Journal:

Article Title: Carbapenem-Resistant Strain of Klebsiella oxytoca Harboring Carbapenem-Hydrolyzing ?-Lactamase KPC-2

doi: 10.1128/AAC.47.12.3881-3889.2003

Figure Lengend Snippet: Antimicrobial susceptibility patterns of K. oxytoca 3127, E. coli DH5α clone, and E. coli HB101 transformant a

Article Snippet: PCR analysis showed that K. oxytoca 3127 and K. pneumoniae ATCC 13883 (the extended-spectrum cephalosporin-susceptible type strain) both carry all three porin genes, ompK35 , ompK36 , and ompK37 (data not shown).

Techniques:

Journal:

Article Title: Carbapenem-Resistant Strain of Klebsiella oxytoca Harboring Carbapenem-Hydrolyzing ?-Lactamase KPC-2

doi: 10.1128/AAC.47.12.3881-3889.2003

Figure Lengend Snippet: (A) Isoelectric focusing patterns of cell lysates from carbapenem-resistant strains. The gel was stained with nitrocefin. Lanes 1 to 4, cell lysates prepared from strains producing TEM-3 (pI 6.3), TEM-1 (pI 5.4), SHV-5 (pI 8.2), and MIR-1 (pI 8.4), respectively; lane 5, the imipenem-resistant E. coli HB101 transformant containing a 70-kb plasmid from 3127; lane 6, an imipenem-resistant E. coli HB101 transconjugant of 3127; lane 7, K. oxytoca 3127. (B) Isoelectric focusing patterns of cell lysates prepared from carbapenem-resistant clones of K. oxytoca 3127. Lane 1, strain producing SHV-46 (pI 8.2); lanes 2 to 3, clones of strain 3127; lane 4, an imipenem-resistant E. coli HB101 transconjugant of K. oxytoca 3127; lane 5, E. coli DH5α containing the blaKPC-1 clone. The pIs of the β-lactamases were calculated by using the known pIs of TEM-1 (5.4), TEM-3 (6. 3), SHV-5 (8.2), KPC-1 (6.7), and MIR-1 (8.4).

Article Snippet: PCR analysis showed that K. oxytoca 3127 and K. pneumoniae ATCC 13883 (the extended-spectrum cephalosporin-susceptible type strain) both carry all three porin genes, ompK35 , ompK36 , and ompK37 (data not shown).

Techniques: Staining, Plasmid Preparation, Clone Assay

Journal:

Article Title: Carbapenem-Resistant Strain of Klebsiella oxytoca Harboring Carbapenem-Hydrolyzing ?-Lactamase KPC-2

doi: 10.1128/AAC.47.12.3881-3889.2003

Figure Lengend Snippet: NuPAGE gel and Western blot analysis of OMPs of K. oxytoca 3127 and a carbapenem-susceptible control strain, K. pneumoniae ATCC 13883. (A) NuPAGE gel analysis of OMPs. Lane 1, molecular mass markers; lane 2, OMPs prepared from K. oxytoca 3127; lane 3, OMPs prepared from K. pneumoniae ATCC 13883. (B) Western blot analysis of OMPs performed with anti-OMPK36 antisera. (C) Western blot analysis of OMPs performed with anti-OMPK35 antisera. Molecular mass is indicated in gels to the left of each panel.

Article Snippet: PCR analysis showed that K. oxytoca 3127 and K. pneumoniae ATCC 13883 (the extended-spectrum cephalosporin-susceptible type strain) both carry all three porin genes, ompK35 , ompK36 , and ompK37 (data not shown).

Techniques: Western Blot

Journal:

Article Title: Curli Fibers Are Highly Conserved between Salmonella typhimurium and Escherichia coli with Respect to Operon Structure and Regulation

doi:

Figure Lengend Snippet: Bacterial strains used in this study

Article Snippet: Recombinant clones were grown on LB medium supplemented with recommended concentrations of antibiotics ( 4 ), if required. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain Relevant genotype Source or reference S. typhimurium ATCC 14028-1s wt1 a American Type Culture Collection SR-11 wt1 a R. Curtiss III LB5010 metA22 metE551 ilv-452 leu-3121 trpD2 xyl-404 galE856 hsdLT6 hsdSA29 hsdSB121 rpsL120 12 146 polA-2 zig214 ::Tn 10 (Tet r ) M. Rhen SF1005 rpoS ::pRR10(Δ trfA )(Amp r ) D. Guiney UMR1 ATCC 14028-1s Nal r This study UMR3 SR-11 Nal r This study MAE1 UMR1 Δ csgA101 ::Km r This study MAE2 UMR3 Δ csgA101 ::Km r This study MAE5 UMR1 Δ csgA101 This study INK1 SR-11 zcg-101 ::Km r This study MAE40 SF1005 × UMR1; UMR1 rpoS ::pRR10(Δ trfA )(Amp r ) This study MAE29 SF1005 × SR-11; SR-11 rpoS ::pRR10 (Δ trfA )(Amp r ) This study MAE46 UMR1 Δ ompR101 ::Amp r This study MAE34 UMR3 Δ ompR101 ::Amp r This study E. coli K-12 DH5α endA1 hsdR17 supE44 thi-1 recA1 gyrA relA1 Δ( lacZYA-argF ) U169 (φ80 lacZΔM15 ) Laboratory collection MC4100 F − araD139 Δ( argF-lac ) U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR Laboratory collection XL1 Blue MR Δ( mcrA ) 183 Δ( mcrCB-hsdSMR-mrr ) 173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Stratagene HLO7 MC4100 Δ( csgG-csgC )::Km r 47a MHR204 MC4100 csgA2 ::Tn 105 Cm r 34 MHR210 MC4100 csgG1 ::Tn 105 Cm r 34 MHR261 MC4100 csgB2 35 MHR426 MC4100 csgF4 36 MHR480 MC4100 Δ csgE3 37 MHR503 MC4100 csgD6 This study Open in a separate window a wt1, wild type 1.

Techniques:

Journal: bioRxiv

Article Title: RhoA activation promotes glucose uptake to elevate proliferation in MAPK inhibitor resistant melanoma cells

doi: 10.1101/2024.01.09.574940

Figure Lengend Snippet: (A) Flow chart to generate MAPKi-resistant melanoma cells. The naïve B-Raf V600E mutant melanoma cells upon exposure to MAPKi show an initial reduction in proliferation. But upon exposure to MAPKi for ∼2 months results in acquisition of resistance. These resistant cells were then cultured in the presence of MAPKi. (B) A375, M481 and UACC62 naïve and resistant cells were compared for proliferation using an imaging based EdU incorporation assay in 2D cell culture conditions. The naïve cells were treated with vehicle control or with MAPKi and the proliferation compared to MAPKi-resistant melanoma cells. The % Proliferation was determined as the ratio of EdU-positive cells to the total cells determined by DAPI counterstain. The scale bar on the image is 50µm. (C) A375 naïve and resistant cells were cultured on glass coverslips. The naive cells were either treated with vehicle control or MAPKi. All the cells were then fixed and stained with phalloidin to evaluate F-actin structures. The scale bar on the image is 10µm. For (B) Three biological repeats were performed with a minimum of 9 images acquired per sample per repeat. The statistical analysis was performed using one-way ANOVA. For (C) Imaging was performed for three biological repeats with a minimum of 10 images acquired per sample per biological repeat. For the statistical significance; ns P ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. The data represented in (B) is mean ± s.e.m.

Article Snippet: A375 cells (human malignant melanoma cells harvested from a 54-year-old female CRL-1619, ATCC, RRID: CVCL_0132), UACC62 cells (human malignant melanoma ABC-TC534S, Accegen, RRID: CVCL_1780), M481 primary cells were acquired from the University of Michigan via Dr. Sean Morrison at UT Southwestern Medical Center, WM164 (metastatic human melanoma cell line established from a metastatic site in a 22-year-old male with stage IV superficial spreading melanoma WM164-01-0001, Rockland, RRID: CVCL_7928), 451Lu (xenograft aggressive metastatic sub-line of WM164; 451Lu-01-0001, Rockland, RRID: CVCL_6357).

Techniques: Mutagenesis, Cell Culture, Imaging, Control, Staining

Journal: bioRxiv

Article Title: RhoA activation promotes glucose uptake to elevate proliferation in MAPK inhibitor resistant melanoma cells

doi: 10.1101/2024.01.09.574940

Figure Lengend Snippet: (A) A375, M481, UACC62, WM164 and 451Lu were evaluated for the metabolic state. The resistant melanoma cells were compared to naïve cells with vehicle control or MAPKi treatment and seahorse assay performed to evaluate ECAR (readout of glycolysis) and OCR (readout for oxidative phosphorylation). The metabolic rates, ECAR and OCR were normalized to per cell through fixation, DAPI staining and cell count using ImageJ after seahorse assay. (B) Glucose uptake in naïve and resistant A375 melanoma cells was evaluated using NBD-glucose. The cells were then imaged, and the image intensity was quantified per cell using ImageJ. (C) To evaluate the role of F-actin structures in metabolism the A375 resistant melanoma cells were treated with Latrunculin-A to evaluate actin depolymerization. (D) Seahorse assay was then performed for the resistant cells treated with Latrunculin-A after 4h of treatment. (E) A375 naïve cells were overexpressed with a PFKP-GFP followed by seahorse assay. All experiments were performed with 3 biological repeats. For (A) A375, M481, and WM164 had 9 data points per repeat. The UACC62 and 451Lu had atleast 3 datapoint per repeat. For (B) Each biological sample in the repeat had at least 12 images. (D) and (E) had 3 data points per repeat. The statistical analysis was performed using one-way ANOVA for (A), (B) and (D). Welch’s t-test was used for (E). For the statistical significance; ns P ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. The data represented in (B) is mean ± s.e.m.

Article Snippet: A375 cells (human malignant melanoma cells harvested from a 54-year-old female CRL-1619, ATCC, RRID: CVCL_0132), UACC62 cells (human malignant melanoma ABC-TC534S, Accegen, RRID: CVCL_1780), M481 primary cells were acquired from the University of Michigan via Dr. Sean Morrison at UT Southwestern Medical Center, WM164 (metastatic human melanoma cell line established from a metastatic site in a 22-year-old male with stage IV superficial spreading melanoma WM164-01-0001, Rockland, RRID: CVCL_7928), 451Lu (xenograft aggressive metastatic sub-line of WM164; 451Lu-01-0001, Rockland, RRID: CVCL_6357).

Techniques: Control, Phospho-proteomics, Staining, Cell Counting

Journal: bioRxiv

Article Title: RhoA activation promotes glucose uptake to elevate proliferation in MAPK inhibitor resistant melanoma cells

doi: 10.1101/2024.01.09.574940

Figure Lengend Snippet: (A) RhoA activation was evaluated in A375, UACC62 and M481 naïve and resistant melanoma cells using the RhoA activation pull down assay. The band intensities were quantified for the rho pull down as a ratio of resistant to naïve cells. (B) The A375 naïve and resistant cells were treated with Y27632 and GSK269962A for 48h followed by EdU incorporation assay to evaluate proliferation. (C) A375 naïve and resistant melanoma cells treated with GSK269962A were evaluated for glucose uptake using NBD-glucose and the intensities quantified per cells using ImageJ. (D) Metabolic rate was quantified for A375 resistant melanoma cells treated with GSK269962A and evaluated for ECAR and OCR. (E) RhoA was activated in naïve A375 and M481 using a constitutively active RhoA (CA-RhoA) and evaluated for glucose uptake using NBD-glucose assay and image intensities quantified per cell which was compared to empty vector (EV). (F) Seahorse assay was performed to evaluate ECAR and OCR in naïve A375 and M481 melanoma cells with EV or CA-RhoA. (G) Proliferation between EV and CA-RhoA was evaluated using EdU incorporation assay for the naïve A375 and M481 melanoma cells. For (A) 4 biological repeats, (B) Three biological repeats were performed with three data points per repeat, (C) (E) and (G) Three biological repeats with at least 10 images per sample per repeat, (D) Three biological repeats with four data points per sample per repeat. (F) Three biological repeats with six data points per repeat. The statistical analysis was performed using one-way ANOVA for (C), and (D). Welch’s t-test was used for (E), (F) and (G). For the statistical significance; ns P ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. The data represented in (B) is mean ± s.e.m.

Article Snippet: A375 cells (human malignant melanoma cells harvested from a 54-year-old female CRL-1619, ATCC, RRID: CVCL_0132), UACC62 cells (human malignant melanoma ABC-TC534S, Accegen, RRID: CVCL_1780), M481 primary cells were acquired from the University of Michigan via Dr. Sean Morrison at UT Southwestern Medical Center, WM164 (metastatic human melanoma cell line established from a metastatic site in a 22-year-old male with stage IV superficial spreading melanoma WM164-01-0001, Rockland, RRID: CVCL_7928), 451Lu (xenograft aggressive metastatic sub-line of WM164; 451Lu-01-0001, Rockland, RRID: CVCL_6357).

Techniques: Activation Assay, Pull Down Assay, Glucose Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: RhoA activation promotes glucose uptake to elevate proliferation in MAPK inhibitor resistant melanoma cells

doi: 10.1101/2024.01.09.574940

Figure Lengend Snippet: (A) The activity of PKA in A375, M481 and UACC62 naïve and resistant melanoma cells was evaluated using the PKA activation assay. (B) To induce PKA activation in A375 resistant cells 8-Bromo-cAMP was used and the metabolic state was evaluated using seahorse assay. (C) Proliferation was evaluated in A375 resistant melanoma cells upon 8-Bromo-cAMP treatment using EdU incorporation assay. For (A) each data point represents a biological repeat. For (B) Three biological repeats were performed with four data points per repeat. For (C) three biological repeats ten data points per repeat. The statistical analysis was performed using one-way ANOVA for (B) and (C). Welch’s t-test was used for (A). For the statistical significance; ns P ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. The data represented in (B) is mean ± s.e.m.

Article Snippet: A375 cells (human malignant melanoma cells harvested from a 54-year-old female CRL-1619, ATCC, RRID: CVCL_0132), UACC62 cells (human malignant melanoma ABC-TC534S, Accegen, RRID: CVCL_1780), M481 primary cells were acquired from the University of Michigan via Dr. Sean Morrison at UT Southwestern Medical Center, WM164 (metastatic human melanoma cell line established from a metastatic site in a 22-year-old male with stage IV superficial spreading melanoma WM164-01-0001, Rockland, RRID: CVCL_7928), 451Lu (xenograft aggressive metastatic sub-line of WM164; 451Lu-01-0001, Rockland, RRID: CVCL_6357).

Techniques: Activity Assay, Activation Assay