Fig 1: Knockdown of STAG2 or STAG3 decreases BRAFi sensitivity in BRAF mutant melanoma cells(a) Viability of A375 cells after treatment with varying concentrations of dabrafenib for 3 d. Experiment was performed 3 times. Data are mean ± s.e.m. (b) A375 cells were treated with dabrafenib for 2 h. Cell lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (c) Viability of SKMEL28 cells after treatment with varying concentrations of vemurafenib for 3 d. Experiment was performed 3 times. Data are mean ± s.e.m. (d) SKMEL28 cells were treated with vemurafenib for 2 h. Cell lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (e) SKMEL30 cells were treated with trametinib for 2 h. Cell lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (f) SKMEL30 cells were treated with trametinib as indicated in clonogenic growth assays. Experiment was performed 3 times. Scale bar: 5 mm. (g) Viability of A375 cells after treatment with varying concentrations of dabrafenib for 3 d. Experiment was performed 3 times. Data are mean ± s.e.m. (h) A375 cells were treated with vemurafenib for 2 h. Cell lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (i) WM902-BR cells stably expressing control vector, FLAG-tagged wild-type STAG2 (WT), Lys1083* (K*) or Asp193Asn (DN) mutants were treated with 3 µM vemurafenib for 2 h. Cell lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (j) WM902-BR cells were used in soft agar assays in the presence or absence of 3 µM vemurafenib. Experiment was performed 3 times. Scale bar: 5 mm.
Fig 2: Knockdown of STAG2 or STAG3 impairs the effects of vemurafenib on inhibiting melanoma xenograft tumor growth in vivo(a) Nude mice bearing xenograft tumors of A375 cells stably expressing pTRIPZ-shSTAG2#60 were treated with vehicle, doxycyline (Dox), vemurafenib (Vem), or both doxycyline and vemurafenib. Unpaired two-tailed Student's t-test was performed to compare between two groups of mice that were treated with vemurafenib (mean ± s.e.m. * P < 0.05). The data variance is similar between groups. n = 5–7. (b) Waterfall plots showing the percent change in tumor volume at day 7 for the individual tumors in each treatment group of the STAG2 knockdown experiment. (c) Representative images of mouse tumor samples from the STAG2 knockdown experiment subjected for various immunohistochemical analyses as indicated. Scale bar: 50 µm. (d) Nude mice bearing xenograft tumors of A375 cells stably expressing pLKO-shSTAG3#69 were treated with control or vemurafenib (Vem) diet. Unpaired two-tailed Student's t-test was performed to compare between two groups of mice that were treated with vemurafenib (mean ± s.e.m. * P < 0.05). The data variance is similar between groups. n = 5–7. (e) Waterfall plots showing the percent change in tumor volume at day 8 for the individual tumors in each treatment group of the STAG3 knockdown experiment. (f) Representative images of mouse tumor samples from the STAG3 knockdown experiment subjected for various immunohistochemical analyses as indicated. Scale bar: 50 µm.
Fig 3: Decreased expression of STAG2 and STAG3 in BRAFi-resistant melanoma primary patient tumors and cell lines(a) Sanger sequencing analysis of the STAG2 locus in pre-treatment (left) and post-relapse (right) biopsies from a patient with recurrent disease following vemurafenib treatment. (b) Expression of cohesion complex components, STAG1, STAG2, STAG3 and RAD21 in a panel of melanoma BRAFi-resistant cell lines and their parental BRAFi-sensitive counterparts was measured by western blotting. GAPDH was used a loading control. P: parental; BR: BRAFi resistant. BMR: BRAFi and MEKi double resistant. (c) Immunohistochemical analyses of STAG2 and STAG3 in pairs of pre-treatment (left) and post-relapse (right) tumor samples from patients treated with BRAFi monotherapy or BRAFi and MEKi combination therapy. Two representative patients are shown for STAG2 and for STAG3. Scale bar: 50 microns.
Fig 4: Chromosome compaction and precocious separation after the knockdown of cohesin components. a Metaphase chromosome images of telomere FISH from ESCs and MEFs expressing a non-targeting siRNA or different siRNAs against SMC3, RAD21, REC8, STAG3, and RAD21-REC8 (siSMC3, siRAD21, siREC8, siSTAG3, siRAD21-siREC8). Telomeric probe signals are shown in red and DAPI-counterstained chromosomes are shown in blue. Scale bars = 10 µm. b Metaphase chromosome images of diverse length. (i) Metaphase chromosomes stained with DAPI and telomere FISH. (ii) Measurement of chromosome length by telomere FISH. Scale bar = 2.5 µm. c Images of a metaphase spread from normal (left) and precocious separation (right) showing proper and defective chromosome separation, respectively. d Quantification of normal chromosomes and precocious separated chromosomes. The bar graph indicates the percentages of cells showing a ratio of normal chromosomes and precocious separated chromosomes among metaphase chromosomes (n = 200 per condition) from three biological replicates. The error bars represent the mean ± SD (n = 3). siR21, siRAD21; siR8, siREC8. e Quantification of chromosome length and percentage of average chromosome length per metaphase cells. Length of chromosomes was measured by calculating the distance between both sides of the telomere probes. Data are shown as mean ± SD value from at least 200 chromosomes per independent experiment (n = 3). f Compaction analysis of chromosomes from the prophase to the metaphase. ESCs expressing histone H2B-GPF were analyzed by fluorescence microscopy. Scale bars = 2.5 µm. g Representative images of ESCs marked with H2B-GFP in metaphase. h Quantification of chromosome compaction. Cell volume and intensity were analyzed using the Nikon NIS software. The X- and Y-axes of the graphs indicate intensity and volume (µm2), respectively. Each biological replicate is color-coded (red and green) and the average of each replicated data is indicated with a larger dot, and black bars indicate the averages of three means (n = 50 for condition). The error bars are the mean ± SD from two biological replicates. **P < 0.01, ***P < 0.001. i Metaphase spreads hybridized with the telomeric probe. The locus-specific probe (“Probe A”) binds to the region of Ch 4 (Chromosome 4: 116,094,264–116,123,690). Telomeric probe and locus-specific probe signals are shown in green and red, respectively. The lengths of chromosomes were measured by calculating the distance between the telomere and locus-specific probes. The chromosome lengths were analyzed using Nikon NIS software. Scale bars are 2.5 µm. j,k Quantification of chromosome lengths in metaphase chromosomes from cells treated or untreated with siRNA specific to the cohesin components for 24 h (n = 30 for condition). The error bars represent the mean ± SD from three independent experiments
Fig 5: STAG2 regulates ERK activity through controlling expression of DUSP6(a) Total RNA from A375 cells were isolated, reverse transcribed, and expression levels of DUSP4 and DUSP6 were analyzed by qPCR. mRNA levels were calculated relative to the scrambled control with housekeeping GAPDH gene as reference. n = 3 biological replicates. Data are mean ± s.e.m. The P values were determined using two-tailed Student's t-test, **** P < 0.0001. The data variance is similar between groups. (b) M14 or A375 cells expressing STAG2 inducible shRNA were cultured in the presence or absence of doxycycline for 5 d before lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (c) Lysates from M14 or A375 cells expressing STAG3 shRNA#71 or scrambled control were used for western blotting with indicated antibodies. Experiment was performed 3 times. (d) Lysates from HEK293 cells transfected with indicated constructs were used for western blotting with indicated antibodies. K*, Lys1083*; DN, Asp193Asn. Experiment was performed 3 times. (e) Genomic structure of DUSP6 gene showing the locations of amplified regions by ChIP-qPCR. R1: CTCF binding region, R2: non-specific region. (f) A375 cells expressing STAG2 inducible shRNA pTRIPZ-shSTAG2#60 were cultured in the presence or absence of doxycycline for 5 d before ChIP-qPCR assays were performed. Chromatins were immuoprecipitated using CTCF antibody or rabbit IgG. IP-ed chromatins were examined using qPCR with primers for R1 and R2 regions of DUSP6 and H19. Results are expressed as fold enrichment relative to the non-specific region (R2). n = 3 biological replicates. Data are mean ± s.e.m. The P values were determined using two-tailed Student's t-test, * P < 0.05. The data variance is similar between groups. (g) Chromatins of LOX-IVMI cells stable expressing FLAG-tagged wild-type STAG2 (WT), Lys1083* (K*) or Asp193Asn (DN) mutants were immuoprecipitated using CTCF antibody or rabbit IgG. n = 3 biological replicates. Data are mean ± s.e.m. The P values were determined using two-tailed Student's t-test, ** P < 0.01; **** P < 0.0001. The data variance is similar between groups. (h) A375 cells expressing STAG2 inducible shRNA pTRIPZ-shSTAG2#60 were infected with lentivirus expressing MYC-DUSP6 or control vector. Cells were cultured in the presence or absence of doxycycline for 5 d and treated with 0.3 µM vemurafenib for 2h before lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (i) A375 cells expressing STAG2 inducible shRNA pTRIPZ-shSTAG2#60 together MYC-tagged DUSP6 or control vector were treated with vemurafenib as indicated in clonogenic growth assays. Experiment was performed 3 times. Scale bar: 5 mm.
Supplier Page from Abcam for Anti-STAG3 antibody