Journal: Nature Communications

Article Title: ERK-mediated NELF-A phosphorylation promotes transcription elongation of immediate-early genes by releasing promoter-proximal pausing of RNA polymerase II

doi: 10.1038/s41467-022-35230-4

Figure Lengend Snippet: a HaCaT cells stably expressing shRNA-resistant Myc-NELF-A (WT or 4A) were depleted of endogenous NELF-A with shRNA. Levels of Myc-NELF-A proteins were confirmed by immunoblotting. Actin, loading control. b , d HaCaT-WT and −4A cells were synchronized at the G1-phase by serum starvation, and stimulated with EGF for the indicated times. In ( b ), phosphorylation of immunoprecipitated Myc-NELF-A (WT and 4A) was probed with anti-phospho-SP/TP antibody. In ( d ), cell lysates were probed for cyclin D1, phosphorylated ERK1/2 (P-ERK) and ERK1/2 by immunoblotting using the appropriate antibodies. c HaCaT-WT and -4A cells were stimulated with or without EGF (60 ng/ml, for 60 min). Total RNA was extracted and analyzed for the indicated mRNA expression using qRT-PCR. All data were normalized to the level of GAPDH expression. e Serum-starved HaCaT-WT and -4A cells were stimulated with EGF for 18 h, and BrdU- positive nuclei were quantified on the Operetta imaging system. More than 5000 cells were counted in each experiment. f The indicated cells were cultured in medium with 10% FBS for 4 days. Cell proliferation was determined using a CCK8 assay. OD, optical density. c , e , f , Error bars, SEM ( n = 3). g – j HaCaT-WT and -4A cells ( g , h ) or Ras-HaCaT-WT and -4A cells ( i , j ) were subcutaneously injected into BALB/c nude mice. In ( g , i ), the tumor volume was monitored on the indicated days. In ( h , j ), the weight of excised xenograft tumors on day 36 ( h ) and on day 35 ( j ) (upper). Photographs of the xenograft tumors excised from individual mice (lower). Error bars, SEM ( n = 7 in ( g ) and ( h ), n = 8 in i and j ). c , e – j , P -values were assessed using a two-tailed Student’s t-test. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: monoclonal anti-NELF-A G-11 (Santa Cruz, sc-365004), anti-NELF-D C-10 (sc-393972), anti-NELF-E F-9 (sc-377052), anti-ERK1/2 C-9 (sc-514302), anti-Myc 9E10 (sc-40), anti-GST B-14 (sc-138), anti-HA F-7 (sc-7392), anti-Pol II 8WG16 (sc-56767), anti-Pol II CTD4H8 (sc-47701), anti-CDK9 D-7 (sc-13130), anti-Elk1 E-5 (sc-365876), anti-CyclinD1 A-12 (sc-8396); anti-Phospho-Rpb1 CTD-Ser5 D9N5I (CST, 13523), anti-RSK1/RSK2/RSK3 32D7 (9355), anti-Phospho-Rpb1 CTD-Ser2 E1Z3G (13499), anti-c-Fos 9F6 (2250); anti-HA 3F10 (Roche, 11867423001); anti-Flag M2 (Sigma, F1804); anti β-Actin (Wako, 010-27841); anti-His-tag (Medical & Biological Laboratories, D291-3); anti-PP2Ac (BD, 610555), anti-DICE1/INTS6 (Santa Cruz Biotechnology, sc-376524), anti-MPM2 (anti-phospho-SP or TP) (Millipore, 05-368); polyclonal anti-ERK1 K-23 (Santa Cruz, sc-94); anti-NELF-A (Protein tech, 10456-1-AP), anti-NELF-B (16418-1-AP); anti-phospho-ERK1/2 (CST, 9101), anti-Phospho-p90RSK T573 (9346); anti-GADD45B (Cloud-Clone, PAL535Hu01), anti-RAS (G12V mutant) (GeneTex, GTX132694).

Techniques: Stable Transfection, Expressing, shRNA, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Imaging, Cell Culture, CCK-8 Assay, Injection, Two Tailed Test

Journal:

Article Title: Differential Sensitivity of Human Epithelial Cells to Pseudomonas aeruginosa Exoenzyme S

doi:

Figure Lengend Snippet: Characteristics of cell lines examined in ExoS coculture studies

Article Snippet: All cells were allowed to grow for 2 to 3 days prior to addition of bacteria. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Cell line Cell type Basal medium Characteristics Source NK77 Primary kidney epithelial DME/F12 a Normal MUSC d NK97612 Primary kidney epithelial DME/F12 a Normal MUSC CFT1 Tracheal epithelial Ham’s F-12 b CFTR ΔF508 mutation Gerald Pier e CFT1-LCFSN Trachael epithelial Ham’s F-12 b CFT1 with normal CFTR Gerald Pier HT29 Colon carcinoma McCoy’s 5A c Normal Ras ATCC HTB 38 LNCaP Prostate carcinoma RPMI 1640 c Normal Ras ATCC CRL 1740 T24 Bladder carcinoma McCoy’s 5A c Mutant H-Ras (G12V) ATCC HTB 4 SW480 Colon carcinoma McCoy’s 5A c Mutant K-Ras (G12V) ATCC CCL 228 SK-N-SH Neuroblastoma DMEM c Mutant N-Ras (Q61K) ATCC HTB 11 Open in a separate window a Supplemented with 5 μg of insulin, 5 μg of transferrin, 5 μg of selenium, 36 ng of hydrocortisone, 4 pg of triiodothyronine, and 10 ng of epidermal growth factor per ml. b Supplemented with 10 μg of insulin per ml, 1 μM hydrocortisone, 3.75 μg of endothelial cell growth supplement per ml, 25 ng of epidermal growth factor per ml, 30 nM triiodothyronine, 5 μg of transferrin per ml, and 10 ng of cholera toxin per ml. c Contains 10% FBS. d MUSC, tissue culture bank, Department of Pathology and Laboratory Medicine, Medical University of South Carolina. e Brigham & Women’s Hospital, Harvard Medical School.

Techniques: Mutagenesis

Journal:

Article Title: Differential Sensitivity of Human Epithelial Cells to Pseudomonas aeruginosa Exoenzyme S

doi:

Figure Lengend Snippet: Characteristics of cell lines examined in ExoS coculture studies

Article Snippet: All cells were allowed to grow for 2 to 3 days prior to addition of bacteria. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Cell line Cell type Basal medium Characteristics Source NK77 Primary kidney epithelial DME/F12 a Normal MUSC d NK97612 Primary kidney epithelial DME/F12 a Normal MUSC CFT1 Tracheal epithelial Ham’s F-12 b CFTR ΔF508 mutation Gerald Pier e CFT1-LCFSN Trachael epithelial Ham’s F-12 b CFT1 with normal CFTR Gerald Pier HT29 Colon carcinoma McCoy’s 5A c Normal Ras ATCC HTB 38 LNCaP Prostate carcinoma RPMI 1640 c Normal Ras ATCC CRL 1740 T24 Bladder carcinoma McCoy’s 5A c Mutant H-Ras (G12V) ATCC HTB 4 SW480 Colon carcinoma McCoy’s 5A c Mutant K-Ras (G12V) ATCC CCL 228 SK-N-SH Neuroblastoma DMEM c Mutant N-Ras (Q61K) ATCC HTB 11 Open in a separate window a Supplemented with 5 μg of insulin, 5 μg of transferrin, 5 μg of selenium, 36 ng of hydrocortisone, 4 pg of triiodothyronine, and 10 ng of epidermal growth factor per ml. b Supplemented with 10 μg of insulin per ml, 1 μM hydrocortisone, 3.75 μg of endothelial cell growth supplement per ml, 25 ng of epidermal growth factor per ml, 30 nM triiodothyronine, 5 μg of transferrin per ml, and 10 ng of cholera toxin per ml. c Contains 10% FBS. d MUSC, tissue culture bank, Department of Pathology and Laboratory Medicine, Medical University of South Carolina. e Brigham & Women’s Hospital, Harvard Medical School.

Techniques: Mutagenesis