecific bands. Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.C. RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection of A549-ACE2 NTC or KO cells with SARS-CoV-2. Cells were treated with BB94 (10 µM) or DMSO for 6 h prior to infection and RNA was isolated at 24 hpi. Two-way ANOVA with Tukey's post-hoc multiple comparison test. Data are normalized to NTC DMSO (N = 3). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.D-G. UMAP of 63,103 cells from BAL fluid derived from infected individuals (D) Individual cell types are indicated in color. (E) Normalized counts of SARS-CoV-2 reads, ADAM10, ADAM17, and TMPRSS2 gene expression levels. (F) Dot plot of ADAM10, ADAM17, and TMPRSS2 expression, grouped by disease severity. Expression levels are color-coded, the percentage of cells expressing the aforementioned genes is size coded. (G) Cells that are infected and additionally express ADAM17 (ADAM17+, red), ADAM10 (ADAM10+, blue), both ADAMs (ADAM10+ ADAM17+, purple), or TMPRSS2 (TMPRSS2+, orange) are indicated.
Figure 4B. Representative images of syncytia formation assay. Large syncytia were observed for NTC and ADAM17 KO cells, but were strongly reduced when ADAM10 was knocked-out, either alone or together with ADAM17 or upon inhibition with BB94 (10 µM). For both ADAMs, the cell lines obtained with gRNA sequence 1 were used. Images show GFP fluorescence and Hoechst staining. Scale bars, 50 µm. C. Quantification of images from fluorescence microscopy (B). Left plot: the area of the fused cells in green (GFP) was quantified and normalized to the Hoechst signal (blue) of the entire image to account for differences in cell density. Right plot: The nuclei within syncytia were determined by calculating the ratio between the Hoechst signal within syncytia and the total Hoechst signal in the entire image. The data are normalized to NTC DMSO (N >= 8). Two-way ANOVA with Tukey's correction for multiple comparisons. All data are represented as means ± 95% CI of at least 3 independent experiments. **p < 0.01, ***p < 0.001.