Journal: Nature Communications

Article Title: USP36 stabilizes nucleolar Snail1 to promote ribosome biogenesis and cancer cell survival upon ribotoxic stress

doi: 10.1038/s41467-023-42257-8

Figure Lengend Snippet: a Immunofluorescence staining assays were performed to examine Snail1 or nucleolus marker B23 in either fixed HCC1806 cells, frozen sections of A549 cell-derived xenograft tumor (A549-CDX), or paraffin sections of two clinical breast tumor samples. Notice that Snail1 was accumulated in the nucleolus in a few cells. b , c HCC1806 cells were treated with ribosome inhibitor HHT (20 ng/mL and hereafter), a Pol I inhibitor CX-5461 (200 nM), mTOR inhibitor rapamycin (Rapa, 20 nM), or ER stress inducer Tunicamycin (Tunica, 2 μg/mL) for 24 h. Cells were subjected to immunofluorescence staining analyses ( b ). The co-localization between Snail1 and B23 (as analyzed by Pearson’s correlation coefficient , ) was quantified and statistically analyzed ( c ). d , e Hs 578T, SUM159, or A549 cells were treated with or without HHT for 24 h. Cells were subjected to immunofluorescence staining analyses ( d ). The co-localization between Snail1 and B23 was quantified and statistically analyzed ( e ). f , g HCC1806 cells were treated with a ribotoxic inducer anisomycin (Aniso, 50 ng/mL), puromycin (Puro, 200 ng/mL), G418 (1 μg/mL), or blasticidin (Blasti, 2 μg/mL) for 24 h. Cells were subjected to immunofluorescence staining analyses ( f ). The co-localization between Snail1 and B23 was quantified and statistically analyzed ( g ). h HCC1806 cells were treated with or without HHT for 24 h followed by cell fractionation and western blot analyses. CF Cellular fraction, CP Cytoplasm, NP Nucleoplasm, No Nucleolus. This experiment has been repeated for three times with similar results. Quantification of the co-localization between Snail1 and B23 using Pearson’s correlation coefficient ( c , e , g ). 40 cells derived from three independent experiments were randomly chosen and subjected to quantification analyses. Data were presented as mean ± SD. Comparisons were performed with unpaired two-tailed Student’s t test. Scale bar, 25 μm.

Article Snippet: HCC1806 (CRL-2335), K-562 (CCL-243), HEK-293 (CRL-1573), and Hs 578T (HTB-126) were obtained from ATCC (Manassas, VA, USA).

Techniques: Immunofluorescence, Staining, Marker, Derivative Assay, Cell Fractionation, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: USP36 stabilizes nucleolar Snail1 to promote ribosome biogenesis and cancer cell survival upon ribotoxic stress

doi: 10.1038/s41467-023-42257-8

Figure Lengend Snippet: a , b Triple-negative breast cancer (SUM159, Hs 578T, and HCC1806) and non-lymphocytic leukemia (K-562, OCI-AML2, and MOLM-13) cells were treated with or without HHT (20 ng/mL and hereafter). Cells were subjected to western blot analyses ( a ) or FACS analyses ( b ). Annexin V + /PI+ cell populations were statistically analyzed ( b , n = 3 biologically independent samples). c , d SUM159, Hs 578T, or HCC1806 cells were treated with or without a JNK inhibitor SP600125 (20 μM and hereafter) in the presence or absence of HHT. Cells were subjected to western blot analyses ( c ) or FACS analyses ( d ). Annexin V + /PI+ cell populations were statistically analyzed ( d , n = 3 biologically independent samples). e , f HCC1806 cells were treated with HHT, anisomycin (Aniso, 50 ng/mL), or blasticidin (Blasti, 2 μg/mL) in the presence or absence of SP600125. Cells were subjected to western blot analyses ( e ) or FACS analyses ( f ). Annexin V + /PI+ cell populations were statistically analyzed ( f , n = 3 biologically independent samples). g – k HCC1806 cells (5 × 10 5 ) were subcutaneously inoculated in 5-week-old female BALB/c nude mice ( n = 5/group). On day 3 after inoculation, mice were intraperitoneally (i.p) injected with SP600125 (15 mg/kg) and/or HHT (1 mg/kg) daily. Dissected tumors were photographed on day 17 after i.p ( g ). Tumor weights ( h ) were presented. The xenograft tumor samples were subjected to western blot analyses ( i ) or immunofluorescence staining ( j ). Pearson’s correlation coefficient was used to qualify the co-localization of Snail1 and B23, using images derived from immunostained tumor samples ( k ). Scale bar, 25 μm. l A model depicts that USP36 stabilizes nucleolar Snail1 to promote ribosome biogenesis and cancer cell survival in response to ribotoxic stress. These experiments have been repeated for three times with similar results ( a , c , e ). Data were presented as mean ± SD ( b , d , f ) or SEM ( h , k ). Comparisons were performed with one-way ANOVA with Tukey’s test ( d , f , k ) and unpaired two-tailed Student’s t test ( b , h ). CC3 Cleaved-Caspase-3, T-JNK total JNK, p-JNK phospho-JNK (Thr183/Tyr185).

Article Snippet: HCC1806 (CRL-2335), K-562 (CCL-243), HEK-293 (CRL-1573), and Hs 578T (HTB-126) were obtained from ATCC (Manassas, VA, USA).

Techniques: Western Blot, Injection, Immunofluorescence, Staining, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: SLC7A11 expression level dictates differential responses to oxidative stress in cancer cells

doi: 10.1038/s41467-023-39401-9

Figure Lengend Snippet: a Protein levels of SLC7A11, SLC7A9, and SLC3A1 in a panel of cancer cell lines. Vinculin was used as the loading control. b Cystine uptake levels in a panel of cancer cell lines. c Protein levels of SLC7A11 (up) and cystine uptake levels (down) in sgCtrl and SLC7A11 knockout T98G cells. d Cell death in response to 1 mM H 2 O 2 treatment (up) or glucose starvation (down) in T98G cells with indicated genotypes for 24 h was measured using PI staining. e Protein levels of SLC7A11 (up) and cystine uptake levels (down) in shCtrl and SLC7A11 knockdown T98G cells. f Cell death in response to 1 mM H 2 O 2 treatment (up) or glucose starvation (down) in T98G cells with indicated genotypes for 24 h was measured using PI staining. g Protein levels of SLC7A11 (up) and cystine uptake levels (down) in sgCtrl and SLC7A11 knockout Hs578T cells. h Cell death in response to 1 mM H 2 O 2 treatment (up) or glucose starvation (down) in Hs578T cells with indicated genotypes for 24 h was measured using PI staining. i Protein levels of SLC7A11 (up) and cystine uptake levels (down) in shCtrl and SLC7A11 knockdown Hs578T cells. j Cell death in response to 1 mM H 2 O 2 treatment (up) or glucose starvation (down) in Hs578T cells with indicated genotypes for 24 h was measured using PI staining. k Protein levels of SLC7A11 (up) and cystine uptake levels (down) in SLC7A11-low, -moderate, and -high H1299 cells. l Cell death in response to 1 mM H 2 O 2 treatment (left) or glucose starvation (right) for 20 h in SLC7A11-low, -moderate, and -high H1299 cells was measured using PI staining. m Protein levels of SLC7A11 (up) and corresponding cystine uptake levels (down) in SLC7A11-low, -moderate, and -high 786-O cells. n Cell death in response to 1 mM H 2 O 2 treatment (left) or glucose starvation (right) in SLC7A11-low, -moderate, and -high 786-O cells for 20 h measured using PI staining. Data were presented as mean ± SD; n = 3. n indicates independent repeats. P value was determined by two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Article Snippet: H1299 (CRL-5803), 786-O (CRL-1932), A498(HTB-44), H226 (CRL-5826), A549(CRL-7909), T98G(CRL-1690), Hs578T(HTB-126), and HEK293T (CRL-3216) cell lines were obtained from ATCC.

Techniques: Knock-Out, Staining, Two Tailed Test

Journal: BMC Cancer

Article Title: Functional proteomic analysis reveals the involvement of KIAA1199 in breast cancer growth, motility and invasiveness

doi: 10.1186/1471-2407-14-194

Figure Lengend Snippet: Knockdown of KIAA1199 inhibits cell migration and proliferation in vitro . A) The wound-healing assay shows significantly lower cell motility in the KIAA1199 knockdown cells (MDA-MB-231-ShA and MDA-MB-231-ShB) compared to the negative controls. B) Trans-well assay shows a decrease in the cell migration rate (migrated/total) for the KIAA1199 knockdown cells (the experiment was performed in three biological replicates). C) The MTT assay demonstrates that both MDA-MB-231 and Hs578T knockdown cells have significantly lower proliferation levels at 72 and 96 h of culture (the experiment was performed in three biological replicates).

Article Snippet: MDA-MB-231 and Hs578T cells (obtained from ATCC (Manassas, VA)) were cultured in DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37˚C in an atmosphere containing 5% CO 2 .

Techniques: Migration, In Vitro, Wound Healing Assay, MTT Assay

Journal: BMC Cancer

Article Title: Functional proteomic analysis reveals the involvement of KIAA1199 in breast cancer growth, motility and invasiveness

doi: 10.1186/1471-2407-14-194

Figure Lengend Snippet: KIAA1199 Knockdown enhanced apoptosis in vitro . A) Flow cytometry analysis shows a large increase in the percentage of cells programmed for apoptosis in MDA-MB-231-ShA, MDA-MB-231-ShB, Hs578T-ShA and Hs578T-ShB cells comparing to the corresponding negative controls. B) Confirmation of the results of Flow cytometry analysis by Western blot (single experiment). Caspase-3 activation is detected in Western blots by the presence of cleavage fragments. The antibody detects both pro (full-length) and active (cleaved) protein. The increased representation of cleaved caspase-3 in KIAA1199 knockdown cells compared to the control cells is qualitatively shown in MDA-MB-231 (left panel) and Hs578T (right panel) cells.

Article Snippet: MDA-MB-231 and Hs578T cells (obtained from ATCC (Manassas, VA)) were cultured in DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37˚C in an atmosphere containing 5% CO 2 .

Techniques: In Vitro, Flow Cytometry, Western Blot, Activation Assay