Journal: Cancer science

Article Title: BRCA1 contributes to transcription-coupled repair of DNA damage through polyubiquitination and degradation of Cockayne syndrome B protein.

doi: 10.1111/j.1349-7006.2011.02037.x

Figure Lengend Snippet: Fig. 1. BRCA1 accumulation at ultraviolet (UV)-irradiated sites is dependent on Cockayne syndrome B (CSB). (a) Saos-2 cells were fixed at the indicated time points after localized UV irradiation and stained with anti-cyclobutane pyrimidine dimers (CPD) and anti-BRCA1 antibodies. (b) Schematic of nucleotide excision repair (NER). (c) XP3BRSV XPG) ⁄ ), XP12ROSV XPA) ⁄ ), XP4PASV XPC) ⁄ ), CS3BESV CSA) ⁄ ), UVs1KOSV CSB) ⁄ ) and HA-CSB ⁄ UVs1KOSV cells were fixed 30 min after UV irradiation and then stained. (d) Saos-2 cells were treated with actinomycin D (Act D) (10 lg ⁄ mL) or a-amanitin (100 lg ⁄ mL) for 1 h and then exposed to UV irradiation. Scale bars, 10 lm. CSA, Cockayne syndrome A; GGR, global genome repair; TCR, transcription-coupled repair.

Article Snippet: Immunocytochemistry was carried out as previously described. (28) An anti-CPD antibody (Medical & Biological Laboratories, Nagoya, Japan) and an anti-BARD1 antibody (H-300; Santa-Cruz Biotechnology, Santa Cruz, CA, USA), a polyclonal anti-BRCA1 antibody specific for residues 397–1080 of BRCA1, or an anti-BRCA1 antibody (C-20; SantaCruz Biotechnology) were used.

Techniques: Irradiation, Staining

Journal: Cancer science

Article Title: BRCA1 contributes to transcription-coupled repair of DNA damage through polyubiquitination and degradation of Cockayne syndrome B protein.

doi: 10.1111/j.1349-7006.2011.02037.x

Figure Lengend Snippet: Fig. 2. siRNA knockdown of BRCA1 impairs transcription-coupled repair (TCR). (a) XP4PASV and UVs1KOSV cells were transfected with control or BRCA1 siRNA. Cell lysates analyzed using western blot with anti-BRCA1 and anti-b-actin antibodies. (b) Colony formation assay for XP4PASV and UVs1KOSV cells. XP4PASV and UVs1KOSV cells were transfected with control or BRCA1 siRNA. Data represent the mean ± standard deviations of four independent experiments. **P < 0.01 versus the corresponding value for control siRNA. (c) Removal of ultraviolet (UV)-induced CPD from the dihydrofolate reductase (DHFR) fragments in XP4PASV cells immediately or 6 h after UV irradiation. The DHFR fragments were analyzed using strand- specific probes recognizing the transcribed (TS) or non-transcribed (NTS) strand.

Article Snippet: Immunocytochemistry was carried out as previously described. (28) An anti-CPD antibody (Medical & Biological Laboratories, Nagoya, Japan) and an anti-BARD1 antibody (H-300; Santa-Cruz Biotechnology, Santa Cruz, CA, USA), a polyclonal anti-BRCA1 antibody specific for residues 397–1080 of BRCA1, or an anti-BRCA1 antibody (C-20; SantaCruz Biotechnology) were used.

Techniques: Knockdown, Transfection, Control, Western Blot, Colony Assay, Irradiation

Journal: Cancer science

Article Title: BRCA1 contributes to transcription-coupled repair of DNA damage through polyubiquitination and degradation of Cockayne syndrome B protein.

doi: 10.1111/j.1349-7006.2011.02037.x

Figure Lengend Snippet: Fig. 3. Cockayne syndrome B (CSB) is associated with BRCA1 and polyubiquitinated for proteasomal degradation after ultraviolet (UV) irradiation. (a) HEK-293T cells were treated with UV irradiation at a dose of 20 J ⁄ m2. One hour after exposure, cell lysates were subjected to immunoprecipitation (IP) with control IgG or anti-CSB antibodies followed by western blotting with anti-BRCA1, anti-CSB or anti-BARD1 antibodies. (b) HEK-293T cells were transfected with Myc-ubiquitin and then UV irradiated. Lysates were subjected to IP using control IgG or anti-Myc antibodies 1 h after UV irradiation. (c) Polyubiquitination of BRCA1 and CSB is enhanced by UV irradiation. (d) CSB polyubiquitination is associated with proteasomal degradation after UV irradiation. HEK-293T cells were treated with or without 50 lM of MG132 after UV irradiation.

Article Snippet: Immunocytochemistry was carried out as previously described. (28) An anti-CPD antibody (Medical & Biological Laboratories, Nagoya, Japan) and an anti-BARD1 antibody (H-300; Santa-Cruz Biotechnology, Santa Cruz, CA, USA), a polyclonal anti-BRCA1 antibody specific for residues 397–1080 of BRCA1, or an anti-BRCA1 antibody (C-20; SantaCruz Biotechnology) were used.

Techniques: Irradiation, Immunoprecipitation, Control, Western Blot, Transfection, Ubiquitin Proteomics

Journal: Cancer science

Article Title: BRCA1 contributes to transcription-coupled repair of DNA damage through polyubiquitination and degradation of Cockayne syndrome B protein.

doi: 10.1111/j.1349-7006.2011.02037.x

Figure Lengend Snippet: Fig. 4. BRCA1 polyubiquitinates Cockayne syndrome B (CSB) and is involved in the Cockayne syndrome A (CSA)-independent resistance to ultraviolet (UV)-irradiation. (a) CS3BESV cells were treated with UV irradiation. One hour after exposure, cell lysates were subjected to IP. (b) HEK-293T cells were transfected with control or BRCA1 siRNA. One hour after exposure, cell lysates were subjected to immunoprecipitation (IP). (c) HEK-293T cells were transfected with wild-type HA-BRCA1 or HA-BRCA1-I26A. One hour after exposure, cell lysates were subjected to IP. (d) BRCA1 polyubiquitinates CSB in vitro. Long and short exposures of the same blot are presented to show that polyubiquitination of CSB is dependent on the amount of BRCA1 ⁄ BARD1 (10 or 20 nM). (e) CS3BESV cells were transfected with control or BRCA1 siRNA. Cells were pre- treated with cycloheximide (CHX) for 1 h and UV irradiated. Cells were incubated with CHX and total cell lysates were prepared at the indicated times for western blot with anti-CSB antibody. (f) Colony formation assay for CS3BESV cells transfected with control or BRCA1 siRNA. The cells were UV irradiatied as indicated. Data represent the mean ± standard deviations of four independent experiments.

Article Snippet: Immunocytochemistry was carried out as previously described. (28) An anti-CPD antibody (Medical & Biological Laboratories, Nagoya, Japan) and an anti-BARD1 antibody (H-300; Santa-Cruz Biotechnology, Santa Cruz, CA, USA), a polyclonal anti-BRCA1 antibody specific for residues 397–1080 of BRCA1, or an anti-BRCA1 antibody (C-20; SantaCruz Biotechnology) were used.

Techniques: Irradiation, Transfection, Control, Immunoprecipitation, In Vitro, Incubation, Western Blot, Colony Assay

Journal: Cancer science

Article Title: BRCA1 contributes to transcription-coupled repair of DNA damage through polyubiquitination and degradation of Cockayne syndrome B protein.

doi: 10.1111/j.1349-7006.2011.02037.x

Figure Lengend Snippet: Fig. 3. Cockayne syndrome B (CSB) is associated with BRCA1 and polyubiquitinated for proteasomal degradation after ultraviolet (UV) irradiation. (a) HEK-293T cells were treated with UV irradiation at a dose of 20 J ⁄ m2. One hour after exposure, cell lysates were subjected to immunoprecipitation (IP) with control IgG or anti-CSB antibodies followed by western blotting with anti-BRCA1, anti-CSB or anti-BARD1 antibodies. (b) HEK-293T cells were transfected with Myc-ubiquitin and then UV irradiated. Lysates were subjected to IP using control IgG or anti-Myc antibodies 1 h after UV irradiation. (c) Polyubiquitination of BRCA1 and CSB is enhanced by UV irradiation. (d) CSB polyubiquitination is associated with proteasomal degradation after UV irradiation. HEK-293T cells were treated with or without 50 lM of MG132 after UV irradiation.

Article Snippet: Immunocytochemistry was carried out as previously described. (28) An anti-CPD antibody (Medical & Biological Laboratories, Nagoya, Japan) and an anti-BARD1 antibody (H-300; Santa-Cruz Biotechnology, Santa Cruz, CA, USA), a polyclonal anti-BRCA1 antibody specific for residues 397–1080 of BRCA1, or an anti-BRCA1 antibody (C-20; SantaCruz Biotechnology) were used.

Techniques: Irradiation, Immunoprecipitation, Control, Western Blot, Transfection, Ubiquitin Proteomics

Journal: Cancer science

Article Title: BRCA1 contributes to transcription-coupled repair of DNA damage through polyubiquitination and degradation of Cockayne syndrome B protein.

doi: 10.1111/j.1349-7006.2011.02037.x

Figure Lengend Snippet: Fig. 4. BRCA1 polyubiquitinates Cockayne syndrome B (CSB) and is involved in the Cockayne syndrome A (CSA)-independent resistance to ultraviolet (UV)-irradiation. (a) CS3BESV cells were treated with UV irradiation. One hour after exposure, cell lysates were subjected to IP. (b) HEK-293T cells were transfected with control or BRCA1 siRNA. One hour after exposure, cell lysates were subjected to immunoprecipitation (IP). (c) HEK-293T cells were transfected with wild-type HA-BRCA1 or HA-BRCA1-I26A. One hour after exposure, cell lysates were subjected to IP. (d) BRCA1 polyubiquitinates CSB in vitro. Long and short exposures of the same blot are presented to show that polyubiquitination of CSB is dependent on the amount of BRCA1 ⁄ BARD1 (10 or 20 nM). (e) CS3BESV cells were transfected with control or BRCA1 siRNA. Cells were pre- treated with cycloheximide (CHX) for 1 h and UV irradiated. Cells were incubated with CHX and total cell lysates were prepared at the indicated times for western blot with anti-CSB antibody. (f) Colony formation assay for CS3BESV cells transfected with control or BRCA1 siRNA. The cells were UV irradiatied as indicated. Data represent the mean ± standard deviations of four independent experiments.

Article Snippet: Immunocytochemistry was carried out as previously described. (28) An anti-CPD antibody (Medical & Biological Laboratories, Nagoya, Japan) and an anti-BARD1 antibody (H-300; Santa-Cruz Biotechnology, Santa Cruz, CA, USA), a polyclonal anti-BRCA1 antibody specific for residues 397–1080 of BRCA1, or an anti-BRCA1 antibody (C-20; SantaCruz Biotechnology) were used.

Techniques: Irradiation, Transfection, Control, Immunoprecipitation, In Vitro, Incubation, Western Blot, Colony Assay

Journal: Breast Cancer: Targets and Therapy

Article Title: Establishment and characterization of a receptor-negative, hormone-nonresponsive breast cancer cell line from an Iraqi patient

doi: 10.2147/bctt.s74509

Figure Lengend Snippet: Figure 8 Immunocytochemistry analysis of AMJ13 cell line. Notes: (A) Negative result for ER (magnification 40×). (B) Negative result for PR (magnification 40×). (C) Weak positive result for Her-neu2 gene expression (magnification 40×). (D) BRCA1-positive nuclear staining of breast cancer epithelial cells (magnification 40×). (E) BRCA2-positive nuclear and cytoplasmic staining of breast cancer cells (magnification 40×). (F) Vimentin marker-positive cells (magnification 40×). (G) Negative control.

Article Snippet: The cell suspension was dropped onto a glass slide and stained with Giemsa dye to examine the slide and in preparation for chromosome G banding. immunocytochemistry AMJ13 cells were cultured to a confluent monolayer on Labtek chamber slides, then fixed in 4% formalin for 10 minutes, rinsed gently in PBS, and incubated in a humid chamber, first with blocking reagents for 1 hour and then with selected primary antibodies, ie, mouse anti human-Her neu2, mouse anti human-PR, mouse anti human-ER BRCA1 and BRAC2, and vimentin (Santa Cruz Biotechnology), overnight at 4°C for detection of markers for breast cancer.

Techniques: Immunocytochemistry, Gene Expression, Staining, Marker, Negative Control

Journal: Breast Cancer : Targets and Therapy

Article Title: Establishment and characterization of a receptor-negative, hormone-nonresponsive breast cancer cell line from an Iraqi patient

doi: 10.2147/BCTT.S74509

Figure Lengend Snippet: Immunocytochemistry analysis of AMJ13 cell line. Notes: ( A ) Negative result for ER (magnification 40×). ( B ) Negative result for PR (magnification 40×). ( C ) Weak positive result for Her-neu2 gene expression (magnification 40×). ( D ) BRCA1-positive nuclear staining of breast cancer epithelial cells (magnification 40×). ( E ) BRCA2-positive nuclear and cytoplasmic staining of breast cancer cells (magnification 40×). ( F ) Vimentin marker-positive cells (magnification 40×). ( G ) Negative control.

Article Snippet: AMJ13 cells were cultured to a confluent monolayer on Labtek chamber slides, then fixed in 4% formalin for 10 minutes, rinsed gently in PBS, and incubated in a humid chamber, first with blocking reagents for 1 hour and then with selected primary antibodies, ie, mouse anti human-Her neu2, mouse anti human-PR, mouse anti human-ER BRCA1 and BRAC2, and vimentin (Santa Cruz Biotechnology), overnight at 4°C for detection of markers for breast cancer.

Techniques: Immunocytochemistry, Gene Expression, Staining, Marker, Negative Control

Journal: Nature methods

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1038/s41592-021-01126-2

Figure Lengend Snippet: a, Confocal microscopic analysis of Lamin B1-GFP reporter cell line displaying dramatic morphological differences in nuclear shape during cell passaging (30 min post-plating). Note the abnormal nuclear morphology of cells undergoing contractions in the presence of DMSO. Although Y-27632 inhibits cell contractions and nuclear morphologies appear more normal, furrow-like constrictions (white arrowheads) indicate that nuclei are affected by ongoing physical stress. In contrast, cells exposed to CEPT maintain normal circular nuclei. Scale bar, 10 μm. b, OCT4 expressing cells were immunoreactive for γH2AX when exposed to DMSO and Y-27632 (red arrowheads) but not when treated with CEPT (3 h post-plating). Scale bar, 10 μm. c, Dramatic cytoskeletal differences during cell passaging (3 h post-plating) as measured by immunocytochemistry against actin and myosin. Stressed cells show blebbing (white arrowheads) in the presence of DMSO or form prominent actin stress fibers at the colony edge when exposed to Y-27632 (white arrowheads). Note the normal morphology and cell attachment in the presence of CEPT. Scale bar, 10 μm. d, Confocal analysis of an iPSC GFP reporter cell line visualizing the tight junction protein TJP1 (ZO-1) before passaging and after cell dissociation. Representative images were taken 3 h post-plating for DMSO, Y-27632, and CEPT. Note the dramatic differences across treatment groups and better recovery of dissociated cells when treated with CEPT. Scale bar, 10 μm. e, Western blot analysis of hESCs (WA09) treated with Y-27632 or CEPT. Several membrane-associated proteins were expressed at higher levels after CEPT treatment (24 h post-plating). Similar results were obtained with iPSCs (see ). f, Western blot analysis of hESCs (WA09) showing strong stress response in cells treated with DMSO or Y-27632 (3 h post-passage). Note the absence of γH2AX and ATF4 in CEPT-treated cells resembling the control group (no passage). Similar results were obtained with iPSCs (see ). g, Puromycin pulse-chase experiment of hESCs (WA09) demonstrates that protein synthesis was strongly impaired during cell passaging and can be rescued by CEPT (3 h post-passage). See also confirming these observations using iPSCs. h, Glutathione levels were significantly higher in hESCs (WA09) passaged with CEPT (3 h post-plating). Data are mean ± s.d. (n = 4 wells for each group), ****p < 0.0001 for both comparison, one-way ANOVA. Similar results were obtained with additional two hESC and three iPSC lines (see ). i, Neutral 96-well comet chip assay was carried out on three different cell lines after treatment with different reagents (6 h post-passage). Note that CEPT-treated cells always displayed the lowest amount of DNA double-strand breaks. Data are mean ± s.d. (n = 5 replicates for each group), WA09, ****p < 0.0001; LiPSC-GR1.1, ****p < 0.0001, ***p = 0.0005, *p = 0.0198; NCMR-5, ****p < 0.0001, **p = 0.0058, one-way ANOVA. j, Representative images showing comets stained with SYBR Gold and imaged by fluorescence microscopy. Comet tails were analyzed with the Comet Analysis Software (Trevigen) and correlated with the amount of DNA damage in single cells. Scale bar, 20 μm. k, Schematic summarizing the various interconnected stress mechanisms that affect cell structure and function. Comparison between stressed (DMSO or Y-27632) and cytoprotected (CEPT) cells. l, Overview of the various applications that can be optimized by using CEPT.

Article Snippet: Cell pellets were lysed by sonication in RIPA lysis buffer (Thermo Fisher Scientific) supplemented with Halt protease inhibitor cocktail (Thermo Fisher Scientific) and centrifuged at 14,000g for 15 min. Etoposide sensitivity was analyzed on the Wes automated Western blotting system (ProteinSimple) using anti-phospho-γH2AX (CST, 9718, 1:50), phospho-BRCA1 (CST, 9009, 1:100), phospho-CHK2 (CST, 2197, 1:50), p21 (CST, 2947, 1:100), phospho-P53 (CST, 9284, 1:50), p53 (Santa Cruz, sc263, 1:50) and H2AX (CST, 2595, 1:50) antibodies.

Techniques: Passaging, Expressing, Immunocytochemistry, Cell Attachment Assay, Western Blot, Membrane, Control, Pulse Chase, Comparison, Staining, Fluorescence, Microscopy, Software

Journal: Nature methods

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1038/s41592-021-01126-2

Figure Lengend Snippet: a, Western blot analysis of iPSCs (LiPSC-GR1.1) showing cellular stress at 3 h post-passage in the presence of DMSO and Y-27632. Note that CEPT protects cells, which are more similar to the condition prior to passage (“no passage” served as control). See also showing similar results using hESCs. b, Western blot analysis of cell membrane-associated proteins. Human iPSCs (LiPSC-GR1.1) were dissociated and exposed to Y-27632 or CEPT for 24 h. Note that all proteins are expressed at higher levels after CEPT treatment. GAPDH was used as a loading control. Similar results were obtained using hESCs (see ). c, Puromycin pulse-chase experiment demonstrating that CEPT-treated iPSCs (LiPSC-GR1.1) show higher protein synthesis capacity than cultures passaged with Y-27632. Note that protein synthesis is completely stalled in the presence of DMSO indicating cellular stress after single-cell dissociation with Accutase. All experiments were performed at 3 h post-passage and samples were collected after 50 min of puromycin exposure. See also showing similar results with hESCs. d, Measurement of glutathione levels in two hESC lines and three iPSC lines. Glutathione levels were consistently higher in CEPT treated cultures compared to DMSO and Y-27632. Data represent mean ± s.d. (n = 4 wells for each group). WA01, ****p < 0.0001 for both comparison; WA07, **p = 0.0071 and ***p = 0.0002; GM23476, ****p < 0.0001 and ***p = 0.0001; GM25256, ****p < 0.0001 and ns = 0.0952; GM26107, ****p < 0.0001 and **p = 0.0075; one-way ANOVA. See also showing similar results for the WA09 cell line. e, Dose-response experiment showing that increasing concentrations of etoposide correlate with enhanced signal for γH2AX in hESCs (WA09) as measured by Western blotting. f, hESCs (WA09) were treated with 340 μM etoposide for 3 h. Note that CEPT treatment reduces γH2AX levels with and without etoposide treatment. g, hESCs (WA09) treated with CEPT show expected physiological stress-response to etoposide-induced DNA damage and strongly induce γH2AX, p21, p53, and phosphorylation of BRCA1, CHK2, and p53. Cells were treated with 340 μM etoposide for 3 h.

Article Snippet: Cell pellets were lysed by sonication in RIPA lysis buffer (Thermo Fisher Scientific) supplemented with Halt protease inhibitor cocktail (Thermo Fisher Scientific) and centrifuged at 14,000g for 15 min. Etoposide sensitivity was analyzed on the Wes automated Western blotting system (ProteinSimple) using anti-phospho-γH2AX (CST, 9718, 1:50), phospho-BRCA1 (CST, 9009, 1:100), phospho-CHK2 (CST, 2197, 1:50), p21 (CST, 2947, 1:100), phospho-P53 (CST, 9284, 1:50), p53 (Santa Cruz, sc263, 1:50) and H2AX (CST, 2595, 1:50) antibodies.

Techniques: Western Blot, Control, Membrane, Pulse Chase, Comparison, Phospho-proteomics