Structured Review

TaKaRa apoptotic cells
Telomere dysfunction induced by MST-312. ( A ) TUNEL assay of MST-312-treated cells. Human lung cancer NCI-H522 [mean telomeric restriction fragment (TRF) length = 3.2 kb] and A549 (mean TRF length = 9.6 kb) cells were treated with 5 μM MST-312 for 48 h and then subjected to TUNEL assay for detection of <t>apoptotic</t> cells ( green ). ( B ) Quantification of apoptotic cells. Cells were treated as in ( A ) and then stained with propidium iodide; the apoptotic sub-G1 fraction was quantitated by flow cytometry. ( C ) Immuno-FISH analysis of NCI-H522 cells treated with 5 μM MST-312 for 48 h. Red : telomere DNA; green : 53BP1; blue : DAPI. Right panels are magnified views of enlarged telomere dysfunction-induced foci (TIF). ( D , E ) Quantitation of TIF. Cells were treated as in ( A ), and telomeres and the indicated proteins were detected by FISH and immunofluorescence staining, respectively. Because the cells with short telomeres gave high background levels of TIF even without MST-312 treatment ( D , upper left ), enlarged TIF were quantitated as a hallmark of the MST-312-induced telomeric DNA damage response. Error bar indicates standard deviation. Asterisk indicates statistical significance in the difference between control and MST-312-treated cells (unpaired two-tailed t test). ALT: alternative lengthening of telomeres. ( F ) Telomere southern blot analysis. Cells were treated with indicated doses of MST-312 for 48 h. HTC75 fibrosarcoma cells were analysed as a control because the telomere length fluctuation of this cell line can be detected by southern blot analysis. ( G ) Cells were treated as in ( A ) and mitotic index was quantitated. ( H – J ) Cells in ( G ) were further incubated with colcemid. Metaphase spreads of chromosomes were subjected to telomere FISH, and chromosome number ( H ), telomeric signal-free ends ( I ) and telomeric fragments ( J ) were quantitated. Red : telomere DNA, blue : DAPI stain of chromosome DNA. Asterisk indicates statistical significance (vs. control, unpaired two-tailed t test).
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Images

1) Product Images from "Cell-based chemical fingerprinting identifies telomeres and lamin A as modifiers of DNA damage response in cancer cells"

Article Title: Cell-based chemical fingerprinting identifies telomeres and lamin A as modifiers of DNA damage response in cancer cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-33139-x

Telomere dysfunction induced by MST-312. ( A ) TUNEL assay of MST-312-treated cells. Human lung cancer NCI-H522 [mean telomeric restriction fragment (TRF) length = 3.2 kb] and A549 (mean TRF length = 9.6 kb) cells were treated with 5 μM MST-312 for 48 h and then subjected to TUNEL assay for detection of apoptotic cells ( green ). ( B ) Quantification of apoptotic cells. Cells were treated as in ( A ) and then stained with propidium iodide; the apoptotic sub-G1 fraction was quantitated by flow cytometry. ( C ) Immuno-FISH analysis of NCI-H522 cells treated with 5 μM MST-312 for 48 h. Red : telomere DNA; green : 53BP1; blue : DAPI. Right panels are magnified views of enlarged telomere dysfunction-induced foci (TIF). ( D , E ) Quantitation of TIF. Cells were treated as in ( A ), and telomeres and the indicated proteins were detected by FISH and immunofluorescence staining, respectively. Because the cells with short telomeres gave high background levels of TIF even without MST-312 treatment ( D , upper left ), enlarged TIF were quantitated as a hallmark of the MST-312-induced telomeric DNA damage response. Error bar indicates standard deviation. Asterisk indicates statistical significance in the difference between control and MST-312-treated cells (unpaired two-tailed t test). ALT: alternative lengthening of telomeres. ( F ) Telomere southern blot analysis. Cells were treated with indicated doses of MST-312 for 48 h. HTC75 fibrosarcoma cells were analysed as a control because the telomere length fluctuation of this cell line can be detected by southern blot analysis. ( G ) Cells were treated as in ( A ) and mitotic index was quantitated. ( H – J ) Cells in ( G ) were further incubated with colcemid. Metaphase spreads of chromosomes were subjected to telomere FISH, and chromosome number ( H ), telomeric signal-free ends ( I ) and telomeric fragments ( J ) were quantitated. Red : telomere DNA, blue : DAPI stain of chromosome DNA. Asterisk indicates statistical significance (vs. control, unpaired two-tailed t test).
Figure Legend Snippet: Telomere dysfunction induced by MST-312. ( A ) TUNEL assay of MST-312-treated cells. Human lung cancer NCI-H522 [mean telomeric restriction fragment (TRF) length = 3.2 kb] and A549 (mean TRF length = 9.6 kb) cells were treated with 5 μM MST-312 for 48 h and then subjected to TUNEL assay for detection of apoptotic cells ( green ). ( B ) Quantification of apoptotic cells. Cells were treated as in ( A ) and then stained with propidium iodide; the apoptotic sub-G1 fraction was quantitated by flow cytometry. ( C ) Immuno-FISH analysis of NCI-H522 cells treated with 5 μM MST-312 for 48 h. Red : telomere DNA; green : 53BP1; blue : DAPI. Right panels are magnified views of enlarged telomere dysfunction-induced foci (TIF). ( D , E ) Quantitation of TIF. Cells were treated as in ( A ), and telomeres and the indicated proteins were detected by FISH and immunofluorescence staining, respectively. Because the cells with short telomeres gave high background levels of TIF even without MST-312 treatment ( D , upper left ), enlarged TIF were quantitated as a hallmark of the MST-312-induced telomeric DNA damage response. Error bar indicates standard deviation. Asterisk indicates statistical significance in the difference between control and MST-312-treated cells (unpaired two-tailed t test). ALT: alternative lengthening of telomeres. ( F ) Telomere southern blot analysis. Cells were treated with indicated doses of MST-312 for 48 h. HTC75 fibrosarcoma cells were analysed as a control because the telomere length fluctuation of this cell line can be detected by southern blot analysis. ( G ) Cells were treated as in ( A ) and mitotic index was quantitated. ( H – J ) Cells in ( G ) were further incubated with colcemid. Metaphase spreads of chromosomes were subjected to telomere FISH, and chromosome number ( H ), telomeric signal-free ends ( I ) and telomeric fragments ( J ) were quantitated. Red : telomere DNA, blue : DAPI stain of chromosome DNA. Asterisk indicates statistical significance (vs. control, unpaired two-tailed t test).

Techniques Used: Microscale Thermophoresis, TUNEL Assay, Staining, Flow Cytometry, Cytometry, Fluorescence In Situ Hybridization, Quantitation Assay, Immunofluorescence, Standard Deviation, Two Tailed Test, Southern Blot, Incubation

Telomere elongation and lamin A expression confers resistance to MST-312. ( A , B ) Telomere elongation and lamin A expression in NCI-H522 cells. Cells were infected with hTERT and/or LMNA expressing retrovirus. Cells were subjected to western blot analysis for detection of hTERT and lamin A expression ( A ) and TRAP assay for detection of telomerase activity ( B ). For ( A ) full-length blots were presented in Supplementary Fig. S5 . ( C ) Telomere southern blot analysis. Genomic DNA was prepared and subjected to southern blot analysis with [ 32 P]-labelled telomeric probe. ( D ) Telomere immuno-FISH analysis. Cells were treated with 5 μM MST-312 for 48 h and subjected to immuno-FISH analysis. Green : 53BP1; red : telomere DNA; blue : DAPI stain of DNA. ( E ) Quantitation of DNA damage foci in ( D ). Cells with more than four 53BP1 foci were counted as positive. ( F ) Quantitation of telomeric DNA damage (TIF and enlarged TIF) in ( D ). Cells with more than four TIF were counted as positive. ( G ) Apoptosis induced by MST-312 and anticancer drugs. Cells were treated with 5 μM MST-312, 3.3 μM cisplatin (CDDP), 10 nM camptothecin (CPT), 1.6 μM etoposide or 5 nM paclitaxel for 96 h. Cells were stained with propidium iodide and the apoptotic sub-G1 fraction of the cell cycle was quantitated by flow cytometry. Error bar indicates standard deviation of four ( E , F ) or at least three ( G ) experiments. Statistical significance was evaluated by Tukey-Kramer test. * P
Figure Legend Snippet: Telomere elongation and lamin A expression confers resistance to MST-312. ( A , B ) Telomere elongation and lamin A expression in NCI-H522 cells. Cells were infected with hTERT and/or LMNA expressing retrovirus. Cells were subjected to western blot analysis for detection of hTERT and lamin A expression ( A ) and TRAP assay for detection of telomerase activity ( B ). For ( A ) full-length blots were presented in Supplementary Fig. S5 . ( C ) Telomere southern blot analysis. Genomic DNA was prepared and subjected to southern blot analysis with [ 32 P]-labelled telomeric probe. ( D ) Telomere immuno-FISH analysis. Cells were treated with 5 μM MST-312 for 48 h and subjected to immuno-FISH analysis. Green : 53BP1; red : telomere DNA; blue : DAPI stain of DNA. ( E ) Quantitation of DNA damage foci in ( D ). Cells with more than four 53BP1 foci were counted as positive. ( F ) Quantitation of telomeric DNA damage (TIF and enlarged TIF) in ( D ). Cells with more than four TIF were counted as positive. ( G ) Apoptosis induced by MST-312 and anticancer drugs. Cells were treated with 5 μM MST-312, 3.3 μM cisplatin (CDDP), 10 nM camptothecin (CPT), 1.6 μM etoposide or 5 nM paclitaxel for 96 h. Cells were stained with propidium iodide and the apoptotic sub-G1 fraction of the cell cycle was quantitated by flow cytometry. Error bar indicates standard deviation of four ( E , F ) or at least three ( G ) experiments. Statistical significance was evaluated by Tukey-Kramer test. * P

Techniques Used: Expressing, Microscale Thermophoresis, Infection, Western Blot, TRAP Assay, Activity Assay, Southern Blot, Fluorescence In Situ Hybridization, Staining, Quantitation Assay, Cycling Probe Technology, Flow Cytometry, Cytometry, Standard Deviation

2) Product Images from "Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels"

Article Title: Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00301

pCt modulates TREK1 and TRAAK single-channel properties. (A,E) Single-channel recordings at +80 and –80 mV, from HEK cells expressing wild-type and mutated channels. Pipette and bath solutions contained 150 mM KCl. (B,F) Single channel current–voltage relationships obtained from one level opening current in symmetrical 150 mM KCl. (C,G) Unitary conductances at +80 and –80 mV. (D,H) Mean open time obtained from channel opening recorded at +80 mV and –80 mV. (C,D,H,G) , Data are presented as mean ± SEM, the number of patches is indicated, ∗ p
Figure Legend Snippet: pCt modulates TREK1 and TRAAK single-channel properties. (A,E) Single-channel recordings at +80 and –80 mV, from HEK cells expressing wild-type and mutated channels. Pipette and bath solutions contained 150 mM KCl. (B,F) Single channel current–voltage relationships obtained from one level opening current in symmetrical 150 mM KCl. (C,G) Unitary conductances at +80 and –80 mV. (D,H) Mean open time obtained from channel opening recorded at +80 mV and –80 mV. (C,D,H,G) , Data are presented as mean ± SEM, the number of patches is indicated, ∗ p

Techniques Used: Expressing, Transferring

Sensitivity to PIP 2 is modulated by a cluster of basic residues in pCt. (A) Sequence alignment of TREK1 and TRAAK pCt. In red and blue the residues that are swapped in TREK1-QRA and TRAAK-RRR. (B,C) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1-QRA (B) and TRAAK-RRR (C) . Same protocol as in Figures 6A , 7A . (D) Current density (%) at 0 mV. (E,F) TREK1-QRA (E) and TRAAK-RRR (F) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (G) Current inhibition (%) at 0 mV. (D,G) Data are presented as mean ± SEM. ∗∗ p
Figure Legend Snippet: Sensitivity to PIP 2 is modulated by a cluster of basic residues in pCt. (A) Sequence alignment of TREK1 and TRAAK pCt. In red and blue the residues that are swapped in TREK1-QRA and TRAAK-RRR. (B,C) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1-QRA (B) and TRAAK-RRR (C) . Same protocol as in Figures 6A , 7A . (D) Current density (%) at 0 mV. (E,F) TREK1-QRA (E) and TRAAK-RRR (F) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (G) Current inhibition (%) at 0 mV. (D,G) Data are presented as mean ± SEM. ∗∗ p

Techniques Used: Sequencing, Patch Clamp, Expressing, Inhibition

Proximal C-ter domain (pCt) modulates TREK1 and TRAAK current amplitudes but not their cellular distribution. (A,B) Current densities at 0 mV of HEK cells expressing wild type or mutated channels. Ct, full cytoplasmic C-ter (A) . pCt, proximal cytoplasmic C-ter (B) . (C) % of the fluorescence sensitive to an extracellular acidification (pH 6) of live HEK cells expressing pHluorin-tagged channels. This % corresponds to the fraction of channels expressed at the plasma membrane. (A–C) Data are presented as mean ± SEM. ∗ p
Figure Legend Snippet: Proximal C-ter domain (pCt) modulates TREK1 and TRAAK current amplitudes but not their cellular distribution. (A,B) Current densities at 0 mV of HEK cells expressing wild type or mutated channels. Ct, full cytoplasmic C-ter (A) . pCt, proximal cytoplasmic C-ter (B) . (C) % of the fluorescence sensitive to an extracellular acidification (pH 6) of live HEK cells expressing pHluorin-tagged channels. This % corresponds to the fraction of channels expressed at the plasma membrane. (A–C) Data are presented as mean ± SEM. ∗ p

Techniques Used: Expressing, Fluorescence

Effect of decoupling pCt and M4 on TREK1 and TRAAK. (A) Representative whole-cell recordings of HEK cells expressing wild-type or mutated channels. Voltage ramps were applied from –100 to 60 mV from a holding potential of –80 mV. (B) Current densities at 0 mV. Data are presented as mean ± SEM. ∗∗∗ p
Figure Legend Snippet: Effect of decoupling pCt and M4 on TREK1 and TRAAK. (A) Representative whole-cell recordings of HEK cells expressing wild-type or mutated channels. Voltage ramps were applied from –100 to 60 mV from a holding potential of –80 mV. (B) Current densities at 0 mV. Data are presented as mean ± SEM. ∗∗∗ p

Techniques Used: Expressing

PIP 2 depletion inhibits TREK1 and TRAAK currents. (A,B) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1 (A) or TRAAK (B) . (C) Average currents measured at +120 mV during VSP-induced PIP 2 depletion. (D,E) , TREK1 (D) and TRAAK (E) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (F) Current inhibition (%) of TREK1 and TRAAK at 0 mV. Data are presented as mean ± SEM. ∗ p
Figure Legend Snippet: PIP 2 depletion inhibits TREK1 and TRAAK currents. (A,B) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1 (A) or TRAAK (B) . (C) Average currents measured at +120 mV during VSP-induced PIP 2 depletion. (D,E) , TREK1 (D) and TRAAK (E) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (F) Current inhibition (%) of TREK1 and TRAAK at 0 mV. Data are presented as mean ± SEM. ∗ p

Techniques Used: Patch Clamp, Expressing, Inhibition

TREK1 and TRAAK currents in HEK293 cells. (A) Representative whole-cell currents from non-transfected cells (NT) and cells transfected with TREK1 and TRAAK channels. Voltage steps were applied from –100 to 60 mV in 20 mV increments from a holding potential of –80 mV. The dotted lines indicate zero current. (B) Current densities at 0 mV. Data are presented as mean ± SEM. ∗∗∗ p
Figure Legend Snippet: TREK1 and TRAAK currents in HEK293 cells. (A) Representative whole-cell currents from non-transfected cells (NT) and cells transfected with TREK1 and TRAAK channels. Voltage steps were applied from –100 to 60 mV in 20 mV increments from a holding potential of –80 mV. The dotted lines indicate zero current. (B) Current densities at 0 mV. Data are presented as mean ± SEM. ∗∗∗ p

Techniques Used: Transfection

Role of pCt in TREK1 and TRAAK differential sensitivities to PIP 2 . (A,B) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1pCt TRAAK (A) and TRAAK pCt TREK1 (B) . (C) Average currents measured at +120 mV during VSP-induced PIP 2 depletion. (D,E) TREK1pCt TRAAK (D) and TRAAKpCt TREK1 (E) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (F) Current inhibition (%) of wild-type and mutated channels at 0 mV. Data are presented as mean ± SEM. ∗ p
Figure Legend Snippet: Role of pCt in TREK1 and TRAAK differential sensitivities to PIP 2 . (A,B) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1pCt TRAAK (A) and TRAAK pCt TREK1 (B) . (C) Average currents measured at +120 mV during VSP-induced PIP 2 depletion. (D,E) TREK1pCt TRAAK (D) and TRAAKpCt TREK1 (E) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (F) Current inhibition (%) of wild-type and mutated channels at 0 mV. Data are presented as mean ± SEM. ∗ p

Techniques Used: Patch Clamp, Expressing, Inhibition

3) Product Images from "MiR‐486 promotes proliferation and suppresses apoptosis in myeloid cells by targeting Cebpa in vitro, et al. MiR‐486 promotes proliferation and suppresses apoptosis in myeloid cells by targeting Cebpa in vitro"

Article Title: MiR‐486 promotes proliferation and suppresses apoptosis in myeloid cells by targeting Cebpa in vitro, et al. MiR‐486 promotes proliferation and suppresses apoptosis in myeloid cells by targeting Cebpa in vitro

Journal: Cancer Medicine

doi: 10.1002/cam4.1694

MiR‐486 inhibits expression of Cebpa . A, Expression of miR‐486 in TM ‐ MDSC s from spleens of both LLC ‐bearing mice and B16 melanoma‐bearing mice was higher than in control groups (their counterparts from spleens of tumor‐free mice) as determined by RT ‐ qPCR . B, Expression of Cebpa , which was predicted as one target of miR‐486 by bioinformatics, was lower in TM ‐ MDSC s from both LLC ‐bearing mice and B16 melanoma‐bearing mice than in control groups. C, Interaction between miR‐486 and Cebpa was detected using the Promega luciferase reporter assay, showing that the miR‐486 mimic, but not control mi RNA , significantly reduced renilla luciferase activity in wide type 3′‐ UTR Cebpa groups; however, neither the miR‐486 mimic, nor control mi RNA , significantly reduced renilla luciferase activity in mutated 3′‐ UTR Cebpa groups. D, Transfection efficiency in murine myeloid cells infected with vehicle lentivirus or miR‐486‐expressing lentivirus observed by a fluorescence microscope. Original magnification ×100. E, Murine myeloid cells were infected with vehicle lentivirus or miR‐486‐expressing lentivirus for 48 h. Expression of Cebpa in these cells was measured by western blot. The results also indicated that miR‐486 reduced the expression of Cebpa in myeloid cells. F, Overexpression of miR‐486 decreased the expression of Cebpa in myeloid cells as shown by RT ‐ qPCR . Three tumor‐bearing mice and five tumor‐free mice were used for each test in A and B. All data are presented as the mean ± SD and repeated three times. Error bars represent SD . Each red scatter plot overlaid onto the solid bar graphs indicates one technical repeat. For A and B, a two‐tailed Student's t test was used to compare data between two groups. For C and F, the P ‐values above the horizontal lines without vertical bars represent the results of one‐way ANOVA for three groups, and other P ‐values represent the results of the Scheff multiple‐comparison test when the P ‐values were lower than 0.05 in the one‐way ANOVA analyses
Figure Legend Snippet: MiR‐486 inhibits expression of Cebpa . A, Expression of miR‐486 in TM ‐ MDSC s from spleens of both LLC ‐bearing mice and B16 melanoma‐bearing mice was higher than in control groups (their counterparts from spleens of tumor‐free mice) as determined by RT ‐ qPCR . B, Expression of Cebpa , which was predicted as one target of miR‐486 by bioinformatics, was lower in TM ‐ MDSC s from both LLC ‐bearing mice and B16 melanoma‐bearing mice than in control groups. C, Interaction between miR‐486 and Cebpa was detected using the Promega luciferase reporter assay, showing that the miR‐486 mimic, but not control mi RNA , significantly reduced renilla luciferase activity in wide type 3′‐ UTR Cebpa groups; however, neither the miR‐486 mimic, nor control mi RNA , significantly reduced renilla luciferase activity in mutated 3′‐ UTR Cebpa groups. D, Transfection efficiency in murine myeloid cells infected with vehicle lentivirus or miR‐486‐expressing lentivirus observed by a fluorescence microscope. Original magnification ×100. E, Murine myeloid cells were infected with vehicle lentivirus or miR‐486‐expressing lentivirus for 48 h. Expression of Cebpa in these cells was measured by western blot. The results also indicated that miR‐486 reduced the expression of Cebpa in myeloid cells. F, Overexpression of miR‐486 decreased the expression of Cebpa in myeloid cells as shown by RT ‐ qPCR . Three tumor‐bearing mice and five tumor‐free mice were used for each test in A and B. All data are presented as the mean ± SD and repeated three times. Error bars represent SD . Each red scatter plot overlaid onto the solid bar graphs indicates one technical repeat. For A and B, a two‐tailed Student's t test was used to compare data between two groups. For C and F, the P ‐values above the horizontal lines without vertical bars represent the results of one‐way ANOVA for three groups, and other P ‐values represent the results of the Scheff multiple‐comparison test when the P ‐values were lower than 0.05 in the one‐way ANOVA analyses

Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Transfection, Infection, Fluorescence, Microscopy, Western Blot, Over Expression, Two Tailed Test

4) Product Images from "Molecular Cloning and Expression of MnGST-1 and MnGST-2 from Oriental River Prawn, Macrobrachium nipponense, in Response to Hypoxia and Reoxygenation"

Article Title: Molecular Cloning and Expression of MnGST-1 and MnGST-2 from Oriental River Prawn, Macrobrachium nipponense, in Response to Hypoxia and Reoxygenation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19103102

Expression of MnGST-1 and MnGST-2 in different tissues (E: eye; Br: brain; H: heart; He: hepatopancreas; G: gill; M: muscle; O: ovary; T: testis; Ag: abdominal ganglion) of Macrobrachium nipponense. Bar charts with different lowercase letters show significant differences in MnGST-1 and MnGST-2 ( p
Figure Legend Snippet: Expression of MnGST-1 and MnGST-2 in different tissues (E: eye; Br: brain; H: heart; He: hepatopancreas; G: gill; M: muscle; O: ovary; T: testis; Ag: abdominal ganglion) of Macrobrachium nipponense. Bar charts with different lowercase letters show significant differences in MnGST-1 and MnGST-2 ( p

Techniques Used: Expressing

Expression of MnGST-1 ( A ) and MnGST-2 ( B ) in the gill of M. nipponense at five time points after hypoxia and reoxygenation (H0: hypoxia for 0 h; H12: hypoxia for 12 h; H24: hypoxia for 24 h; R12: reoxygenation for 12 h; R24: reoxygenation for 24 h). Data indicated with asterisks are significantly different ( p
Figure Legend Snippet: Expression of MnGST-1 ( A ) and MnGST-2 ( B ) in the gill of M. nipponense at five time points after hypoxia and reoxygenation (H0: hypoxia for 0 h; H12: hypoxia for 12 h; H24: hypoxia for 24 h; R12: reoxygenation for 12 h; R24: reoxygenation for 24 h). Data indicated with asterisks are significantly different ( p

Techniques Used: Expressing

Phylogenetic tree based on GST sequences generated using the neighbor-joining method in the MEGA 5.10 program with 1000 bootstrap replicates. MnGST-1 and MnGST-2 were highlighted in red boxes.
Figure Legend Snippet: Phylogenetic tree based on GST sequences generated using the neighbor-joining method in the MEGA 5.10 program with 1000 bootstrap replicates. MnGST-1 and MnGST-2 were highlighted in red boxes.

Techniques Used: Generated

Expression of MnGST-1 ( A ) and MnGST-2 ( B ) in the hepatopancreas (He), gill (G) and muscle (M) of M. nipponense in response to chronic hypoxia stress experiment. Values are means ± SE for triplicate samples. Bars with different letters indicate significant differences ( p
Figure Legend Snippet: Expression of MnGST-1 ( A ) and MnGST-2 ( B ) in the hepatopancreas (He), gill (G) and muscle (M) of M. nipponense in response to chronic hypoxia stress experiment. Values are means ± SE for triplicate samples. Bars with different letters indicate significant differences ( p

Techniques Used: Expressing

Expression of MnGST-1 ( A ) and MnGST-2 ( B ) in the hepatopancreas of M. nipponense at five time points after hypoxia and reoxygenation (H0: hypoxia for 0 h; H12: hypoxia for 12 h; H24: hypoxia for 24 h; R12: reoxygenation for 12 h; R24: reoxygenation for 24 h). Data indicated with asterisks are significantly different ( p
Figure Legend Snippet: Expression of MnGST-1 ( A ) and MnGST-2 ( B ) in the hepatopancreas of M. nipponense at five time points after hypoxia and reoxygenation (H0: hypoxia for 0 h; H12: hypoxia for 12 h; H24: hypoxia for 24 h; R12: reoxygenation for 12 h; R24: reoxygenation for 24 h). Data indicated with asterisks are significantly different ( p

Techniques Used: Expressing

Expression of MnGST-1 ( A ) and MnGST-2 ( B ) in the muscle of M. nipponense at five time points after hypoxia and reoxygenation (H0: hypoxia for 0 h; H12: hypoxia for 12 h; H24: hypoxia for 24 h; R12: reoxygenation for 12 h; R24: reoxygenation for 24 h). Data indicated with asterisks are significantly different ( p
Figure Legend Snippet: Expression of MnGST-1 ( A ) and MnGST-2 ( B ) in the muscle of M. nipponense at five time points after hypoxia and reoxygenation (H0: hypoxia for 0 h; H12: hypoxia for 12 h; H24: hypoxia for 24 h; R12: reoxygenation for 12 h; R24: reoxygenation for 24 h). Data indicated with asterisks are significantly different ( p

Techniques Used: Expressing

5) Product Images from "Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome"

Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome

Journal: Clinical Epigenetics

doi: 10.1186/s13148-018-0546-4

CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies anti-GFP and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged DNMT1 protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein
Figure Legend Snippet: CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies anti-GFP and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged DNMT1 protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein

Techniques Used: Western Blot, Purification, Generated, Mutagenesis

6) Product Images from "GSK3β-mediated Ser156 phosphorylation modulates a BH3-like domain in BCL2L12 during TMZ-induced apoptosis and autophagy in glioma cells"

Article Title: GSK3β-mediated Ser156 phosphorylation modulates a BH3-like domain in BCL2L12 during TMZ-induced apoptosis and autophagy in glioma cells

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3672

Proposed mechanism of Ser156 phosphorylation as an allosteric site to modulate a BH3-like domain on BCL2L12 in glioma cells. When BCL2/BCL-XL interacts with the Beclin-1 BH3 domain, autophagy is inhibited. Overexpression of BCL2L12 may displace Beclin-1 in integrating with BCL2/BCL-XL via its BH3-like domain, leading to release of Beclin-1 and initiation of the autophagy process. In addition, since BCL2L12 occupies the hydrophobic groove of BCL2/BCL-XL, BH3 only BCL2 activator or sensitizer is unable to gain access, and the gross result of anti-apoptosis is observed. The BH3 domain mimetic agent, ABT-737, also binds to BCL2/BCL-XL, and hence competes and disrupts the interaction between BCL2/BCL-XL and BCL2L12, making tumor cells more vulnerable to apoptosis. Of note, GSK3β-mediated BCL2L12 S156 phosphorylation may affect BH3 domain function in glioma cells. BCL2L12, BCL2-like 12; BCL2, BCL2 apoptosis regulator; BCL-XL, BCL-extra large; GSK3β, glycogen synthase kinase 3β.
Figure Legend Snippet: Proposed mechanism of Ser156 phosphorylation as an allosteric site to modulate a BH3-like domain on BCL2L12 in glioma cells. When BCL2/BCL-XL interacts with the Beclin-1 BH3 domain, autophagy is inhibited. Overexpression of BCL2L12 may displace Beclin-1 in integrating with BCL2/BCL-XL via its BH3-like domain, leading to release of Beclin-1 and initiation of the autophagy process. In addition, since BCL2L12 occupies the hydrophobic groove of BCL2/BCL-XL, BH3 only BCL2 activator or sensitizer is unable to gain access, and the gross result of anti-apoptosis is observed. The BH3 domain mimetic agent, ABT-737, also binds to BCL2/BCL-XL, and hence competes and disrupts the interaction between BCL2/BCL-XL and BCL2L12, making tumor cells more vulnerable to apoptosis. Of note, GSK3β-mediated BCL2L12 S156 phosphorylation may affect BH3 domain function in glioma cells. BCL2L12, BCL2-like 12; BCL2, BCL2 apoptosis regulator; BCL-XL, BCL-extra large; GSK3β, glycogen synthase kinase 3β.

Techniques Used: Over Expression

7) Product Images from "Silkworm genetic sexing through W chromosome-linked, targeted gene integration"

Article Title: Silkworm genetic sexing through W chromosome-linked, targeted gene integration

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1810945115

Schematic representation of the B. mori W chromosome and donor plasmids. ( A ) Schematic representation of female-specific RAPD markers on the W chromosome (not to scale). The gray region represents the W chromosome. The black rectangles are the female-specific RAPD markers. A 50-bp fragment located in the Rikishi RAPD marker is the targeted site. The sequences in blue and underlined are the targets of the TALENs. A 17-bp sequence serves as a spacer between the two sites. ( B ) Schematic representation of the genomic target site and donor plasmid A. Donor A contains a cassette expressing DsRed2 under the control of the HR5-IE1 promoter and the SV40 polyadenylation site. Two homologous DNA fragments (L-homo and R-homo), each 1,000 bp in length at the 5′- and 3′-ends of the TALEN sites, were cloned separately into the left and right sides of the DsRed2 cassette. The two homologous fragments were both flanked by two of the 50-bp TALEN sites (blue). ( C ) Donor B contains two cassettes expressing EGFP under the control of an HR5-IE1 promoter and Cas9 driven by the embryo-specific nos promoter. Two homologous DNA fragments (L-homo and R-homo), each 1,000 bp in length at the 5′- and 3′-ends of the TALEN site, were cloned separately to the left of the Cas9 cassette and to the right of the EGFP cassette. All other descriptions are the same as in B .
Figure Legend Snippet: Schematic representation of the B. mori W chromosome and donor plasmids. ( A ) Schematic representation of female-specific RAPD markers on the W chromosome (not to scale). The gray region represents the W chromosome. The black rectangles are the female-specific RAPD markers. A 50-bp fragment located in the Rikishi RAPD marker is the targeted site. The sequences in blue and underlined are the targets of the TALENs. A 17-bp sequence serves as a spacer between the two sites. ( B ) Schematic representation of the genomic target site and donor plasmid A. Donor A contains a cassette expressing DsRed2 under the control of the HR5-IE1 promoter and the SV40 polyadenylation site. Two homologous DNA fragments (L-homo and R-homo), each 1,000 bp in length at the 5′- and 3′-ends of the TALEN sites, were cloned separately into the left and right sides of the DsRed2 cassette. The two homologous fragments were both flanked by two of the 50-bp TALEN sites (blue). ( C ) Donor B contains two cassettes expressing EGFP under the control of an HR5-IE1 promoter and Cas9 driven by the embryo-specific nos promoter. Two homologous DNA fragments (L-homo and R-homo), each 1,000 bp in length at the 5′- and 3′-ends of the TALEN site, were cloned separately to the left of the Cas9 cassette and to the right of the EGFP cassette. All other descriptions are the same as in B .

Techniques Used: Marker, TALENs, Sequencing, Plasmid Preparation, Expressing, Clone Assay

8) Product Images from "Microbial Diversity in Sediments from the Bottom of the Challenger Deep, the Mariana Trench"

Article Title: Microbial Diversity in Sediments from the Bottom of the Challenger Deep, the Mariana Trench

Journal: Microbes and Environments

doi: 10.1264/jsme2.ME17194

Matrices of UniFrac distances and Bray-Curtis and Jaccard dissimilarity indices of SSU rRNA gene communities obtained by tag sequencing for trench bottom sediments in the Challenger Deep, the Mariana Trench, shown by colored bars.
Figure Legend Snippet: Matrices of UniFrac distances and Bray-Curtis and Jaccard dissimilarity indices of SSU rRNA gene communities obtained by tag sequencing for trench bottom sediments in the Challenger Deep, the Mariana Trench, shown by colored bars.

Techniques Used: Sequencing

Composition of SSU rRNA gene tags of microbial communities from hadal water and trench bottom sediment (sediment core #AB11) in the Challenger Deep, the Mariana Trench.
Figure Legend Snippet: Composition of SSU rRNA gene tags of microbial communities from hadal water and trench bottom sediment (sediment core #AB11) in the Challenger Deep, the Mariana Trench.

Techniques Used:

Profiles of direct cell counts (A) and copy numbers of whole prokaryotic and archaeal SSU rRNA genes (B), amoA genes (C), and SSU rRNA genes of nitrite oxidizers (D) in sediment core #AB11 taken from the Challenger Deep, the Mariana Trench. Groups D and A and Beta (C) indicate the archaeal amoA of groups D and A and betaproteobacterial amoA , respectively. SFNLG (D) indicates the potential nitrite-oxidizing Subseafloor Nitrospina -Like Group.
Figure Legend Snippet: Profiles of direct cell counts (A) and copy numbers of whole prokaryotic and archaeal SSU rRNA genes (B), amoA genes (C), and SSU rRNA genes of nitrite oxidizers (D) in sediment core #AB11 taken from the Challenger Deep, the Mariana Trench. Groups D and A and Beta (C) indicate the archaeal amoA of groups D and A and betaproteobacterial amoA , respectively. SFNLG (D) indicates the potential nitrite-oxidizing Subseafloor Nitrospina -Like Group.

Techniques Used:

9) Product Images from "Expression of heterologous oxalate decarboxylase in HEK293 cells confers protection against oxalate induced oxidative stress as a therapeutic approach for calcium oxalate stone disease"

Article Title: Expression of heterologous oxalate decarboxylase in HEK293 cells confers protection against oxalate induced oxidative stress as a therapeutic approach for calcium oxalate stone disease

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

doi: 10.1080/14756366.2016.1256884

(A) Expression of oxdC gene in HEK 293 cells, confirmed by Semi-quantitative PCR. Lane M, 1 kb ladder; lane 1, Empty vector; lanes 2, 3, 4, 5, 1.2 kb amplified product of oxdC . (B) The results of protein immunoblot analysis. Lane 1, HEK293 cells, lane 2, HEK293/pcDNA cells, lane 3, HEK293/pcDNAOXDC expressed protein corresponding to size 44 kDa detected in the stably transfected cells. (C) HEK293 cells were transfected with pEGFP-N1 (empty vector) and (D) pOXDC-EGFP. At 48 h, pEGFP-N1 (empty vector) and pOXDC-EGFP transfected cells exhibited bright green fluorescence and fusion proteins (OXDC-EGFP) were located in the cytoplasm. (E) Cell viability of HEK293/pcDNA and HEK293/pcDNAOXDC cells exposed to oxalate (750 μM for 18 h) was determined by MTT assay. (F) Trypan Blue Exclusion assay for determination of cytotoxicity effect of oxalate stress. (G) Live and dead cell population of HEK293/pcDNAOXDC following exposure to oxalate stress by flow cytometry analysis. (H) Bar diagram shows the % of live and dead cells following oxalate treatment. Each experiment was repeated a minimum of three independent times. Continuous line – HEK293/pcDNA (No stress); Dotted line – HEK293/pcDNA (Oxalate stress); Dashed line – HEK293/pcDNAOXDC (Oxalate stress). a* – significant difference from HEK293/pcDNA cells without oxalate stress. b* – significant difference from HEK293/pcDNA cells with oxalate stress. Values are statistically significant at p
Figure Legend Snippet: (A) Expression of oxdC gene in HEK 293 cells, confirmed by Semi-quantitative PCR. Lane M, 1 kb ladder; lane 1, Empty vector; lanes 2, 3, 4, 5, 1.2 kb amplified product of oxdC . (B) The results of protein immunoblot analysis. Lane 1, HEK293 cells, lane 2, HEK293/pcDNA cells, lane 3, HEK293/pcDNAOXDC expressed protein corresponding to size 44 kDa detected in the stably transfected cells. (C) HEK293 cells were transfected with pEGFP-N1 (empty vector) and (D) pOXDC-EGFP. At 48 h, pEGFP-N1 (empty vector) and pOXDC-EGFP transfected cells exhibited bright green fluorescence and fusion proteins (OXDC-EGFP) were located in the cytoplasm. (E) Cell viability of HEK293/pcDNA and HEK293/pcDNAOXDC cells exposed to oxalate (750 μM for 18 h) was determined by MTT assay. (F) Trypan Blue Exclusion assay for determination of cytotoxicity effect of oxalate stress. (G) Live and dead cell population of HEK293/pcDNAOXDC following exposure to oxalate stress by flow cytometry analysis. (H) Bar diagram shows the % of live and dead cells following oxalate treatment. Each experiment was repeated a minimum of three independent times. Continuous line – HEK293/pcDNA (No stress); Dotted line – HEK293/pcDNA (Oxalate stress); Dashed line – HEK293/pcDNAOXDC (Oxalate stress). a* – significant difference from HEK293/pcDNA cells without oxalate stress. b* – significant difference from HEK293/pcDNA cells with oxalate stress. Values are statistically significant at p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Amplification, Western Blot, Stable Transfection, Transfection, Fluorescence, MTT Assay, Trypan Blue Exclusion Assay, Flow Cytometry, Cytometry

10) Product Images from "Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression"

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression

Journal: Molecular Cancer

doi: 10.1186/s12943-019-0969-3

circAKT3 expression is increased in CDDP-resistant GC cells and tissues. a Validated expression of 10 circRNAs in the tissues from 44 GC patients using RT-qPCR. b Expression levels of circAKT3 in CDDP-resistant and their matched sensitive parental cell lines (SGC7901CDDP, BGC823CDDP, SGC7901 and BGC823) normalized to GAPDH expression. c The existence of circAKT3 was validated by Sanger sequencing. The red arrow shows the “head-to-tail” splicing sites of circAKT3. d The existence of circAKT3 was validated in SGC7901CDDP and BGC823CDDP cell lines by RT-PCR. Divergent primers amplified circAKT3 in cDNA but not in genomic DNA (gDNA). GAPDH served as a negative control. e RNA from SGC7901CDDP and BGC823CDDP cells was treated with or without RNase R for RT-qPCR. The relative levels of circAKT3 and AKT3 mRNA were normalized to the values measured in the mock-treated cells. f Levels of small nucleolar RNA (U6, as a positive control for the nuclear fraction), GAPDH (positive control for cytoplasmic fraction), AKT3 mRNA and circRNAs from the nuclear and cytoplasmic fractions of SGC7901CDDP cells. g RNA stability of circular and linear transcripts of AKT3 and of 18S rRNA in SGC7901CDDP cells. h Representative images of RNA FISH of circAKT3 expression in SGC7901CDDP cells, which show that circAKT3 is predominantly localized to the cytoplasm. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P
Figure Legend Snippet: circAKT3 expression is increased in CDDP-resistant GC cells and tissues. a Validated expression of 10 circRNAs in the tissues from 44 GC patients using RT-qPCR. b Expression levels of circAKT3 in CDDP-resistant and their matched sensitive parental cell lines (SGC7901CDDP, BGC823CDDP, SGC7901 and BGC823) normalized to GAPDH expression. c The existence of circAKT3 was validated by Sanger sequencing. The red arrow shows the “head-to-tail” splicing sites of circAKT3. d The existence of circAKT3 was validated in SGC7901CDDP and BGC823CDDP cell lines by RT-PCR. Divergent primers amplified circAKT3 in cDNA but not in genomic DNA (gDNA). GAPDH served as a negative control. e RNA from SGC7901CDDP and BGC823CDDP cells was treated with or without RNase R for RT-qPCR. The relative levels of circAKT3 and AKT3 mRNA were normalized to the values measured in the mock-treated cells. f Levels of small nucleolar RNA (U6, as a positive control for the nuclear fraction), GAPDH (positive control for cytoplasmic fraction), AKT3 mRNA and circRNAs from the nuclear and cytoplasmic fractions of SGC7901CDDP cells. g RNA stability of circular and linear transcripts of AKT3 and of 18S rRNA in SGC7901CDDP cells. h Representative images of RNA FISH of circAKT3 expression in SGC7901CDDP cells, which show that circAKT3 is predominantly localized to the cytoplasm. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P

Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Fluorescence In Situ Hybridization, Staining

11) Product Images from "The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning"

Article Title: The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning

Journal: eLife

doi: 10.7554/eLife.39748

Effect of modified forms of the Svb protein on epidermal trichome formation. UAS-GFP (control) ( A,A’ ), UAS-Svb-ACT ( B,B’ ) and UAS-Svb-3Kmut ( C,C’ ) were expressed in the embryonic epidermis under the control of the ptc-Gal4 driver. Top rows show whole embryo cuticles ( A–C ), the bottom row shows close-ups in the ventral region of the third abdominal segment ( A’–C’ ). Svb-ACT, which lacks the N-terminal repressor domain and thus mimics the processed form of Svb, acts as a constitutive activator of transcription and triggers the production of ectopic trichomes. In contrast, Svb-3Kmut -bearing mutations on the 3 Lysines ubiquitinated by Ubr3 in response to Tal peptides- behaves as a repressor and counteracts endogenous Svb activity, resulting in loss of trichomes.
Figure Legend Snippet: Effect of modified forms of the Svb protein on epidermal trichome formation. UAS-GFP (control) ( A,A’ ), UAS-Svb-ACT ( B,B’ ) and UAS-Svb-3Kmut ( C,C’ ) were expressed in the embryonic epidermis under the control of the ptc-Gal4 driver. Top rows show whole embryo cuticles ( A–C ), the bottom row shows close-ups in the ventral region of the third abdominal segment ( A’–C’ ). Svb-ACT, which lacks the N-terminal repressor domain and thus mimics the processed form of Svb, acts as a constitutive activator of transcription and triggers the production of ectopic trichomes. In contrast, Svb-3Kmut -bearing mutations on the 3 Lysines ubiquitinated by Ubr3 in response to Tal peptides- behaves as a repressor and counteracts endogenous Svb activity, resulting in loss of trichomes.

Techniques Used: Modification, Activated Clotting Time Assay, Activity Assay

Schematic representation of Tc-shavenbaby locus ( A ) and transcript ( B ), showing the site at which a GFP-containing marker plasmid was inserted by CRISPR/cas9 genome editing (see also Materials and methods). The mutagenic cassette is inserted into exon 2, that is within the open reading frame upstream of the region encoding the DNA-binding zinc finger domain. Gene disruption leads to mRNA truncation after the insertion site, since Tc-svb expression is absent in homozygous mutants. In addition to segmentation defects and transformation toward thoracic identity, other phenotypes observed in Tc-svb CRISPR mutants include incipient spiracles (possibly a secondary effect of cuticle thinning leading to a defect in the development of tracheal rings); sensory bristles that are shorter and thicker; leg segment boundaries that are not clearly defined; missing leg bristles; unsclerotized pretarsi with soft, rounded apices; and antennae lacking the terminal setae. Therefore, late functions of Tc-svb in epidermal and appendage differentiation are strongly affected in Tc-svb CRISPR mutant embryos, while the segmentation phenotype is milder that Tc-svb -RNAi knockdown due to maternal contribution of Tc-svb (Ray et al., in preparation).
Figure Legend Snippet: Schematic representation of Tc-shavenbaby locus ( A ) and transcript ( B ), showing the site at which a GFP-containing marker plasmid was inserted by CRISPR/cas9 genome editing (see also Materials and methods). The mutagenic cassette is inserted into exon 2, that is within the open reading frame upstream of the region encoding the DNA-binding zinc finger domain. Gene disruption leads to mRNA truncation after the insertion site, since Tc-svb expression is absent in homozygous mutants. In addition to segmentation defects and transformation toward thoracic identity, other phenotypes observed in Tc-svb CRISPR mutants include incipient spiracles (possibly a secondary effect of cuticle thinning leading to a defect in the development of tracheal rings); sensory bristles that are shorter and thicker; leg segment boundaries that are not clearly defined; missing leg bristles; unsclerotized pretarsi with soft, rounded apices; and antennae lacking the terminal setae. Therefore, late functions of Tc-svb in epidermal and appendage differentiation are strongly affected in Tc-svb CRISPR mutant embryos, while the segmentation phenotype is milder that Tc-svb -RNAi knockdown due to maternal contribution of Tc-svb (Ray et al., in preparation).

Techniques Used: Marker, Plasmid Preparation, CRISPR, Binding Assay, Expressing, Transformation Assay, Mutagenesis

12) Product Images from "Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum"

Article Title: Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum

Journal: Molecules

doi: 10.3390/molecules200610110

The detection of recombinant in the two transformations by PCR amplification. ( a ) The detection of recombinant in the first transformation by PCR amplification with primers Cm-F/R. M: Trans DNA Marker II. 1: Negative control, the PCR amplification band of Cm r gene with the genomic DNA of A. limacinum OUC88 as the template. 2: The PCR amplification band of Cm r gene with the genomic DNA of A. limacinum OUC_CG as the template. 3: Positive control, the PCR amplification band of Cm r gene with the plasmid pACYCDuet-1 as the template. B: Blank control; ( b ) The detection of recombinant in the second transformation by PCR amplification with primers 18S+-F/P pgk -R. M: Trans 15K DNA Marker. 1: The PCR amplification band with genomic DNA of A. limacinum OUC88 as the template; 2: The PCR amplification band (3655 bp) of 18S+- lox P- Cm r - lox P-P pgk with genomic DNA of A. limacinum OUC_CG as the template; 3: The PCR amplification band (2185 bp) of 18S+- lox P- lox P-P pgk with genomic DNA of A. limacinum OUC_EG as the template; B: Blank control.
Figure Legend Snippet: The detection of recombinant in the two transformations by PCR amplification. ( a ) The detection of recombinant in the first transformation by PCR amplification with primers Cm-F/R. M: Trans DNA Marker II. 1: Negative control, the PCR amplification band of Cm r gene with the genomic DNA of A. limacinum OUC88 as the template. 2: The PCR amplification band of Cm r gene with the genomic DNA of A. limacinum OUC_CG as the template. 3: Positive control, the PCR amplification band of Cm r gene with the plasmid pACYCDuet-1 as the template. B: Blank control; ( b ) The detection of recombinant in the second transformation by PCR amplification with primers 18S+-F/P pgk -R. M: Trans 15K DNA Marker. 1: The PCR amplification band with genomic DNA of A. limacinum OUC88 as the template; 2: The PCR amplification band (3655 bp) of 18S+- lox P- Cm r - lox P-P pgk with genomic DNA of A. limacinum OUC_CG as the template; 3: The PCR amplification band (2185 bp) of 18S+- lox P- lox P-P pgk with genomic DNA of A. limacinum OUC_EG as the template; B: Blank control.

Techniques Used: Recombinant, Polymerase Chain Reaction, Amplification, Transformation Assay, Marker, Negative Control, Positive Control, Plasmid Preparation

Southern blotting analysis of egfp in the genomic DNA of transformants ( a ) The results of total DNA digested by restriction enzymes. Molecular-size of markers (bp) is shown on the left. The genomic DNA of A. limacinum OUC88 (WT), A. limacinum OUC_CG and A. limacinum OUC_EG were double digested with either Bam HI/ Eco RI or Hin dIII/ Xba I; ( b ) The southern-blot hybridization results for the egfp gene. The number of fragment copies as deduced from the comparison of the hybridization bands are depicted for the transformants.
Figure Legend Snippet: Southern blotting analysis of egfp in the genomic DNA of transformants ( a ) The results of total DNA digested by restriction enzymes. Molecular-size of markers (bp) is shown on the left. The genomic DNA of A. limacinum OUC88 (WT), A. limacinum OUC_CG and A. limacinum OUC_EG were double digested with either Bam HI/ Eco RI or Hin dIII/ Xba I; ( b ) The southern-blot hybridization results for the egfp gene. The number of fragment copies as deduced from the comparison of the hybridization bands are depicted for the transformants.

Techniques Used: Southern Blot, Hybridization

13) Product Images from "Development of a Novel Antimicrobial Screening System Targeting the Pyoverdine-Mediated Iron Acquisition System and Xenobiotic Efflux Pumps"

Article Title: Development of a Novel Antimicrobial Screening System Targeting the Pyoverdine-Mediated Iron Acquisition System and Xenobiotic Efflux Pumps

Journal: Molecules

doi: 10.3390/molecules20057790

Absorption spectra of the culture supernatant. Cells were grown in casamino acid medium overnight at 37 °C under aerobic conditions and removed by the centrifugation at 13,700× g for 10 min. The clear supernatant fractions were scanned with a spectrophotometer. ( A ), PAO4290 (wild-type); ( B ), TNP072 (Δ oprM ); ( C ), TNP091 (Δ oprM , fpvA :: oprM ); ( D ), TNP092 (Δ oprM , fpvA :: oprM , Δ pvdS ).
Figure Legend Snippet: Absorption spectra of the culture supernatant. Cells were grown in casamino acid medium overnight at 37 °C under aerobic conditions and removed by the centrifugation at 13,700× g for 10 min. The clear supernatant fractions were scanned with a spectrophotometer. ( A ), PAO4290 (wild-type); ( B ), TNP072 (Δ oprM ); ( C ), TNP091 (Δ oprM , fpvA :: oprM ); ( D ), TNP092 (Δ oprM , fpvA :: oprM , Δ pvdS ).

Techniques Used: Centrifugation, Spectrophotometry

Western blot analysis of the cells grown under iron-replete and iron-depleted conditions. Cells were grown in the absence (lanes 1 to 4) or presence (lanes 5 to 9) of 0.5 mM dipyridyl and the whole cell lysate was subjected to SDS-polyacrylamide gel (10%) electrophoresis followed by visualization with an anti-OprM antibody. The position of OprM is indicated by an arrowhead. Lanes 1 and 5, PAO4290 (wild-type); lanes 2 and 6, TNP072 (Δ oprM ); lanes 3 and 7, TNP091 (Δ oprM , fpvA :: oprM ); lanes 4 and 8, TNP092 (Δ oprM , fpvA :: oprM , Δ pvdS ); lane 9, TNP092 (pPvdS).
Figure Legend Snippet: Western blot analysis of the cells grown under iron-replete and iron-depleted conditions. Cells were grown in the absence (lanes 1 to 4) or presence (lanes 5 to 9) of 0.5 mM dipyridyl and the whole cell lysate was subjected to SDS-polyacrylamide gel (10%) electrophoresis followed by visualization with an anti-OprM antibody. The position of OprM is indicated by an arrowhead. Lanes 1 and 5, PAO4290 (wild-type); lanes 2 and 6, TNP072 (Δ oprM ); lanes 3 and 7, TNP091 (Δ oprM , fpvA :: oprM ); lanes 4 and 8, TNP092 (Δ oprM , fpvA :: oprM , Δ pvdS ); lane 9, TNP092 (pPvdS).

Techniques Used: Western Blot, Electrophoresis

14) Product Images from "Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators"

Article Title: Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators

Journal: Nature Communications

doi: 10.1038/s41467-018-08150-5

Cell line panel screen of drug sensitivity identifies BCL2L1 expression as a marker of insensitivity to E7107. a Top five genes whose mRNA expression positively correlated with maximum effect of cell killing (Emax) of E7107 profiled in 478 cancer cell lines. Pearson’s correlation coefficient R and p values were calculated using R package Hmisc 4.1-0. Lower Emax value indicates more robust cell killing activity. b Heatmap demonstrating the positive correlation between Emax of E7107 and BCL2L1 mRNA expression in 478 cancer cell lines. The heatmap was sorted by Emax of E7107 in cancer cell lines from high (less sensitive) to low (more sensitive). c Box plots showing the distribution of Emax (%) of E7107 in two groups of profiled cell lines defined by expression levels of each BCL2 family genes. For each BCL2 family genes, cell lines with expression level higher than the third quartile are classified into “high” and cell lines with expression level lower than the first quartile are classified into “low.” p Values were calculated using Student’s t test. d Growth-inhibitory activity of E7107 in lung cancer cell lines A549 and NCIH1568 upon doxycycline-induced shRNA knockdown of BCL2L1 . Left, Western blot analysis of BCLxL(encoded by BCL2L1 ) knockdown; Right, Growth curves of two cell lines measured by CellTiter-Glo. Data represent means ± SD of biological triplicates. e Growth-inhibitory activity of E7107 in lung cancer cell lines NCIH2110 and NCIH1568 upon stable cDNA expression of BCL2L1 (BCLxL). Left, Western blot analysis of BCLxL(encoded by BCL2L1 ) overexpression; Right, Growth curves of two cell lines measured by CellTiter-Glo. Data represent means ± SD of biological triplicates
Figure Legend Snippet: Cell line panel screen of drug sensitivity identifies BCL2L1 expression as a marker of insensitivity to E7107. a Top five genes whose mRNA expression positively correlated with maximum effect of cell killing (Emax) of E7107 profiled in 478 cancer cell lines. Pearson’s correlation coefficient R and p values were calculated using R package Hmisc 4.1-0. Lower Emax value indicates more robust cell killing activity. b Heatmap demonstrating the positive correlation between Emax of E7107 and BCL2L1 mRNA expression in 478 cancer cell lines. The heatmap was sorted by Emax of E7107 in cancer cell lines from high (less sensitive) to low (more sensitive). c Box plots showing the distribution of Emax (%) of E7107 in two groups of profiled cell lines defined by expression levels of each BCL2 family genes. For each BCL2 family genes, cell lines with expression level higher than the third quartile are classified into “high” and cell lines with expression level lower than the first quartile are classified into “low.” p Values were calculated using Student’s t test. d Growth-inhibitory activity of E7107 in lung cancer cell lines A549 and NCIH1568 upon doxycycline-induced shRNA knockdown of BCL2L1 . Left, Western blot analysis of BCLxL(encoded by BCL2L1 ) knockdown; Right, Growth curves of two cell lines measured by CellTiter-Glo. Data represent means ± SD of biological triplicates. e Growth-inhibitory activity of E7107 in lung cancer cell lines NCIH2110 and NCIH1568 upon stable cDNA expression of BCL2L1 (BCLxL). Left, Western blot analysis of BCLxL(encoded by BCL2L1 ) overexpression; Right, Growth curves of two cell lines measured by CellTiter-Glo. Data represent means ± SD of biological triplicates

Techniques Used: Expressing, Marker, Activity Assay, shRNA, Western Blot, Over Expression

15) Product Images from "Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic Chlamydomonas reinhardtii"

Article Title: Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic Chlamydomonas reinhardtii

Journal: Marine Drugs

doi: 10.3390/md16120493

mRNA expression level of klk1 in transgenic C. reinhardtii (sgk) cells under heat shock and intense light conditions. The relative transcription levels of klk1 were determined after 0, 30, 60, 90, 120, 150, and 180 min by qRTPCR. β-actin was used as a reference gene and the values were normalized to the transcript levels in the control. Data are averages of biological triplicate, and the error bars represent standard deviation. One asterisk, p
Figure Legend Snippet: mRNA expression level of klk1 in transgenic C. reinhardtii (sgk) cells under heat shock and intense light conditions. The relative transcription levels of klk1 were determined after 0, 30, 60, 90, 120, 150, and 180 min by qRTPCR. β-actin was used as a reference gene and the values were normalized to the transcript levels in the control. Data are averages of biological triplicate, and the error bars represent standard deviation. One asterisk, p

Techniques Used: Expressing, Transgenic Assay, Standard Deviation

PCR-electrophoresis result for the sgk genome ( klk1 genome with upstream signal peptide genome) detection. Marker, DL-2000; 1, non-transfected cc-503; 2, sgk-1; 3, sgk-2; 4, sgk-3; 5, negative control (water).
Figure Legend Snippet: PCR-electrophoresis result for the sgk genome ( klk1 genome with upstream signal peptide genome) detection. Marker, DL-2000; 1, non-transfected cc-503; 2, sgk-1; 3, sgk-2; 4, sgk-3; 5, negative control (water).

Techniques Used: Polymerase Chain Reaction, Electrophoresis, Marker, Transfection, Negative Control

Construction of pHSgk124 plasmid. Abbreviations: sg , signal peptide; klk1 , human tissue kallikrein 1 gene; ble , bleomycin resistance gene; PmaC I, enzyme cleavage point 1; Bgl II, enzyme cleavage point 2.
Figure Legend Snippet: Construction of pHSgk124 plasmid. Abbreviations: sg , signal peptide; klk1 , human tissue kallikrein 1 gene; ble , bleomycin resistance gene; PmaC I, enzyme cleavage point 1; Bgl II, enzyme cleavage point 2.

Techniques Used: Plasmid Preparation

16) Product Images from "Molecular characterisation of the NDM-1-encoding plasmid p2189-NDM in an Escherichia coli ST410 clinical isolate from Ghana"

Article Title: Molecular characterisation of the NDM-1-encoding plasmid p2189-NDM in an Escherichia coli ST410 clinical isolate from Ghana

Journal: PLoS ONE

doi: 10.1371/journal.pone.0209623

S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) of the donor strain EC2189, trans-conjugant TcEC2189 and recipient C600. (A) PFGE of genomic DNA digested with S1-nuclease. (B) Southern blot hybridisation of the PFGE gel with a bla NDM-1 specific probe. Lane M: Lambda ladder; Lane 1: EC2189; Lane 2: TcEC2189; Lane 3: C600.
Figure Legend Snippet: S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) of the donor strain EC2189, trans-conjugant TcEC2189 and recipient C600. (A) PFGE of genomic DNA digested with S1-nuclease. (B) Southern blot hybridisation of the PFGE gel with a bla NDM-1 specific probe. Lane M: Lambda ladder; Lane 1: EC2189; Lane 2: TcEC2189; Lane 3: C600.

Techniques Used: Pulsed-Field Gel, Electrophoresis, Southern Blot, Hybridization

17) Product Images from "A plasma membrane localized protein phosphatase in Toxoplasma gondii, PPM5C, regulates attachment to host cells"

Article Title: A plasma membrane localized protein phosphatase in Toxoplasma gondii, PPM5C, regulates attachment to host cells

Journal: Scientific Reports

doi: 10.1038/s41598-019-42441-1

Schematic of five putative membrane associated protein phosphatases. Predicted functional domains and protein modification sites are shown for PPM2A (TGGT1_232340), PPM2B (TGGT1_267100), PPM3D (TGGT1_202610), PPM5C (TGGT1_281580), PPM11C (TGGT1_304955). Below PPM11C are the two putative proteins encoded genomic locus of TGGT1_304955. M, myristoylation; P, palmitoylation; SP, signal peptide; TM, transmembrane domain; PP2Cc, PP2C phosphatase catalytic domain.
Figure Legend Snippet: Schematic of five putative membrane associated protein phosphatases. Predicted functional domains and protein modification sites are shown for PPM2A (TGGT1_232340), PPM2B (TGGT1_267100), PPM3D (TGGT1_202610), PPM5C (TGGT1_281580), PPM11C (TGGT1_304955). Below PPM11C are the two putative proteins encoded genomic locus of TGGT1_304955. M, myristoylation; P, palmitoylation; SP, signal peptide; TM, transmembrane domain; PP2Cc, PP2C phosphatase catalytic domain.

Techniques Used: Functional Assay, Modification

18) Product Images from "Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells"

Article Title: Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

Journal: Protein & Cell

doi: 10.1007/s13238-019-0608-1

Generation and characterization of WT iPSCs and CADASIL iPSCs. (A) Schematic procedures for establishing iPSC-based CADASIL disease model. Fibroblasts obtained from one CADASIL patient and two healthy controls were reprogrammed into iPSCs. The iPSCs were then differentiated to generate VSMCs and VECs. Changes in disease-associated transcriptional profiling and cellular phenotypes were analyzed. (B) Confirmation of the heterozygous mutation of NOTCH3 (c.3226C > T, p.R1076C) in CADASIL iPSCs by DNA sequencing (right). Phase-contrast images of fibroblasts (left) and fibroblast-derived iPSCs (middle). Scale bar of fibroblasts, 50 μm; Scale bar of iPSCs, 100 μm. (C) RT-PCR of pluripotency markers, SOX2 , OCT4 , and NANOG . Human ESCs (hESCs) were used as positive controls and human fibroblasts as negative controls. (D) Immunofluorescence staining of pluripotency markers, NANOG, SOX2, and OCT4. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. (E) Immunofluorescence staining of TUJ1 (ectoderm), α-SMA (mesoderm), and FOXA2 (endoderm) in teratomas derived from WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 50 μm. (F) DNA methylation analysis of the OCT4 promoter in WT and CADASIL iPSCs. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively ( n = 7). (G) Karyotyping analysis of WT and CADASIL iPSCs. (H) Clonal expansion analysis of WT and CADASIL iPSCs. Representative images of crystal violet staining are shown to the left. The statistical analyses of relative clonal expansion abilities are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (I) Immunofluorescence staining of Ki67 in WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. The relative percentages of Ki67-positive cells are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (J) Cell cycle analysis of WT and CADASIL iPSCs. Data are presented as mean ± SD, n = 3. NS, not significant
Figure Legend Snippet: Generation and characterization of WT iPSCs and CADASIL iPSCs. (A) Schematic procedures for establishing iPSC-based CADASIL disease model. Fibroblasts obtained from one CADASIL patient and two healthy controls were reprogrammed into iPSCs. The iPSCs were then differentiated to generate VSMCs and VECs. Changes in disease-associated transcriptional profiling and cellular phenotypes were analyzed. (B) Confirmation of the heterozygous mutation of NOTCH3 (c.3226C > T, p.R1076C) in CADASIL iPSCs by DNA sequencing (right). Phase-contrast images of fibroblasts (left) and fibroblast-derived iPSCs (middle). Scale bar of fibroblasts, 50 μm; Scale bar of iPSCs, 100 μm. (C) RT-PCR of pluripotency markers, SOX2 , OCT4 , and NANOG . Human ESCs (hESCs) were used as positive controls and human fibroblasts as negative controls. (D) Immunofluorescence staining of pluripotency markers, NANOG, SOX2, and OCT4. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. (E) Immunofluorescence staining of TUJ1 (ectoderm), α-SMA (mesoderm), and FOXA2 (endoderm) in teratomas derived from WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 50 μm. (F) DNA methylation analysis of the OCT4 promoter in WT and CADASIL iPSCs. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively ( n = 7). (G) Karyotyping analysis of WT and CADASIL iPSCs. (H) Clonal expansion analysis of WT and CADASIL iPSCs. Representative images of crystal violet staining are shown to the left. The statistical analyses of relative clonal expansion abilities are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (I) Immunofluorescence staining of Ki67 in WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. The relative percentages of Ki67-positive cells are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (J) Cell cycle analysis of WT and CADASIL iPSCs. Data are presented as mean ± SD, n = 3. NS, not significant

Techniques Used: Mutagenesis, DNA Sequencing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, DNA Methylation Assay, Methylation, Cell Cycle Assay

19) Product Images from "ZafA Gene Is Important for Trichophyton mentagrophytes Growth and Pathogenicity"

Article Title: ZafA Gene Is Important for Trichophyton mentagrophytes Growth and Pathogenicity

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20040848

In vitro biodegradation of hair. The hair biodegradation test examined the susceptibility of different animal hair to the test strains of T. mentagrophytes . No obvious traces of pathological changes or decomposition were found in human and dog hair. The fox and feline hair were highly susceptible to T. mentagrophytes , and the rabbit hair was slightly susceptible to T. mentagrophytes . The decomposition significantly reduced on fox and cat’s hair in ZafA-hph, the pathological changes or decomposition on fox and cat’s hair have no obvious difference in ZafA+bar and the wild-type T. mentagrophytes strain.
Figure Legend Snippet: In vitro biodegradation of hair. The hair biodegradation test examined the susceptibility of different animal hair to the test strains of T. mentagrophytes . No obvious traces of pathological changes or decomposition were found in human and dog hair. The fox and feline hair were highly susceptible to T. mentagrophytes , and the rabbit hair was slightly susceptible to T. mentagrophytes . The decomposition significantly reduced on fox and cat’s hair in ZafA-hph, the pathological changes or decomposition on fox and cat’s hair have no obvious difference in ZafA+bar and the wild-type T. mentagrophytes strain.

Techniques Used: In Vitro

( A ): The polymerase chain reaction results of hph gene (1200 bp) amplification; the inserted hph gene was detected with primers hph -F and hph -R. Lane 1: pDHt/ZafA::hph , Lane 2: ZafA-hph, Lane 3: ZafA+bar, Lane 4: The wild-type T. mentagrophytes strain. ( B ): The polymerase chain reaction results of bar (900 bp) gene amplification, the inserted bar gene was detected with primers bar- F and bar -R. Lane 1: pDHt/ZafA-bar , Lane 2: ZafA+bar, Lane 3: pDHt/ZafA::hph , Lane 4: ZafA-hph, Lane 5: The wild-type T. mentagrophytes strain. ( C ): The polymerase chain reaction results of ZafA gene (400 bp) amplification, the inserted ZafA gene was detected with primers ZafA q-F and ZafA q-R. Lane 1: The wild-type T. mentagrophytes strain, Lane 2: pDHt/ZafA-bar , Lane 3: ZafA+bar, Lane 4: pDHt/ZafA::hph , Lane 5: ZafA-hph. ( D ): The Southern blot results of ZafA gene, in which a partial ZafA gene fragment (304 bp) was used as a hybridization probe for detecting the deleted gene fragment. Lane 1: The wild-type T. mentagrophytes strain, Lane 2: pDHt/ZafA::hph , Lane 3: ZafA-hph, Lane M: DNA Molecular-Weight Marker, Lane 4: The wild-type T. mentagrophytes strain, Lane 5: pDHt/ZafA-bar , Lane 6: ZafA+bar, Lane 7: pDHt/ZafA::hph , Lane 8: ZafA-hph, Lane C: Positive fragments.
Figure Legend Snippet: ( A ): The polymerase chain reaction results of hph gene (1200 bp) amplification; the inserted hph gene was detected with primers hph -F and hph -R. Lane 1: pDHt/ZafA::hph , Lane 2: ZafA-hph, Lane 3: ZafA+bar, Lane 4: The wild-type T. mentagrophytes strain. ( B ): The polymerase chain reaction results of bar (900 bp) gene amplification, the inserted bar gene was detected with primers bar- F and bar -R. Lane 1: pDHt/ZafA-bar , Lane 2: ZafA+bar, Lane 3: pDHt/ZafA::hph , Lane 4: ZafA-hph, Lane 5: The wild-type T. mentagrophytes strain. ( C ): The polymerase chain reaction results of ZafA gene (400 bp) amplification, the inserted ZafA gene was detected with primers ZafA q-F and ZafA q-R. Lane 1: The wild-type T. mentagrophytes strain, Lane 2: pDHt/ZafA-bar , Lane 3: ZafA+bar, Lane 4: pDHt/ZafA::hph , Lane 5: ZafA-hph. ( D ): The Southern blot results of ZafA gene, in which a partial ZafA gene fragment (304 bp) was used as a hybridization probe for detecting the deleted gene fragment. Lane 1: The wild-type T. mentagrophytes strain, Lane 2: pDHt/ZafA::hph , Lane 3: ZafA-hph, Lane M: DNA Molecular-Weight Marker, Lane 4: The wild-type T. mentagrophytes strain, Lane 5: pDHt/ZafA-bar , Lane 6: ZafA+bar, Lane 7: pDHt/ZafA::hph , Lane 8: ZafA-hph, Lane C: Positive fragments.

Techniques Used: Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization, Molecular Weight, Marker

Animal skin inoculation test. Histopathological analysis of sections of skin infected with three strains. Sections were stained with hematoxylin–eosin (HE). Compared to normal rabbit skin, there were different degrees of thickening of the stratum corneum and the stratum spinosum layers (as shown by the black line), with an initial infiltration of inflammatory cells (as shown by the black arrow) following the inoculation of the wild-type T. mentagrophytes strain and ZafA+bar. However, the rabbit skin had a very slight lesion following inoculation with ZafA-hph, while there was no obvious difference with the normal skin.
Figure Legend Snippet: Animal skin inoculation test. Histopathological analysis of sections of skin infected with three strains. Sections were stained with hematoxylin–eosin (HE). Compared to normal rabbit skin, there were different degrees of thickening of the stratum corneum and the stratum spinosum layers (as shown by the black line), with an initial infiltration of inflammatory cells (as shown by the black arrow) following the inoculation of the wild-type T. mentagrophytes strain and ZafA+bar. However, the rabbit skin had a very slight lesion following inoculation with ZafA-hph, while there was no obvious difference with the normal skin.

Techniques Used: Infection, Staining

The growth ability of wild-type T. mentagrophytes strain, ZafA-hph, and ZafA+bar. Sabouraud dextrose agar (SDA) medium with one mM of N,N,N′,N′-tetrakis (2-pyridinylmethyl)-1,2-ethanediamine (TPEN) was supplemented to generate zinc deficient SDA medium, which was named SDA-Zn. The wild-type T. mentagrophytes strain, ZafA-hph, and ZafA+bar were inoculated to SDA medium (grouped into Norm) and SDA-Zn medium with 200 μM, 400 μM, 600 μM, 800 μM, and 1000 μM of zinc sulfate (grouped into Zn200, Zn400, Zn600, Zn800, and Zn1000) respectively. In SDA-Zn medium, there was no significant difference between the wild-type T. mentagrophytes strain and ZafA+bar in the growth performance (size of the colony), the number of spores, and the quality of mycelium. However, the growth performance (size of the colony) and reproduction ability of ZafA-hph were significantly lower than the wild-type T. mentagrophytes strain and ZafA+bar. The spores and mycelium are stained with lactate gossypol blue. The blue dots are spores, as shown at the black arrow. The denser of the blue dots, the more spores, and the more blue lines, the more mycelium.
Figure Legend Snippet: The growth ability of wild-type T. mentagrophytes strain, ZafA-hph, and ZafA+bar. Sabouraud dextrose agar (SDA) medium with one mM of N,N,N′,N′-tetrakis (2-pyridinylmethyl)-1,2-ethanediamine (TPEN) was supplemented to generate zinc deficient SDA medium, which was named SDA-Zn. The wild-type T. mentagrophytes strain, ZafA-hph, and ZafA+bar were inoculated to SDA medium (grouped into Norm) and SDA-Zn medium with 200 μM, 400 μM, 600 μM, 800 μM, and 1000 μM of zinc sulfate (grouped into Zn200, Zn400, Zn600, Zn800, and Zn1000) respectively. In SDA-Zn medium, there was no significant difference between the wild-type T. mentagrophytes strain and ZafA+bar in the growth performance (size of the colony), the number of spores, and the quality of mycelium. However, the growth performance (size of the colony) and reproduction ability of ZafA-hph were significantly lower than the wild-type T. mentagrophytes strain and ZafA+bar. The spores and mycelium are stained with lactate gossypol blue. The blue dots are spores, as shown at the black arrow. The denser of the blue dots, the more spores, and the more blue lines, the more mycelium.

Techniques Used: Staining

( A ): The growth weight of wild-type T. mentagrophytes strain, ZafA-hph, and ZafA+bar. In Sabouraud glucose liquid medium, there was no significant growth weight difference in ZafA+bar and the wild-type T. mentagrophytes strain. However, the difference between ZafA-hph and the wild-type T. mentagrophytes strain was significant in growth weight. ** p
Figure Legend Snippet: ( A ): The growth weight of wild-type T. mentagrophytes strain, ZafA-hph, and ZafA+bar. In Sabouraud glucose liquid medium, there was no significant growth weight difference in ZafA+bar and the wild-type T. mentagrophytes strain. However, the difference between ZafA-hph and the wild-type T. mentagrophytes strain was significant in growth weight. ** p

Techniques Used:

20) Product Images from "Actinomyces denticolens as a causative agent of actinomycosis in animals"

Article Title: Actinomyces denticolens as a causative agent of actinomycosis in animals

Journal: The Journal of Veterinary Medical Science

doi: 10.1292/jvms.18-0207

Phylogenetic tree derived from 16S rRNA gene sequences, reconstructed using the maximum-likelihood method. The sequence of Trueperella pyogenes was used as an outgroup. Numbers at the branch points are percentages of 1,000 bootstrap replicates. Bar=0.02 substitutions per site.
Figure Legend Snippet: Phylogenetic tree derived from 16S rRNA gene sequences, reconstructed using the maximum-likelihood method. The sequence of Trueperella pyogenes was used as an outgroup. Numbers at the branch points are percentages of 1,000 bootstrap replicates. Bar=0.02 substitutions per site.

Techniques Used: Derivative Assay, Sequencing

21) Product Images from "Putative Interaction Proteins of the Ubiquitin Ligase Hrd1 inMagnaporthe oryzae"

Article Title: Putative Interaction Proteins of the Ubiquitin Ligase Hrd1 inMagnaporthe oryzae

Journal: Evolutionary Bioinformatics Online

doi: 10.1177/1176934318810990

Western blots of total proteins (total) and proteins eluted (elution) from the anti-GFP agarose beads from the wild type constitutively expressing GFP and transformant expressing MoHrd1::GFP were probed with the anti-GFP (GFP-Tag 7G9 Mouse mAb; Abmart, Shanghai, China) antibody. GFP indicates green fluorescent protein.
Figure Legend Snippet: Western blots of total proteins (total) and proteins eluted (elution) from the anti-GFP agarose beads from the wild type constitutively expressing GFP and transformant expressing MoHrd1::GFP were probed with the anti-GFP (GFP-Tag 7G9 Mouse mAb; Abmart, Shanghai, China) antibody. GFP indicates green fluorescent protein.

Techniques Used: Western Blot, Expressing

22) Product Images from "Marek’s Disease Virus Activates the PI3K/Akt Pathway Through Interaction of Its Protein Meq With the P85 Subunit of PI3K to Promote Viral Replication"

Article Title: Marek’s Disease Virus Activates the PI3K/Akt Pathway Through Interaction of Its Protein Meq With the P85 Subunit of PI3K to Promote Viral Replication

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02547

PI3K specific inhibitor LY294002 inhibits MDV replication. CEFs were pre-incubated with LY294002 (20 μM), LY303511 (30 μM) or wortmannin (500 μM) for 1 h, infected with MDV-LZ1309 at 0.1 MOI and analyzed for virus replication at 24, 48, 72, and 96 hpi. (A) MDV plaque quantification. (B) TaqMan real-time PCR detection of viral DNA. The data represent the mean ± SD of three independent experiments. One-way ANOVA, ∗ P
Figure Legend Snippet: PI3K specific inhibitor LY294002 inhibits MDV replication. CEFs were pre-incubated with LY294002 (20 μM), LY303511 (30 μM) or wortmannin (500 μM) for 1 h, infected with MDV-LZ1309 at 0.1 MOI and analyzed for virus replication at 24, 48, 72, and 96 hpi. (A) MDV plaque quantification. (B) TaqMan real-time PCR detection of viral DNA. The data represent the mean ± SD of three independent experiments. One-way ANOVA, ∗ P

Techniques Used: Incubation, Infection, Real-time Polymerase Chain Reaction

MDV induces phosphorylation of signaling proteins involved in the PI3K/Akt pathway. (A) CEFs cells were pre-incubated or not with LY294002 (20 μM) for 1 h and infected with MDV-LZ1309 at 0.1 MOI. Cells were collected at 0, 0.5, 1, 2, 4, and 6 hpi and cell lysates examined for phospho-p85 (Tyr458), total p85, phospho-Akt (Ser473), phospho-Akt (Thr308), total Akt, phospho-GSK-3β (Ser9), total GSK-3β, phospho-mTOR (Ser2448), total mTOR, and GAPDH by western blotting. (B) Quantification of relative phosphorylated protein band intensities to total protein in (A) . The data represent the mean ± SD of three independent experiments. Two-way ANOVA, ∗ P
Figure Legend Snippet: MDV induces phosphorylation of signaling proteins involved in the PI3K/Akt pathway. (A) CEFs cells were pre-incubated or not with LY294002 (20 μM) for 1 h and infected with MDV-LZ1309 at 0.1 MOI. Cells were collected at 0, 0.5, 1, 2, 4, and 6 hpi and cell lysates examined for phospho-p85 (Tyr458), total p85, phospho-Akt (Ser473), phospho-Akt (Thr308), total Akt, phospho-GSK-3β (Ser9), total GSK-3β, phospho-mTOR (Ser2448), total mTOR, and GAPDH by western blotting. (B) Quantification of relative phosphorylated protein band intensities to total protein in (A) . The data represent the mean ± SD of three independent experiments. Two-way ANOVA, ∗ P

Techniques Used: Incubation, Infection, Western Blot

Inhibition of PI3K activity results in the apoptosis of MDV-infected CEF cells. (A) CEFs were pre-treated or not with LY294002 (20 μM), for 1 h, infected with MDV-LZ1309, and analyzed for apoptosis at 24, 48, and 72 hpi by flow cytometry after Annexin V-FITC/PI double staining. (B) Quantification of the numbers of apoptotic cells of (A) . (C) CEFs were pre-treated or not with LY294002 (20 μM), LY303511 (30 μM) or wortmannin (500 μM) for 1 h, infected with MDV-LZ1309, and analyzed for apoptosis at 72 hpi by flow cytometry after Annexin V-FITC/PI double staining; (D) Quantification of the numbers of apoptotic cells of (C) . The data represent the mean ± SD of three independent experiments. Two-way ANOVA, ∗ P
Figure Legend Snippet: Inhibition of PI3K activity results in the apoptosis of MDV-infected CEF cells. (A) CEFs were pre-treated or not with LY294002 (20 μM), for 1 h, infected with MDV-LZ1309, and analyzed for apoptosis at 24, 48, and 72 hpi by flow cytometry after Annexin V-FITC/PI double staining. (B) Quantification of the numbers of apoptotic cells of (A) . (C) CEFs were pre-treated or not with LY294002 (20 μM), LY303511 (30 μM) or wortmannin (500 μM) for 1 h, infected with MDV-LZ1309, and analyzed for apoptosis at 72 hpi by flow cytometry after Annexin V-FITC/PI double staining; (D) Quantification of the numbers of apoptotic cells of (C) . The data represent the mean ± SD of three independent experiments. Two-way ANOVA, ∗ P

Techniques Used: Inhibition, Activity Assay, Infection, Flow Cytometry, Cytometry, Double Staining

MDV infection activates Akt phosphorylation in CEFs. Serum-starved CEFs were infected with vvMDV Md5, vvMDV LZ1309, or mMDV 814 at 0.1 MOI for 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 24, 36, 48, or 60 h, mock group (uninfected CEFs) and analyzed for Akt phosphorylation at Ser473 (pAkt 473) and total Akt expression by western blotting; GAPDH was used as a loading control. (A) vvMDV Md5 infection significantly increased Akt phosphorylation in CEFs at 2–6 hpi. (B) vvMDV LZ1309 infection significantly increased Akt phosphorylation in CEFs at 4–6 hpi. (C) mMDV 814 infection significantly increased Akt phosphorylation in CEFs at 4–6 hpi. (D) MDV infection reduced Akt phosphorylation in CEFs at 8–48 hpi and slightly induced it at 60 hpi. (E) Quantification of relative pAkt band intensities to Akt in (A–D) . The data represent the mean ± SD of three independent experiments. Two-way ANOVA, ∗ P
Figure Legend Snippet: MDV infection activates Akt phosphorylation in CEFs. Serum-starved CEFs were infected with vvMDV Md5, vvMDV LZ1309, or mMDV 814 at 0.1 MOI for 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 24, 36, 48, or 60 h, mock group (uninfected CEFs) and analyzed for Akt phosphorylation at Ser473 (pAkt 473) and total Akt expression by western blotting; GAPDH was used as a loading control. (A) vvMDV Md5 infection significantly increased Akt phosphorylation in CEFs at 2–6 hpi. (B) vvMDV LZ1309 infection significantly increased Akt phosphorylation in CEFs at 4–6 hpi. (C) mMDV 814 infection significantly increased Akt phosphorylation in CEFs at 4–6 hpi. (D) MDV infection reduced Akt phosphorylation in CEFs at 8–48 hpi and slightly induced it at 60 hpi. (E) Quantification of relative pAkt band intensities to Akt in (A–D) . The data represent the mean ± SD of three independent experiments. Two-way ANOVA, ∗ P

Techniques Used: Infection, Expressing, Western Blot

23) Product Images from "The Shieldin complex mediates 53BP1-dependent DNA repair"

Article Title: The Shieldin complex mediates 53BP1-dependent DNA repair

Journal: Nature

doi: 10.1038/s41586-018-0340-7

Supporting data that Shieldin localizes to DSB sites a, Representative micrographs of the experiments quantitated in Fig 3c . b, Representative micrographs of the experiments quantitated in Fig 3e . c, Quantitation of mRNAs for SHLD1 , SHLD2 and SHLD3 . RPE1 cells were transfected with siCTRL (non-targeting control siRNA) or siRNA targeting the indicated Shieldin subunits. 48 h post-transfection, mRNA was purified and reverse transcribed before being assayed by quantitative real-time PCR. Data were normalized to the amount of GAPDH mRNA and expressed relative to the corresponding value for cells transfected with siCTRL. Data is presented as the mean ± SD (n=3, independent siRNA transfections). d, Whole cell extracts from RPE1 WT cells transfected with the indicated siRNAs were processed for immunoblotting with the indicated antibodies. Tubulin is used as a loading control (n=1 experiment; siRNA efficiency is also monitored by immunofluorescence). e, Quantitation of 53BP1 and RIF1 recruitment to IR-induced DSBs (1 h post-irradiation with 10 Gy) following depletion of the indicated Shieldin components. Data is represented as the mean ± SD (n=3, independent siRNA transfections). f, Representative micrographs of the experiments quantitated in ED Fig 4e.
Figure Legend Snippet: Supporting data that Shieldin localizes to DSB sites a, Representative micrographs of the experiments quantitated in Fig 3c . b, Representative micrographs of the experiments quantitated in Fig 3e . c, Quantitation of mRNAs for SHLD1 , SHLD2 and SHLD3 . RPE1 cells were transfected with siCTRL (non-targeting control siRNA) or siRNA targeting the indicated Shieldin subunits. 48 h post-transfection, mRNA was purified and reverse transcribed before being assayed by quantitative real-time PCR. Data were normalized to the amount of GAPDH mRNA and expressed relative to the corresponding value for cells transfected with siCTRL. Data is presented as the mean ± SD (n=3, independent siRNA transfections). d, Whole cell extracts from RPE1 WT cells transfected with the indicated siRNAs were processed for immunoblotting with the indicated antibodies. Tubulin is used as a loading control (n=1 experiment; siRNA efficiency is also monitored by immunofluorescence). e, Quantitation of 53BP1 and RIF1 recruitment to IR-induced DSBs (1 h post-irradiation with 10 Gy) following depletion of the indicated Shieldin components. Data is represented as the mean ± SD (n=3, independent siRNA transfections). f, Representative micrographs of the experiments quantitated in ED Fig 4e.

Techniques Used: Quantitation Assay, Transfection, Purification, Real-time Polymerase Chain Reaction, Immunofluorescence, Irradiation

24) Product Images from "Molecular and Functional Characterization of Odorant Binding Protein 7 From the Oriental Fruit Moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae)"

Article Title: Molecular and Functional Characterization of Odorant Binding Protein 7 From the Oriental Fruit Moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2018.01762

Binding curve of 1-NPN and Scatchard plots for recombinant GmolOBP7. A 2 μM solution of protein in 20 mM Tris-HCl buffer (pH 7.4) was titrated with 1 mM 1-NPN solution to final concentrations of 1 to 18 μM, and the emission spectra were recorded between 370 and 550 nm. The dissociation constant (K d ) of GmolOBP7 was 2.30 μM.
Figure Legend Snippet: Binding curve of 1-NPN and Scatchard plots for recombinant GmolOBP7. A 2 μM solution of protein in 20 mM Tris-HCl buffer (pH 7.4) was titrated with 1 mM 1-NPN solution to final concentrations of 1 to 18 μM, and the emission spectra were recorded between 370 and 550 nm. The dissociation constant (K d ) of GmolOBP7 was 2.30 μM.

Techniques Used: Binding Assay, Recombinant

Electrophysiological response of Grapholita molesta to nine stimulants after RNAi knockdown. (A) male and (B) female moths. dsGmolOBP7 and dsGFP indicates treated moths injected with GmolOBP7 dsRNA and GFP dsRNA, respectively. Each treatment included 8 months, the antennae were stimulated with 15 μL volatile compound dissolved in liquid paraffin (ck), and ck and ( Z )-3-hexenyl acetate were used to stimulate the antennae before and after a group of volatiles stimulation. Different letters indicate significant differences between dsRNA-treated moths and non-injected moths (independent t -test, α = 0.05).
Figure Legend Snippet: Electrophysiological response of Grapholita molesta to nine stimulants after RNAi knockdown. (A) male and (B) female moths. dsGmolOBP7 and dsGFP indicates treated moths injected with GmolOBP7 dsRNA and GFP dsRNA, respectively. Each treatment included 8 months, the antennae were stimulated with 15 μL volatile compound dissolved in liquid paraffin (ck), and ck and ( Z )-3-hexenyl acetate were used to stimulate the antennae before and after a group of volatiles stimulation. Different letters indicate significant differences between dsRNA-treated moths and non-injected moths (independent t -test, α = 0.05).

Techniques Used: Injection

Expression level of GmolOBP7 in dsRNA-treated and no-injected moths at different ages of adult moths. (A) male moths; (B) female moths. About 240 ng (69 nL) of GmolOBP7 dsRNA and dsGFP were injected into 5-day-old pupae, respectively. Asterisk Different letters indicate significantly different expression levels of GmolOBP7 between dsRNA-treated moths and non-injected moths (independent t -test, α = 0.05).
Figure Legend Snippet: Expression level of GmolOBP7 in dsRNA-treated and no-injected moths at different ages of adult moths. (A) male moths; (B) female moths. About 240 ng (69 nL) of GmolOBP7 dsRNA and dsGFP were injected into 5-day-old pupae, respectively. Asterisk Different letters indicate significantly different expression levels of GmolOBP7 between dsRNA-treated moths and non-injected moths (independent t -test, α = 0.05).

Techniques Used: Expressing, Injection

Phylogenetic tree of OBPs from Grapholita molesta and other Lepidoptera via the neighbor-joining method with a bootstrap replication of 1,000. The unrooted tree was constructed using MEGA 6.0, based on the sequence alignment produced using ClustalX 1.83 software. The species and GenBank accession numbers of the sequence are as follows: A grotis ipsilon ( AipsGOBP1 , AFM36759.1 ; AipsGOBP2, AFM36760.1; AipsPBP2, JQ822241; Aips PBP3, JQ822242; AipsOBP6, AGR39569.1); Antheraea yamamai (AyamOBP7, ADO95155.1); Chilo suppressalis (CsupOBP1, AGK24577.1; CsupOBP4, AGK24580.1); Choristoneura fumiferana (CfumPBP1, AAF06127.1); Cnaphalocrocis medinalis (CmedOBP2, AFG73000.1; CmedOBP11, AFG72998.1; CmedOBP13, ALT31643.1); Danaus plexippus (DpleOBP8, OWR42851.1); Dendrolimus punctatus (DpunOBP19, ARO70178.1; DpunOBP31, ARO70190.1; DpunOBP32, ARO70191.1); Ectropis obliqua (EoblOBP1, ANA75015.1; EoblOBP3, ANA75017.1; EoblOBP7, ALS03855.1; EoblOBP14, ALS03862.1); Eogystia hippophaecolus (EhipPBP1, AOG12881.1; EhipOBP, AOG12871.1); Graphita molesta (GmolGOBP1, JN857939; GmolGOBP2, JN857940; GmolPBP1, MF066363; GmolPBP2, KF365878; GmolPBP3, KF365879; GmolOBP7, MF066359; GmolOBP14. MF066361); Helicoverpa armigera (HarmGOBP1, AAL09821.1; HarmGOBP2, CAC08211.1; HarmPBP2, AEB54583.1; HarmPBP3, AAO16091.1; HarmOBP8, AEB54589.1; HarmOBP16, AFI57165.1; HarmOBP17, AFI57166.1;HarmOBP18, AFI57167.1); Helicoverpa assulta (HassOBP16, AGC92791.1; HassOBP35, ASA40073.1); Heliothis virescens (HvirPBP2, CAL48346.1; HvirOBP, ACX53795.1); Lobesia botrana (LbotPBP1, AXF48748.1; LbotOBP37, AXF48734.1; LbotOBP38, AXF48735.1; LbotOBP45, AXF48742.1); Manduca sexta (MsexPBP3, AAF16703.1); Plutella xylostella (PxylOBP19, ANC60176.1); Spodoptera litura (SlitPBP, ABQ84981.1; SlitGOBP2, AKI87961.1; SlitPBP3, GU082321; SlitOBP8, AKI87969.1); Cydia pomonella (CpomGOBP1, AFP66957.1; CpomGOBP2, AFP66958.1; CpomPBP2, AFL91693.1) and Synanthedon exitiosa (SexiPBP1, AAF06142; SexiOBP17, AKT26495.1).
Figure Legend Snippet: Phylogenetic tree of OBPs from Grapholita molesta and other Lepidoptera via the neighbor-joining method with a bootstrap replication of 1,000. The unrooted tree was constructed using MEGA 6.0, based on the sequence alignment produced using ClustalX 1.83 software. The species and GenBank accession numbers of the sequence are as follows: A grotis ipsilon ( AipsGOBP1 , AFM36759.1 ; AipsGOBP2, AFM36760.1; AipsPBP2, JQ822241; Aips PBP3, JQ822242; AipsOBP6, AGR39569.1); Antheraea yamamai (AyamOBP7, ADO95155.1); Chilo suppressalis (CsupOBP1, AGK24577.1; CsupOBP4, AGK24580.1); Choristoneura fumiferana (CfumPBP1, AAF06127.1); Cnaphalocrocis medinalis (CmedOBP2, AFG73000.1; CmedOBP11, AFG72998.1; CmedOBP13, ALT31643.1); Danaus plexippus (DpleOBP8, OWR42851.1); Dendrolimus punctatus (DpunOBP19, ARO70178.1; DpunOBP31, ARO70190.1; DpunOBP32, ARO70191.1); Ectropis obliqua (EoblOBP1, ANA75015.1; EoblOBP3, ANA75017.1; EoblOBP7, ALS03855.1; EoblOBP14, ALS03862.1); Eogystia hippophaecolus (EhipPBP1, AOG12881.1; EhipOBP, AOG12871.1); Graphita molesta (GmolGOBP1, JN857939; GmolGOBP2, JN857940; GmolPBP1, MF066363; GmolPBP2, KF365878; GmolPBP3, KF365879; GmolOBP7, MF066359; GmolOBP14. MF066361); Helicoverpa armigera (HarmGOBP1, AAL09821.1; HarmGOBP2, CAC08211.1; HarmPBP2, AEB54583.1; HarmPBP3, AAO16091.1; HarmOBP8, AEB54589.1; HarmOBP16, AFI57165.1; HarmOBP17, AFI57166.1;HarmOBP18, AFI57167.1); Helicoverpa assulta (HassOBP16, AGC92791.1; HassOBP35, ASA40073.1); Heliothis virescens (HvirPBP2, CAL48346.1; HvirOBP, ACX53795.1); Lobesia botrana (LbotPBP1, AXF48748.1; LbotOBP37, AXF48734.1; LbotOBP38, AXF48735.1; LbotOBP45, AXF48742.1); Manduca sexta (MsexPBP3, AAF16703.1); Plutella xylostella (PxylOBP19, ANC60176.1); Spodoptera litura (SlitPBP, ABQ84981.1; SlitGOBP2, AKI87961.1; SlitPBP3, GU082321; SlitOBP8, AKI87969.1); Cydia pomonella (CpomGOBP1, AFP66957.1; CpomGOBP2, AFP66958.1; CpomPBP2, AFL91693.1) and Synanthedon exitiosa (SexiPBP1, AAF06142; SexiOBP17, AKT26495.1).

Techniques Used: Construct, Sequencing, Produced, Software

Expression profiles of GmolOBP 7 in different tissues (A) and developmental stages (B) of male and female moths. An, antennae; He, heads; Th, thoraces; Ab, abdomens; Le, Legs; Wi, Wings; 1st, first-instar larvae; 2nd, second-instar larvae; 3rd, third-instar larvae; 4th, fourth-instar larvae; 5th, fifth-instar larvae; Pup, prepupae; Later Pup, 5-d-old pupae; 1♂, 1-d-old adult males; 1♀, 1-d-old adult females; 3♂,3-d-old adult males; 3♀, 3-d-old adult females; 5♂, 5-d-old adult males; 5♀, 5-d-old adult females. Different lowercase and capital letters indicate significantly different expression levels among different tissues of female and male, respectively (Tukey's test, α = 0.05). Asterisks indicate significant different expression levels of GmolOBP7 between two sexes in the same tissue (Independent t -test, α = 0.05).
Figure Legend Snippet: Expression profiles of GmolOBP 7 in different tissues (A) and developmental stages (B) of male and female moths. An, antennae; He, heads; Th, thoraces; Ab, abdomens; Le, Legs; Wi, Wings; 1st, first-instar larvae; 2nd, second-instar larvae; 3rd, third-instar larvae; 4th, fourth-instar larvae; 5th, fifth-instar larvae; Pup, prepupae; Later Pup, 5-d-old pupae; 1♂, 1-d-old adult males; 1♀, 1-d-old adult females; 3♂,3-d-old adult males; 3♀, 3-d-old adult females; 5♂, 5-d-old adult males; 5♀, 5-d-old adult females. Different lowercase and capital letters indicate significantly different expression levels among different tissues of female and male, respectively (Tukey's test, α = 0.05). Asterisks indicate significant different expression levels of GmolOBP7 between two sexes in the same tissue (Independent t -test, α = 0.05).

Techniques Used: Expressing

SDS-PAGE analysis of recombinant GmolOBP7. (A) Expression and purification of GmolOBP7; (B) Purified GmolOBP7 protein after removed the His-tags. M1 and M2: standard protein maker; 1. Noninduced pET32a(+)/GmolOBP7; 2. Induced pET32a(+)/GmolOBP7; 3. Supernatant-induced pET32a(+)/GmolOBP7; 4. Precipitate of induced pET32a(+)/GmolOBP7; 5. Purified protein of pET32a(+)/GmolOBP7; 6. Digestion products of the purified pET32a(+)/GmolOBP7 using enterokinase; 7. Re-purification of GmolOBP7 after the removal of His-tags.
Figure Legend Snippet: SDS-PAGE analysis of recombinant GmolOBP7. (A) Expression and purification of GmolOBP7; (B) Purified GmolOBP7 protein after removed the His-tags. M1 and M2: standard protein maker; 1. Noninduced pET32a(+)/GmolOBP7; 2. Induced pET32a(+)/GmolOBP7; 3. Supernatant-induced pET32a(+)/GmolOBP7; 4. Precipitate of induced pET32a(+)/GmolOBP7; 5. Purified protein of pET32a(+)/GmolOBP7; 6. Digestion products of the purified pET32a(+)/GmolOBP7 using enterokinase; 7. Re-purification of GmolOBP7 after the removal of His-tags.

Techniques Used: SDS Page, Recombinant, Expressing, Purification

Binding curves of recombinant GmolOBP7 to a series of tested ligands. (A) sex pheromones; (B) aldehydes; (C) alcohols; (D) esters; (E) terpenes; (F) nitriles. The protein was diluted to a fixed concentration of 2 μM and then titrated with 1 mM of each competing ligand to a concentration of 0–14 μM for sex pheromones and 0–35 μM for host-plant volatiles. Fluorescence intensities are displayed as the percentage of the initial fluorescence. The calculated dissociation constants for all the ligands are listed in Table 2 .
Figure Legend Snippet: Binding curves of recombinant GmolOBP7 to a series of tested ligands. (A) sex pheromones; (B) aldehydes; (C) alcohols; (D) esters; (E) terpenes; (F) nitriles. The protein was diluted to a fixed concentration of 2 μM and then titrated with 1 mM of each competing ligand to a concentration of 0–14 μM for sex pheromones and 0–35 μM for host-plant volatiles. Fluorescence intensities are displayed as the percentage of the initial fluorescence. The calculated dissociation constants for all the ligands are listed in Table 2 .

Techniques Used: Binding Assay, Recombinant, Concentration Assay, Fluorescence

Sequence alignment of GmolOBP7 and other OBPs from Lepidopteran insects. The names and GeneBank accession numbers of five OBPs are as follows: Graphita molesta (GmolOBP7, MF066359); Ectropis obliqua (EoblOBP1, ANA75015.1; EoblOBP14, ALS03862.1); Spodoptera exigua (SexiOBP11, AGP03457.1); and Cnaphalocrocis medinalis (CmedOBP13, ALT31643.1). The six conserved cysteines were marked by a triangle with a black background.
Figure Legend Snippet: Sequence alignment of GmolOBP7 and other OBPs from Lepidopteran insects. The names and GeneBank accession numbers of five OBPs are as follows: Graphita molesta (GmolOBP7, MF066359); Ectropis obliqua (EoblOBP1, ANA75015.1; EoblOBP14, ALS03862.1); Spodoptera exigua (SexiOBP11, AGP03457.1); and Cnaphalocrocis medinalis (CmedOBP13, ALT31643.1). The six conserved cysteines were marked by a triangle with a black background.

Techniques Used: Sequencing

25) Product Images from "Combined use of cutinase and high-resolution mass-spectrometry to query the molecular architecture of cutin"

Article Title: Combined use of cutinase and high-resolution mass-spectrometry to query the molecular architecture of cutin

Journal: Plant Methods

doi: 10.1186/s13007-018-0384-6

a Relative ratio of dansylated and non-dansylated cutin-monomers recovered from cutinase hydrolysis of dansylated intact cutin. b Recovery of alkylated and nonalkylated cutin-monomers generated by the hydrolysis of benzyl- O -alkylated intact cutin. c Relative ratio of DmPA-derivatized and non-derivatized cutin-monomers recovered from hydrolysis of DmPA-treated intact cutin. The calculation of the standard error in panels a , b is described in the Materials and methods
Figure Legend Snippet: a Relative ratio of dansylated and non-dansylated cutin-monomers recovered from cutinase hydrolysis of dansylated intact cutin. b Recovery of alkylated and nonalkylated cutin-monomers generated by the hydrolysis of benzyl- O -alkylated intact cutin. c Relative ratio of DmPA-derivatized and non-derivatized cutin-monomers recovered from hydrolysis of DmPA-treated intact cutin. The calculation of the standard error in panels a , b is described in the Materials and methods

Techniques Used: Generated

Spatial distribution of cutinase-generated cutin fragments visualized by MALDI-based mass spectrometry. False color intensity of the relative abundance of a 9,10,18-trihydroxy-18:0/9,10,18-trihydroxy-18:1 ( m/z 651.43) dimer (blue); b 9,10,18-trihydroxy-18:0/9,10,18-trihydroxy-18:0 ( m/z 653.43) dimer (green); c 9,10,18-trihydroxy-18:0/9,10,18-trihydroxy-18:0/9,10,18-trihydroxy-18:0 ( m/z 973.68) trimer (purple). d The three molecular images shown in panels a – c , are overlayed on the visual image of the apple skin section with the cutinase containing droplets (highlighted by the yellow circles)
Figure Legend Snippet: Spatial distribution of cutinase-generated cutin fragments visualized by MALDI-based mass spectrometry. False color intensity of the relative abundance of a 9,10,18-trihydroxy-18:0/9,10,18-trihydroxy-18:1 ( m/z 651.43) dimer (blue); b 9,10,18-trihydroxy-18:0/9,10,18-trihydroxy-18:0 ( m/z 653.43) dimer (green); c 9,10,18-trihydroxy-18:0/9,10,18-trihydroxy-18:0/9,10,18-trihydroxy-18:0 ( m/z 973.68) trimer (purple). d The three molecular images shown in panels a – c , are overlayed on the visual image of the apple skin section with the cutinase containing droplets (highlighted by the yellow circles)

Techniques Used: Generated, Mass Spectrometry

Schematic structures of cutinase-generated cutin dimers ( a – d ), trimers ( e – h ) and tetramers ( i – k ). The digits within the black-filled rectangle represent the nature of the acyl moiety in the standard short-hand fatty acid nomenclature. The red-shaded “PAC-MAN” symbol represents the carboxyl-group of each acyl-chain. The digits in the blue-shaded triangles represent the positions of hydroxyl groups on the acyl-chain, and the digits in the green-shaded circles represent the positions of epoxy-groups on the acyl-chain. The blue-shaded triangle juxtaposed in the red PAC-MAN schematic represents the ester bond between adjoining acyl-monomers
Figure Legend Snippet: Schematic structures of cutinase-generated cutin dimers ( a – d ), trimers ( e – h ) and tetramers ( i – k ). The digits within the black-filled rectangle represent the nature of the acyl moiety in the standard short-hand fatty acid nomenclature. The red-shaded “PAC-MAN” symbol represents the carboxyl-group of each acyl-chain. The digits in the blue-shaded triangles represent the positions of hydroxyl groups on the acyl-chain, and the digits in the green-shaded circles represent the positions of epoxy-groups on the acyl-chain. The blue-shaded triangle juxtaposed in the red PAC-MAN schematic represents the ester bond between adjoining acyl-monomers

Techniques Used: Generated

Hierarchical phylogenetic analysis of 1243 cutinase sequences clustered in 211 different clades (color coded). The arrows indicate the 43 cutinases selected from each clade for recombinant expression in E. coli
Figure Legend Snippet: Hierarchical phylogenetic analysis of 1243 cutinase sequences clustered in 211 different clades (color coded). The arrows indicate the 43 cutinases selected from each clade for recombinant expression in E. coli

Techniques Used: Recombinant, Expressing

Chemical derivatization of non-esterified-hydroxyl- and carboxyl groups in intact cutin. a LC–MS/MS characterization of a cutinase hydrolysis product obtained from dansylated cutin; identification of the dansyl-derivative of 16-hydroxy-16:0 ( m/z 506.30). b GC/MS characterization of a cutinase hydrolysis product obtained from benzyl- O -alkylated cutin; identification of 18- O -benzyl-derivative of 9,10,18-trihydroxy-18:0. c LC–MS/MS characterization of a cutinase hydrolysis product obtained from DmPA-derivatized cutin; identification of DmPA-derivatized 16-hydroxy-16:0 ( m/z 434.38)
Figure Legend Snippet: Chemical derivatization of non-esterified-hydroxyl- and carboxyl groups in intact cutin. a LC–MS/MS characterization of a cutinase hydrolysis product obtained from dansylated cutin; identification of the dansyl-derivative of 16-hydroxy-16:0 ( m/z 506.30). b GC/MS characterization of a cutinase hydrolysis product obtained from benzyl- O -alkylated cutin; identification of 18- O -benzyl-derivative of 9,10,18-trihydroxy-18:0. c LC–MS/MS characterization of a cutinase hydrolysis product obtained from DmPA-derivatized cutin; identification of DmPA-derivatized 16-hydroxy-16:0 ( m/z 434.38)

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Gas Chromatography-Mass Spectrometry

Exemplary MS/MS characterization of cutinase-generated cutin hydrolysis products. a Identification of the monomer, 9,10,18-trihydroxyoctadecanoic acid (9,10,18-trihdroxy-18:0). b Identification of the dimer, 16-hydroxy-16:0/9,10,18-trihydroxy-18:0 ( m/z 585.47). c Identification of the trimer, 18-hydroxy-9,10-epoxy-18:1/10,16-dihydroxy-16:0/18-hydroxy-18:3 ( m/z 857.65). d Identification of the tetramer, 18-hydroxy-18:3/18-hydroxy-9,10-epoxy-18:0/18-hydroxy-18:2/18-hydroxy-9,10-epoxy-18:0 ( m/z 1161.83)
Figure Legend Snippet: Exemplary MS/MS characterization of cutinase-generated cutin hydrolysis products. a Identification of the monomer, 9,10,18-trihydroxyoctadecanoic acid (9,10,18-trihdroxy-18:0). b Identification of the dimer, 16-hydroxy-16:0/9,10,18-trihydroxy-18:0 ( m/z 585.47). c Identification of the trimer, 18-hydroxy-9,10-epoxy-18:1/10,16-dihydroxy-16:0/18-hydroxy-18:3 ( m/z 857.65). d Identification of the tetramer, 18-hydroxy-18:3/18-hydroxy-9,10-epoxy-18:0/18-hydroxy-18:2/18-hydroxy-9,10-epoxy-18:0 ( m/z 1161.83)

Techniques Used: Mass Spectrometry, Generated

26) Product Images from "Development of the VIGS System in the Dioecious Plant Silene latifolia"

Article Title: Development of the VIGS System in the Dioecious Plant Silene latifolia

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20051031

Virus-induced gene silencing (VIGS) of SlPDS gene in S. latifolia plants. ( A ) Schematic representation of ALSV- Sl PDS vectors used for VIGS. Four partial SlPDS fragments shown below, which differ in positions and length, were inserted into the artificial processing site of ALSV vector; ( B ) photographs of S. latifolia plants infected with ALSV- Sl PDS vectors at 20 dpi. Healthy represents a S. latifolia plant without any virus infection, and wtALSV represents a S. latifolia plant infected with wild-type ALSV vector without an SlPDS insert; ( C ) relative chlorophyll contents measured by the Soil Plant Analysis Development (SPAD) values in leaves infected with wtALSV and ALSV- Sl PDS vectors. SPAD values represent mean ± SE from three leaves of each plant ( n = 3 to 7). Asterisks indicate a significant difference between plants infected with wtALSV and those infected with each ALSV- Sl PDS vector at p
Figure Legend Snippet: Virus-induced gene silencing (VIGS) of SlPDS gene in S. latifolia plants. ( A ) Schematic representation of ALSV- Sl PDS vectors used for VIGS. Four partial SlPDS fragments shown below, which differ in positions and length, were inserted into the artificial processing site of ALSV vector; ( B ) photographs of S. latifolia plants infected with ALSV- Sl PDS vectors at 20 dpi. Healthy represents a S. latifolia plant without any virus infection, and wtALSV represents a S. latifolia plant infected with wild-type ALSV vector without an SlPDS insert; ( C ) relative chlorophyll contents measured by the Soil Plant Analysis Development (SPAD) values in leaves infected with wtALSV and ALSV- Sl PDS vectors. SPAD values represent mean ± SE from three leaves of each plant ( n = 3 to 7). Asterisks indicate a significant difference between plants infected with wtALSV and those infected with each ALSV- Sl PDS vector at p

Techniques Used: Plasmid Preparation, Infection

27) Product Images from "Molecular Characterization of Two Mitogen-Activated Protein Kinases: p38 MAP Kinase and Ribosomal S6 Kinase From Bombyx mori (Lepidoptera: Bombycidae), and Insight Into Their Roles in Response to BmNPV Infection"

Article Title: Molecular Characterization of Two Mitogen-Activated Protein Kinases: p38 MAP Kinase and Ribosomal S6 Kinase From Bombyx mori (Lepidoptera: Bombycidae), and Insight Into Their Roles in Response to BmNPV Infection

Journal: Journal of Insect Science

doi: 10.1093/jisesa/iey134

The dynamic proliferation of BmNPV in the midgut of Bombyx mori -resistant BC9 and -susceptible P50 strains analyzed by RT-qPCR. A pair of primers against BmNPV GP41 gene were used. Data were normalized using BmGAPDH and represented as mean ± SEM from three independent experiments. Relative expression levels were calculated using the 2 −ΔΔCt method.
Figure Legend Snippet: The dynamic proliferation of BmNPV in the midgut of Bombyx mori -resistant BC9 and -susceptible P50 strains analyzed by RT-qPCR. A pair of primers against BmNPV GP41 gene were used. Data were normalized using BmGAPDH and represented as mean ± SEM from three independent experiments. Relative expression levels were calculated using the 2 −ΔΔCt method.

Techniques Used: Quantitative RT-PCR, Expressing

28) Product Images from "N-acetylcysteine decreases malignant characteristics of glioblastoma cells by inhibiting Notch2 signaling"

Article Title: N-acetylcysteine decreases malignant characteristics of glioblastoma cells by inhibiting Notch2 signaling

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-1016-8

NAC induces apoptosis in GBM cells via targeting Notch2 signaling. a Total cellular GSH was measured in U87 and U251 cells under pre-treatment of BSO (2 mM, 12 h), followed by NAC (20 mM, 24 h). * P
Figure Legend Snippet: NAC induces apoptosis in GBM cells via targeting Notch2 signaling. a Total cellular GSH was measured in U87 and U251 cells under pre-treatment of BSO (2 mM, 12 h), followed by NAC (20 mM, 24 h). * P

Techniques Used:

NAC inhibits migration and invasion of GBM cells by suppressing Notch2 pathway. a Migration rate was measured by wound healing assay. Scale bar: 500 μm. b Transwell invasion assays of U87 and U251 cells. U87 and U251 cells were electroporated with pcDNA3.1-Notch2 or pcDNA3.1-EV, pcDNA3.1-EV served as a control, followed by BSO (1 mM, 6 h) and NAC (10 mM, 12 h) treatment. Scale bar: 200 μm. All data are presented as means ± SD of three independent experiments. * P
Figure Legend Snippet: NAC inhibits migration and invasion of GBM cells by suppressing Notch2 pathway. a Migration rate was measured by wound healing assay. Scale bar: 500 μm. b Transwell invasion assays of U87 and U251 cells. U87 and U251 cells were electroporated with pcDNA3.1-Notch2 or pcDNA3.1-EV, pcDNA3.1-EV served as a control, followed by BSO (1 mM, 6 h) and NAC (10 mM, 12 h) treatment. Scale bar: 200 μm. All data are presented as means ± SD of three independent experiments. * P

Techniques Used: Migration, Wound Healing Assay

NAC decreases protein levels of Notch2, Notch3 signaling and restrains cell proliferation of GBM. a , Cell viability was analyzed by CCK8 at 450 nm. U87 and U251 cells were treated with NAC (2, 5, 10 or 20 mM) for 24 h. b and c The protein levels of Notch2, Notch3, Hes1 and Hey1 were analyzed by western blot. U87 and U251 cells were treated with NAC (2, 5, 10 or 20 mM) for 24 h (b) and NAC (10 mM) for 3, 6, 12, 24 or 48 h ( c ) respectively. d The protein levels of Notch1 were detected in U87 and U251 cells by western blot after NAC (10 mM) treatment for 24 h. β-actin was used as a loading control. e and f Cell viability was analyzed by CCK8 at 450 nm. U87 ( e ) and U251 ( f ) cells were transfected with Scramble, si-Notch2 or si-Notch3 (10 μM) respectively for 24 h and 48 h. Scramble served as a control. All data are presented as means ± SD of three independent experiments. * P
Figure Legend Snippet: NAC decreases protein levels of Notch2, Notch3 signaling and restrains cell proliferation of GBM. a , Cell viability was analyzed by CCK8 at 450 nm. U87 and U251 cells were treated with NAC (2, 5, 10 or 20 mM) for 24 h. b and c The protein levels of Notch2, Notch3, Hes1 and Hey1 were analyzed by western blot. U87 and U251 cells were treated with NAC (2, 5, 10 or 20 mM) for 24 h (b) and NAC (10 mM) for 3, 6, 12, 24 or 48 h ( c ) respectively. d The protein levels of Notch1 were detected in U87 and U251 cells by western blot after NAC (10 mM) treatment for 24 h. β-actin was used as a loading control. e and f Cell viability was analyzed by CCK8 at 450 nm. U87 ( e ) and U251 ( f ) cells were transfected with Scramble, si-Notch2 or si-Notch3 (10 μM) respectively for 24 h and 48 h. Scramble served as a control. All data are presented as means ± SD of three independent experiments. * P

Techniques Used: Western Blot, Transfection

NAC diminishes Notch2 depending on lysosomal degradation. a , The mRNA analysis of Notch2 following dose-dependent treatment of NAC. U87 and U251 cells were treated with NAC (5, 10 or 20 mM) for 24 h. β-actin was used as a housekeeping gene. b The western blot analysis of Notch2 under proteasome (MG132, 10 μM) or lysosome (NH 4 Cl, 100 μM) inhibition and NAC (10 mM) treatment in U87 and U251 cells. c Notch2 and Itch were examined by western blot in U87 and U251 cells after NAC treatment (10 mM) for 24 h. d and e The western blot analysis of Itch and Notch2 after Itch silencing ( d ) or after NAC (10 mM) treatment in the presence of si-Itch ( e ) in U87 and U251 cells. f CRMP5 was analyzed by western blot in U87 and U251 cells after NAC treatment (10 mM) for 24 h. β-actin was used as a loading control. All data are presented as means ± SD of three independent experiments. * P
Figure Legend Snippet: NAC diminishes Notch2 depending on lysosomal degradation. a , The mRNA analysis of Notch2 following dose-dependent treatment of NAC. U87 and U251 cells were treated with NAC (5, 10 or 20 mM) for 24 h. β-actin was used as a housekeeping gene. b The western blot analysis of Notch2 under proteasome (MG132, 10 μM) or lysosome (NH 4 Cl, 100 μM) inhibition and NAC (10 mM) treatment in U87 and U251 cells. c Notch2 and Itch were examined by western blot in U87 and U251 cells after NAC treatment (10 mM) for 24 h. d and e The western blot analysis of Itch and Notch2 after Itch silencing ( d ) or after NAC (10 mM) treatment in the presence of si-Itch ( e ) in U87 and U251 cells. f CRMP5 was analyzed by western blot in U87 and U251 cells after NAC treatment (10 mM) for 24 h. β-actin was used as a loading control. All data are presented as means ± SD of three independent experiments. * P

Techniques Used: Western Blot, Inhibition

NAC attenuates proliferation of GBM cells through Notch2 signaling. a ,Notch2 was analyzed by western blot. b and c Cell viability was analyzed by CCK8 at 450 nm. d and e The cell cycle analysis was measured by the percentage of cells in each phase in U87 and U251 cells. f The expression levels of P21, cyclin E and CDK2 in U87 and U251 cells. All cells were electroporated with pcDNA3.1-Notch2 or pcDNA3.1-EV, pcDNA3.1-EV served as a control, followed by BSO (1 mM, 12 h) and NAC (10 mM, 24 h) treatment. β-actin was used as a loading control. All data are presented as means ± SD of three independent experiments. * P
Figure Legend Snippet: NAC attenuates proliferation of GBM cells through Notch2 signaling. a ,Notch2 was analyzed by western blot. b and c Cell viability was analyzed by CCK8 at 450 nm. d and e The cell cycle analysis was measured by the percentage of cells in each phase in U87 and U251 cells. f The expression levels of P21, cyclin E and CDK2 in U87 and U251 cells. All cells were electroporated with pcDNA3.1-Notch2 or pcDNA3.1-EV, pcDNA3.1-EV served as a control, followed by BSO (1 mM, 12 h) and NAC (10 mM, 24 h) treatment. β-actin was used as a loading control. All data are presented as means ± SD of three independent experiments. * P

Techniques Used: Western Blot, Cell Cycle Assay, Expressing

Independent of GSH in NAC induces Notch2 decrease. a The ROS levels were analyzed by flow cytometry using DCFH-DA (10 μM) following the treatments. U87 cells were treated with NAC (10 mM), GSH (20 mM) or Ebselen (10 μM) or for 6, 12 and 24 h respectively. b and c The western blot analysis of Notch2 under the treatment as a described. d Total cellular GSH was measured in U87 and U251 cells under pre-treatment of BSO (1 mM, 12 h), followed by NAC (10 mM, 24 h). e Effect of GSH depletion caused by BSO on Notch2 expression using western blot analysis. β-actin was used as a loading control. F and G, Cell viability was analyzed by CCK8 at 450 nm. U87 ( f ) and U251 ( g ) cells were treated as d described. All data are presented as means ± SD of three independent experiments. * P
Figure Legend Snippet: Independent of GSH in NAC induces Notch2 decrease. a The ROS levels were analyzed by flow cytometry using DCFH-DA (10 μM) following the treatments. U87 cells were treated with NAC (10 mM), GSH (20 mM) or Ebselen (10 μM) or for 6, 12 and 24 h respectively. b and c The western blot analysis of Notch2 under the treatment as a described. d Total cellular GSH was measured in U87 and U251 cells under pre-treatment of BSO (1 mM, 12 h), followed by NAC (10 mM, 24 h). e Effect of GSH depletion caused by BSO on Notch2 expression using western blot analysis. β-actin was used as a loading control. F and G, Cell viability was analyzed by CCK8 at 450 nm. U87 ( f ) and U251 ( g ) cells were treated as d described. All data are presented as means ± SD of three independent experiments. * P

Techniques Used: Flow Cytometry, Cytometry, Western Blot, Expressing

29) Product Images from "Monoclonal anti-double-stranded DNA autoantibody stimulates the expression and release of IL-1?, IL-6, IL-8, IL-10 and TNF-? from normal human mononuclear cells involving in the lupus pathogenesis"

Article Title: Monoclonal anti-double-stranded DNA autoantibody stimulates the expression and release of IL-1?, IL-6, IL-8, IL-10 and TNF-? from normal human mononuclear cells involving in the lupus pathogenesis

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00970.x

The effect of monoclonal anti-dsDNA antibody (9D7 and 12B3), NS-1 supernatant and purified mouse IgG on IL-1β, TNF-α and IL-8 cytokine release from normal human mononuclear cells (MNC) detected by ELISA after incubation for 3 days. The concentration of different proteins: 9D7 = 12·8 µg/ml, 12B3 = 11·04 µg/ml, mouse IgG = 20 µg/ml and NS-1 = 2·5× dilution. * denotes P
Figure Legend Snippet: The effect of monoclonal anti-dsDNA antibody (9D7 and 12B3), NS-1 supernatant and purified mouse IgG on IL-1β, TNF-α and IL-8 cytokine release from normal human mononuclear cells (MNC) detected by ELISA after incubation for 3 days. The concentration of different proteins: 9D7 = 12·8 µg/ml, 12B3 = 11·04 µg/ml, mouse IgG = 20 µg/ml and NS-1 = 2·5× dilution. * denotes P

Techniques Used: Purification, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay

30) Product Images from "B7x: A widely expressed B7 family member that inhibits T cell activation"

Article Title: B7x: A widely expressed B7 family member that inhibits T cell activation

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1434299100

B7x inhibits T cell activation. ( A ) Purified T cells and CD4 + /CD8 + subsets from BALB/c mice were stimulated with plate-bound anti-CD3 (0.25 μg/ml for CD4 + and total T cells, 2 μg/ml for CD8 + T cells) and plate-bound B7x-Ig (▾) or control Ig (•). Aliquots of supernatants were collected 48 h after initiation of cultures, cytokines were measured by ELISA, and proliferation was measured after 72 h by thymidine incorporation. Error bars indicate SD of triplicate cultures. These data are representative of three independent experiments. ( B ) Purified T cells were stimulated with plate-bound anti-CD3 and CHO transfectants expressing GFP (•), B7-2 (○), or B7x (▾). Cytokines and proliferation were measured as above. These data are representative of five independent experiments. ( C ) T cells were labeled with CFSE and stimulated with or without plate-bound anti-CD3 and CHO transfectants expressing GFP or B7x. On day 4, cells were harvested, stained with PE-anti-CD4 or PE-anti-CD8, and analyzed by flow cytometry. Percentages refer to fraction of cells in the nondividing peak or cells that have divided more than twice. These data are representative of three independent experiments.
Figure Legend Snippet: B7x inhibits T cell activation. ( A ) Purified T cells and CD4 + /CD8 + subsets from BALB/c mice were stimulated with plate-bound anti-CD3 (0.25 μg/ml for CD4 + and total T cells, 2 μg/ml for CD8 + T cells) and plate-bound B7x-Ig (▾) or control Ig (•). Aliquots of supernatants were collected 48 h after initiation of cultures, cytokines were measured by ELISA, and proliferation was measured after 72 h by thymidine incorporation. Error bars indicate SD of triplicate cultures. These data are representative of three independent experiments. ( B ) Purified T cells were stimulated with plate-bound anti-CD3 and CHO transfectants expressing GFP (•), B7-2 (○), or B7x (▾). Cytokines and proliferation were measured as above. These data are representative of five independent experiments. ( C ) T cells were labeled with CFSE and stimulated with or without plate-bound anti-CD3 and CHO transfectants expressing GFP or B7x. On day 4, cells were harvested, stained with PE-anti-CD4 or PE-anti-CD8, and analyzed by flow cytometry. Percentages refer to fraction of cells in the nondividing peak or cells that have divided more than twice. These data are representative of three independent experiments.

Techniques Used: Activation Assay, Purification, Mouse Assay, Enzyme-linked Immunosorbent Assay, Expressing, Labeling, Staining, Flow Cytometry, Cytometry

31) Product Images from "A Two-Component Multidrug Efflux Pump, EbrAB, in Bacillus subtilis"

Article Title: A Two-Component Multidrug Efflux Pump, EbrAB, in Bacillus subtilis

Journal: Journal of Bacteriology

doi:

Construction of plasmids carrying ebrA and/or ebrB . The plasmid pBET52 carries normal ebrA and ebrB . Plasmid pBET51 carries intact ebrB and defective ebrA in which two nucleotides were removed from the Cla I site. Plasmid pTS93 carries intact ebrA . Both pBET52 and pBET51 are derivatives of pUC19, and pTS93 is a derivative of pACYC184.
Figure Legend Snippet: Construction of plasmids carrying ebrA and/or ebrB . The plasmid pBET52 carries normal ebrA and ebrB . Plasmid pBET51 carries intact ebrB and defective ebrA in which two nucleotides were removed from the Cla I site. Plasmid pTS93 carries intact ebrA . Both pBET52 and pBET51 are derivatives of pUC19, and pTS93 is a derivative of pACYC184.

Techniques Used: Plasmid Preparation

32) Product Images from "Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle"

Article Title: Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0409885102

Amino acid residues 1556–1612 in the proximal C terminus of Ca V 1.1 bind the distal C terminus. ( Left ) Schematic map of the proximal C-terminal constructs of Ca V 1.1 that were cloned into the pAS2.1 vector and cotransformed with the distal C terminus (1753–1873 pACT2) or pACT2 alone into the Y190 yeast strain. Numbers correspond to the amino acid residues in Ca V 1.1. ( Right ) Representative growth of yeast cotransformed with the Ca V 1.1 proximal and distal C-terminal plasmids depicted on the left and the resulting β-galactosidase assay (+ or –).
Figure Legend Snippet: Amino acid residues 1556–1612 in the proximal C terminus of Ca V 1.1 bind the distal C terminus. ( Left ) Schematic map of the proximal C-terminal constructs of Ca V 1.1 that were cloned into the pAS2.1 vector and cotransformed with the distal C terminus (1753–1873 pACT2) or pACT2 alone into the Y190 yeast strain. Numbers correspond to the amino acid residues in Ca V 1.1. ( Right ) Representative growth of yeast cotransformed with the Ca V 1.1 proximal and distal C-terminal plasmids depicted on the left and the resulting β-galactosidase assay (+ or –).

Techniques Used: Construct, Clone Assay, Plasmid Preparation

Amino acid residues 1802–1841 in the distal C terminus of Ca V 1.1 bind the proximal C terminus. ( Left ) Schematic map of the distal C-terminal constructs of Ca V 1.1 that were cloned into the pACT2 vector and cotransformed with the proximal C terminus (1556–1612 pAS2.1) or pAS2.1 alone into the Y190 yeast strain. Numbers correspond to the amino acid residues in Ca V 1.1. ( Right ) Representative growth of yeast cotransformed with the Ca V 1.1 proximal and distal C-terminal plasmids depicted on the left and the resulting β-galactosidase assay (+ or –).
Figure Legend Snippet: Amino acid residues 1802–1841 in the distal C terminus of Ca V 1.1 bind the proximal C terminus. ( Left ) Schematic map of the distal C-terminal constructs of Ca V 1.1 that were cloned into the pACT2 vector and cotransformed with the proximal C terminus (1556–1612 pAS2.1) or pAS2.1 alone into the Y190 yeast strain. Numbers correspond to the amino acid residues in Ca V 1.1. ( Right ) Representative growth of yeast cotransformed with the Ca V 1.1 proximal and distal C-terminal plasmids depicted on the left and the resulting β-galactosidase assay (+ or –).

Techniques Used: Construct, Clone Assay, Plasmid Preparation

Coimmunoprecipitation of the separately expressed distal C terminus with truncated Ca V 1.1 channels in tsA-201 cells. ( A ) Immunoblot showing that expression of Ca V 1.1 channels by transfection of full-length cDNA in tsA-201 cells yields only full-length α 1 -subunit proteins, as detected by an anti-peptide antibody against the final C-terminal peptide (anti-CP1). ( B ) Schematic diagram of truncated Ca V 1.1 channels and the separately expressed distal C terminus used in coimmunoprecipitation experiments. ( C ) Lysates of tsA-201 cells transfected with Ca V 1.1 1684 and the distal C terminus (1685–1873) were immunoprecipitated with anti-CP11 (lane 2) or control IgG (lane 3). Immunoblots were probed with anti-CP11 ( Upper ) or anti-myc ( Lower ). Positive control for immunoblotting was 20 μl of lysate (lane 1).
Figure Legend Snippet: Coimmunoprecipitation of the separately expressed distal C terminus with truncated Ca V 1.1 channels in tsA-201 cells. ( A ) Immunoblot showing that expression of Ca V 1.1 channels by transfection of full-length cDNA in tsA-201 cells yields only full-length α 1 -subunit proteins, as detected by an anti-peptide antibody against the final C-terminal peptide (anti-CP1). ( B ) Schematic diagram of truncated Ca V 1.1 channels and the separately expressed distal C terminus used in coimmunoprecipitation experiments. ( C ) Lysates of tsA-201 cells transfected with Ca V 1.1 1684 and the distal C terminus (1685–1873) were immunoprecipitated with anti-CP11 (lane 2) or control IgG (lane 3). Immunoblots were probed with anti-CP11 ( Upper ) or anti-myc ( Lower ). Positive control for immunoblotting was 20 μl of lysate (lane 1).

Techniques Used: Expressing, Transfection, Immunoprecipitation, Western Blot, Positive Control

33) Product Images from "Identification of Colletotrichum spp. isolated from strawberry in Zhejiang Province and Shanghai City, China *"

Article Title: Identification of Colletotrichum spp. isolated from strawberry in Zhejiang Province and Shanghai City, China *

Journal: Journal of Zhejiang University. Science. B

doi: 10.1631/jzus.B0900174

PCR amplification of a specific fragment from Colletotrichum species using the C. acutatum specific primer Ca Int2 in conjunction with the primer ITS4 Lanes 1–3 correspond to representative C. acutatum isolates; Lanes 4–6 are the representative C. gloeosporioides isolates; Lanes 7–9 correspond to representative C. fragariae isolates; Lane 10 is a negative control ( Trichoderma virens genomic DNA); Lane M corresponds to the 100–3 000 bp molecular weight marker (Bio Basic Inc., Canada)
Figure Legend Snippet: PCR amplification of a specific fragment from Colletotrichum species using the C. acutatum specific primer Ca Int2 in conjunction with the primer ITS4 Lanes 1–3 correspond to representative C. acutatum isolates; Lanes 4–6 are the representative C. gloeosporioides isolates; Lanes 7–9 correspond to representative C. fragariae isolates; Lane 10 is a negative control ( Trichoderma virens genomic DNA); Lane M corresponds to the 100–3 000 bp molecular weight marker (Bio Basic Inc., Canada)

Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control, Molecular Weight, Marker

34) Product Images from "Novel Promoter and Alternate Transcription Start Site of the Human Serotonin Reuptake Transporter (SERT) in Intestinal Mucosa"

Article Title: Novel Promoter and Alternate Transcription Start Site of the Human Serotonin Reuptake Transporter (SERT) in Intestinal Mucosa

Journal: Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society

doi: 10.1111/j.1365-2982.2008.01247.x

A) Schematic of the location of promoter construct inserts relative to the human SERT gene used in secreted alkaline phosphatase reporter assays. Promoter activity, as measured by the activity of secreted alkaline phosphatase, of five constructs transfected into Caco-2, human intestinal epithelial cells (B) or JAR, human choriocarcinoma cells (C). Data are the mean (± SEM) of four independent experiments with triplicate values for each experiment. Construct 1, which is upstream of exon 1a, demonstrated the highest activity in both Caco-2 cells and JAR cells. In JAR cells, this activity was over 90 times greater than the promoterless basic vector. In Caco-2 cells, this activity was nearly 8 times greater than the promoterless basic vector. Constructs 2, 3, and 4 all contain various lengths of genomic DNA upstream of exon 2, and demonstrated activity in Caco-2 cells that was 5, 6, and 7 times greater than the promoterless basic vector, respectively. Construct 5, however, demonstrated no significant activity above the promoterless basic vector in the Caco cells. Conversely in the JAR cells, constructs 2, 3 and 4 demonstrated no significant activity in comparsion to the promoterless vector, while construct 5 had levels of activity 5 fold greater than the promoterless vector. *P
Figure Legend Snippet: A) Schematic of the location of promoter construct inserts relative to the human SERT gene used in secreted alkaline phosphatase reporter assays. Promoter activity, as measured by the activity of secreted alkaline phosphatase, of five constructs transfected into Caco-2, human intestinal epithelial cells (B) or JAR, human choriocarcinoma cells (C). Data are the mean (± SEM) of four independent experiments with triplicate values for each experiment. Construct 1, which is upstream of exon 1a, demonstrated the highest activity in both Caco-2 cells and JAR cells. In JAR cells, this activity was over 90 times greater than the promoterless basic vector. In Caco-2 cells, this activity was nearly 8 times greater than the promoterless basic vector. Constructs 2, 3, and 4 all contain various lengths of genomic DNA upstream of exon 2, and demonstrated activity in Caco-2 cells that was 5, 6, and 7 times greater than the promoterless basic vector, respectively. Construct 5, however, demonstrated no significant activity above the promoterless basic vector in the Caco cells. Conversely in the JAR cells, constructs 2, 3 and 4 demonstrated no significant activity in comparsion to the promoterless vector, while construct 5 had levels of activity 5 fold greater than the promoterless vector. *P

Techniques Used: Construct, Activity Assay, Transfection, Plasmid Preparation

35) Product Images from "Expression of the gene for Dec2, a basic helix-loop-helix transcription factor, is regulated by a molecular clock system"

Article Title: Expression of the gene for Dec2, a basic helix-loop-helix transcription factor, is regulated by a molecular clock system

Journal: Biochemical Journal

doi: 10.1042/BJ20031760

Repression of Dec2 promoter activity by Dec2 and Dec1 ( A ) C2C12 cells were transfected with expression vectors for Dec2 , Clock (Cl) and/or Bmal1 (Bm1). At 48 h after transfection, total RNA was prepared to examine the expression levels of endogenous Dec2 mRNA by RT-PCR analysis. ( B ) Dose-dependent repression of Dec2 promoter activity by Dec2 and Dec1. NIH3T3 cells were transfected with 50 ng of the p-1596-Luc construct and the indicated amounts of mDec2 or mDec1 expression vector, with or without Clock (Cl) and Bmal1 (Bm1) expression vectors. The total amount of transfected DNA was adjusted to a constant value with an empty vector. Promoter activities in the absence of expression vectors are given a value of 1. ( C ) Effects of HDAC inhibitors on Dec2-mediated transcriptional repression. The p-1596-Luc reporter construct was co-transfected with or without 10 ng of Dec2 expression vector. TSA (trichostatin A; 100 nM) or Scriptaid (8 μM) was added 3 h after transfection, and incubation was continued for 21 h. Luciferase activity is expressed as percentage of that in the absence of Dec2. ( D ) Involvement of E-box sites in Dec2- and Dec1-mediated transcriptional repression. An artificial promoter construct (pE1-TK-Luc or pE2-TK-Luc) containing three repeats of E-box1 or E-box2 was used as a reporter plasmid for the luciferase assay. Expression vectors were co-transfected into NIH3T3 cells. Promoter activities in the absence of expression vectors are given a value of 1. All data presented are means±S.D. for four different experiments; * P
Figure Legend Snippet: Repression of Dec2 promoter activity by Dec2 and Dec1 ( A ) C2C12 cells were transfected with expression vectors for Dec2 , Clock (Cl) and/or Bmal1 (Bm1). At 48 h after transfection, total RNA was prepared to examine the expression levels of endogenous Dec2 mRNA by RT-PCR analysis. ( B ) Dose-dependent repression of Dec2 promoter activity by Dec2 and Dec1. NIH3T3 cells were transfected with 50 ng of the p-1596-Luc construct and the indicated amounts of mDec2 or mDec1 expression vector, with or without Clock (Cl) and Bmal1 (Bm1) expression vectors. The total amount of transfected DNA was adjusted to a constant value with an empty vector. Promoter activities in the absence of expression vectors are given a value of 1. ( C ) Effects of HDAC inhibitors on Dec2-mediated transcriptional repression. The p-1596-Luc reporter construct was co-transfected with or without 10 ng of Dec2 expression vector. TSA (trichostatin A; 100 nM) or Scriptaid (8 μM) was added 3 h after transfection, and incubation was continued for 21 h. Luciferase activity is expressed as percentage of that in the absence of Dec2. ( D ) Involvement of E-box sites in Dec2- and Dec1-mediated transcriptional repression. An artificial promoter construct (pE1-TK-Luc or pE2-TK-Luc) containing three repeats of E-box1 or E-box2 was used as a reporter plasmid for the luciferase assay. Expression vectors were co-transfected into NIH3T3 cells. Promoter activities in the absence of expression vectors are given a value of 1. All data presented are means±S.D. for four different experiments; * P

Techniques Used: Activity Assay, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Construct, Plasmid Preparation, Incubation, Luciferase

Both Dec2 and Dec1 bind to two CACGTG E-box sites in the Dec2 promoter ( A ) Sense-strand sequences of double-stranded oligonucleotides used as probes and competitors in electrophoretic mobility shift assays. The position of each E-box is underlined, and the mutant nucleotides are shown in lower-case letters. 32 P-labelled oligonucleotides encompassing the E-box1 ( B ) or E-box2 ( C ) sites were incubated with reticulocyte lysates in the absence (lane 1; negative control) or presence of Dec proteins. For competition assays, a 100-fold excess of unlabelled oligonucleotides was added. W1, wild-type oligonucleotide identical to the E-box1 probe; M1, mutant oligonucleotide of E-box1 probe; W2, wild-type oligonucleotide identical to the E-box2 probe; M2, mutant oligonucleotide of E-box2 probe.
Figure Legend Snippet: Both Dec2 and Dec1 bind to two CACGTG E-box sites in the Dec2 promoter ( A ) Sense-strand sequences of double-stranded oligonucleotides used as probes and competitors in electrophoretic mobility shift assays. The position of each E-box is underlined, and the mutant nucleotides are shown in lower-case letters. 32 P-labelled oligonucleotides encompassing the E-box1 ( B ) or E-box2 ( C ) sites were incubated with reticulocyte lysates in the absence (lane 1; negative control) or presence of Dec proteins. For competition assays, a 100-fold excess of unlabelled oligonucleotides was added. W1, wild-type oligonucleotide identical to the E-box1 probe; M1, mutant oligonucleotide of E-box1 probe; W2, wild-type oligonucleotide identical to the E-box2 probe; M2, mutant oligonucleotide of E-box2 probe.

Techniques Used: Electrophoretic Mobility Shift Assay, Mutagenesis, Incubation, Negative Control

36) Product Images from "Identification and Characterization of hsa, the Gene Encoding the Sialic Acid-Binding Adhesin of Streptococcus gordonii DL1"

Article Title: Identification and Characterization of hsa, the Gene Encoding the Sialic Acid-Binding Adhesin of Streptococcus gordonii DL1

Journal: Infection and Immunity

doi: 10.1128/IAI.70.3.1209-1218.2002

Plasmid deletion analysis of the Hs-coding region of S. gordonii DL1 in E. coli. Thick lines represent inserts of S. gordonii DL1 DNA in plasmid pIRH801 and different deletion mutants. Vertical lines identify the 1.6-kb region associated with Hs immunoreactivity, which was determined by colony immunoblotting.
Figure Legend Snippet: Plasmid deletion analysis of the Hs-coding region of S. gordonii DL1 in E. coli. Thick lines represent inserts of S. gordonii DL1 DNA in plasmid pIRH801 and different deletion mutants. Vertical lines identify the 1.6-kb region associated with Hs immunoreactivity, which was determined by colony immunoblotting.

Techniques Used: Plasmid Preparation

37) Product Images from "hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage"

Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.24.8635-8647.2002

hSnm1 focus formation occurs normally in Nijmegen breakage syndrome cells. GM7166 primary fibroblasts were fixed 9 h after mock treatment or after exposure to 15 Gray of ionizing radiation (IR) and subjected to indirect immunofluorescence with anti-hSnm1. (a) Mock-treated cells; (b) after ionizing radiation exposure.
Figure Legend Snippet: hSnm1 focus formation occurs normally in Nijmegen breakage syndrome cells. GM7166 primary fibroblasts were fixed 9 h after mock treatment or after exposure to 15 Gray of ionizing radiation (IR) and subjected to indirect immunofluorescence with anti-hSnm1. (a) Mock-treated cells; (b) after ionizing radiation exposure.

Techniques Used: Immunofluorescence

Colocalization of hSnm1 foci with hMre11 and BRCA1 but not hRad51, as determined by indirect immunofluorescence. HT-1080 cells were irradiated with 10 Gray and stained 2 h later with (a) anti-hSnm1 and (b) anti-hRad51; (c) merged fields after DAPI staining. HT-1080 cells were irradiated with 15 Gray and stained 9 h later with (d) anti-hSnm1 and (e) anti-hMre11; (f) merged fields after DAPI staining. Partial colocalization with BRCA1 ionizing radiation-induced foci was observed 5 h after 10 Gray, as shown by (g) anti-hSnm1 and (h) anti-BRCA1; (i) both fields merged with DAPI staining.
Figure Legend Snippet: Colocalization of hSnm1 foci with hMre11 and BRCA1 but not hRad51, as determined by indirect immunofluorescence. HT-1080 cells were irradiated with 10 Gray and stained 2 h later with (a) anti-hSnm1 and (b) anti-hRad51; (c) merged fields after DAPI staining. HT-1080 cells were irradiated with 15 Gray and stained 9 h later with (d) anti-hSnm1 and (e) anti-hMre11; (f) merged fields after DAPI staining. Partial colocalization with BRCA1 ionizing radiation-induced foci was observed 5 h after 10 Gray, as shown by (g) anti-hSnm1 and (h) anti-BRCA1; (i) both fields merged with DAPI staining.

Techniques Used: Immunofluorescence, Irradiation, Staining

hSnm1 is localized to the nucleus. Indirect immunofluorescence of MCF-7 cells probed with hSnm1 affinity-purified polyclonal antibodies displaying (a) diffuse nuclear staining and hSnm1 bodies (as indicated by white arrow) or (b) multiple nuclear foci. Epifluorescence of MCF-7 cells expressing EGFP-hSnm1 fusion protein showing localization to (c) a nuclear body and (d) foci (transfected cell indicated by white arrow). An undamaged HT-1080 cell transiently expressing the Flag-hSnm1 fusion construct was stained with (e) anti-hSnm1 antibodies and (f) anti-Flag M2 monoclonal antibodies. (g) The two images are shown merged after DAPI staining.
Figure Legend Snippet: hSnm1 is localized to the nucleus. Indirect immunofluorescence of MCF-7 cells probed with hSnm1 affinity-purified polyclonal antibodies displaying (a) diffuse nuclear staining and hSnm1 bodies (as indicated by white arrow) or (b) multiple nuclear foci. Epifluorescence of MCF-7 cells expressing EGFP-hSnm1 fusion protein showing localization to (c) a nuclear body and (d) foci (transfected cell indicated by white arrow). An undamaged HT-1080 cell transiently expressing the Flag-hSnm1 fusion construct was stained with (e) anti-hSnm1 antibodies and (f) anti-Flag M2 monoclonal antibodies. (g) The two images are shown merged after DAPI staining.

Techniques Used: Immunofluorescence, Affinity Purification, Staining, Expressing, Transfection, Construct

Colocalization of EGFP-hSnm1 protein with 53BP1. MCF-7 cells were transfected with pEGFP-hSnm1 followed by staining with anti-53BP1 and Toto-3. Localization of (a) EGFP-hSnm1 and (b) 53BP1 to a nuclear body in the same cell; (c) the two fields merged with Toto-3.
Figure Legend Snippet: Colocalization of EGFP-hSnm1 protein with 53BP1. MCF-7 cells were transfected with pEGFP-hSnm1 followed by staining with anti-53BP1 and Toto-3. Localization of (a) EGFP-hSnm1 and (b) 53BP1 to a nuclear body in the same cell; (c) the two fields merged with Toto-3.

Techniques Used: Transfection, Staining

Coimmunoprecipitation of 53BP1 and hSnm1. (A) HeLa cell extracts were incubated with beads only, preimmune serum, or affinity-purified anti-hSnm1 antibodies. Polyclonal anti-53BP1 antibodies were used to detect 53BP1 by immunoblotting. (B) HEK293 nuclear extracts were prepared with or without infection with EGFP-hSnm1-expressing adenovirus and immunoblotted with monoclonal antibodies against EGFP or polyclonal antibodies against 53BP1. Immunoprecipitations (IP) were performed with these extracts with anti-hSnm1 and anti-53BP1 antibodies as shown.
Figure Legend Snippet: Coimmunoprecipitation of 53BP1 and hSnm1. (A) HeLa cell extracts were incubated with beads only, preimmune serum, or affinity-purified anti-hSnm1 antibodies. Polyclonal anti-53BP1 antibodies were used to detect 53BP1 by immunoblotting. (B) HEK293 nuclear extracts were prepared with or without infection with EGFP-hSnm1-expressing adenovirus and immunoblotted with monoclonal antibodies against EGFP or polyclonal antibodies against 53BP1. Immunoprecipitations (IP) were performed with these extracts with anti-hSnm1 and anti-53BP1 antibodies as shown.

Techniques Used: Incubation, Affinity Purification, Infection, Expressing

Deletion analysis of hSNM1. (A) Various truncations and in-frame deletions were constructed in the hSNM1 segment of the EGFP-hSNM1 fusion gene. The solid black region indicates EGFP, and vertical lines indicate the conserved metallo-β-lactamase domain. Each construct was transfected into HT-1080 cells, and the location of the EGFP signal was determined 24 h later. (B) Expression of EGFP-hSnm1 deletion mutants in HT-1080 cells.
Figure Legend Snippet: Deletion analysis of hSNM1. (A) Various truncations and in-frame deletions were constructed in the hSNM1 segment of the EGFP-hSNM1 fusion gene. The solid black region indicates EGFP, and vertical lines indicate the conserved metallo-β-lactamase domain. Each construct was transfected into HT-1080 cells, and the location of the EGFP signal was determined 24 h later. (B) Expression of EGFP-hSnm1 deletion mutants in HT-1080 cells.

Techniques Used: Construct, Transfection, Expressing

(A) Northern blot of mRNAs from various tissues was probed with hSnm1 cDNA. A probe for β-actin was used to control for loading. (B) In vitro-translated Flag-hSnm1 protein labeled with [ 35 S]methionine was precipitated by anti-hSnm1-protein A-agarose beads, protein A beads only, or preimmune-protein A beads. B and SN indicate beads and supernatant, respectively.
Figure Legend Snippet: (A) Northern blot of mRNAs from various tissues was probed with hSnm1 cDNA. A probe for β-actin was used to control for loading. (B) In vitro-translated Flag-hSnm1 protein labeled with [ 35 S]methionine was precipitated by anti-hSnm1-protein A-agarose beads, protein A beads only, or preimmune-protein A beads. B and SN indicate beads and supernatant, respectively.

Techniques Used: Northern Blot, In Vitro, Labeling

Quantitative analysis of hSnm1 focus induction after DNA damage in MCF-7 cells by indirect immunofluorescence. hSnm1 antibodies were used to probe cells at various times after treatment with either ionizing radiation (IR) or 4HC. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (open bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 100 nuclei for each time point. Reported here are the averages and standard deviations of three data sets.
Figure Legend Snippet: Quantitative analysis of hSnm1 focus induction after DNA damage in MCF-7 cells by indirect immunofluorescence. hSnm1 antibodies were used to probe cells at various times after treatment with either ionizing radiation (IR) or 4HC. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (open bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 100 nuclei for each time point. Reported here are the averages and standard deviations of three data sets.

Techniques Used: Immunofluorescence, Staining

Quantitative analysis of hSnm1 nuclear staining during the cell cycle. (A) Histogram displaying the DNA content of asynchronous untreated MCF-7 cells analyzed by laser scanning cytometry and the observed frequencies of the different hSnm1 staining patterns corresponding to the G 1 , S, and G 2 subpopulations. PI, propidium iodide. (B) Similar data obtained from MCF-7 cells taken 5 h after treatment with 10 Gray of ionizing radiation. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (white bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 150 nuclei for each time point.
Figure Legend Snippet: Quantitative analysis of hSnm1 nuclear staining during the cell cycle. (A) Histogram displaying the DNA content of asynchronous untreated MCF-7 cells analyzed by laser scanning cytometry and the observed frequencies of the different hSnm1 staining patterns corresponding to the G 1 , S, and G 2 subpopulations. PI, propidium iodide. (B) Similar data obtained from MCF-7 cells taken 5 h after treatment with 10 Gray of ionizing radiation. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (white bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 150 nuclei for each time point.

Techniques Used: Staining, Cytometry

Colocalization of hSnm1 and 53BP1 in foci as detected by indirect immunofluorescence. MCF-7 cells were mock treated (a, b, and c) or treated with 10 Gray of ionizing radiation and fixed after 30 min (d, e, and f), after 90 min (g, h, and i), or after 5 h (j, k, and l). (a, d, g, and j) Polyclonal anti-hSnm1 staining with fluorescein isothiocyanate; (b, e, h, and k) monoclonal anti-53BP1 staining with tetramethyl rhodamine isocyanate; (c, f, i, and l) merged fields plus DAPI staining.
Figure Legend Snippet: Colocalization of hSnm1 and 53BP1 in foci as detected by indirect immunofluorescence. MCF-7 cells were mock treated (a, b, and c) or treated with 10 Gray of ionizing radiation and fixed after 30 min (d, e, and f), after 90 min (g, h, and i), or after 5 h (j, k, and l). (a, d, g, and j) Polyclonal anti-hSnm1 staining with fluorescein isothiocyanate; (b, e, h, and k) monoclonal anti-53BP1 staining with tetramethyl rhodamine isocyanate; (c, f, i, and l) merged fields plus DAPI staining.

Techniques Used: Immunofluorescence, Staining

38) Product Images from "Recognition of enzymes lacking bound cofactor by protein quality control"

Article Title: Recognition of enzymes lacking bound cofactor by protein quality control

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1611994113

Riboflavin deficiency affects the function of NQO1. The efficiency of HSP90 inhibition by 17-AAG ( A ) or radicicol ( B ) in riboflavin-containing (+Ribo, white bars) and riboflavin-deficient (−Ribo, gray bars) medium was analyzed by measuring the degradation of the HSP90 client kinase B-Raf (V600E mutant) using Western blotting ( n = 3, mean ± SD). The amount of B-Raf in untreated cells was set as 1. Western blotting results from one representative experiment are shown.
Figure Legend Snippet: Riboflavin deficiency affects the function of NQO1. The efficiency of HSP90 inhibition by 17-AAG ( A ) or radicicol ( B ) in riboflavin-containing (+Ribo, white bars) and riboflavin-deficient (−Ribo, gray bars) medium was analyzed by measuring the degradation of the HSP90 client kinase B-Raf (V600E mutant) using Western blotting ( n = 3, mean ± SD). The amount of B-Raf in untreated cells was set as 1. Western blotting results from one representative experiment are shown.

Techniques Used: Inhibition, Mutagenesis, Western Blot

39) Product Images from "Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity"

Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Journal: The EMBO Journal

doi: 10.1093/emboj/cdf402

Fig. 7. Co-localization of FGF-1 and CK2 in U2OS cells transfected with GFP–FGF-1. U2OS cells grown on coverslips were transfected with pEGFP-FGF-1 and pRc/CMV-CK2α-HA or pEGFP-FGF-1 and pcDNA3-CK2β, fixed in 3% paraformaldehyde, permeabilized and labelled with either a polyclonal antibody against CK2α or a monoclonal antibody against CK2β. The cells were subsequently stained with anti-rabbit-Cy3 (CK2α) or anti-mouse–lissamine–rhodamine (CK2β) secondary antibodies and analysed by confocal microscopy.
Figure Legend Snippet: Fig. 7. Co-localization of FGF-1 and CK2 in U2OS cells transfected with GFP–FGF-1. U2OS cells grown on coverslips were transfected with pEGFP-FGF-1 and pRc/CMV-CK2α-HA or pEGFP-FGF-1 and pcDNA3-CK2β, fixed in 3% paraformaldehyde, permeabilized and labelled with either a polyclonal antibody against CK2α or a monoclonal antibody against CK2β. The cells were subsequently stained with anti-rabbit-Cy3 (CK2α) or anti-mouse–lissamine–rhodamine (CK2β) secondary antibodies and analysed by confocal microscopy.

Techniques Used: Transfection, Staining, Confocal Microscopy

Fig. 8. FGF-1 interacts with CK2α in vivo . HeLa cells were transfected with either pcDNA3-myc-FGF-1 containing a signal for targeting to peroxisomes (myc-FGF-1 pts ) and pECFP-CK2α ( A – F ) or pcDNA3-myc-FGF-1 and pECFP-CK2α ( G – L ). After depletion of the cytosol with digitonin, the cells were fixed, permeabilized with Triton X-100 and double stained with rabbit anti-catalase and mouse anti-c-Myc antibodies followed by staining with anti-rabbit Cy5 (catalase) and anti-mouse lissamine–rhodamine (c-Myc). ( M ) Quantitation of peroxisomes containing both FGF-1 pts and CK2α. The number of peroxisomes that stained for both FGF-1 pts and CK2α was counted in a number of cells (20) both by accurately merging the pictures taken of FGF-1 pts and CK2α and by merging them nine pixels askew. The number of peroxisomes containing both FGF-1 pts and CK2α in the image merged askew is taken as the amount of unspecific co-localization.
Figure Legend Snippet: Fig. 8. FGF-1 interacts with CK2α in vivo . HeLa cells were transfected with either pcDNA3-myc-FGF-1 containing a signal for targeting to peroxisomes (myc-FGF-1 pts ) and pECFP-CK2α ( A – F ) or pcDNA3-myc-FGF-1 and pECFP-CK2α ( G – L ). After depletion of the cytosol with digitonin, the cells were fixed, permeabilized with Triton X-100 and double stained with rabbit anti-catalase and mouse anti-c-Myc antibodies followed by staining with anti-rabbit Cy5 (catalase) and anti-mouse lissamine–rhodamine (c-Myc). ( M ) Quantitation of peroxisomes containing both FGF-1 pts and CK2α. The number of peroxisomes that stained for both FGF-1 pts and CK2α was counted in a number of cells (20) both by accurately merging the pictures taken of FGF-1 pts and CK2α and by merging them nine pixels askew. The number of peroxisomes containing both FGF-1 pts and CK2α in the image merged askew is taken as the amount of unspecific co-localization.

Techniques Used: In Vivo, Transfection, Staining, Quantitation Assay

Fig. 4. Binding of FGF-1 to free subunits of CK2. ( A ) MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated for 90 min with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing amounts of CK2β as indicated. The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. ( B ) The same conditions as described in (A), but MBP–CK2β was bound to CNBr-activated Sepharose and incubated with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing concentrations of FGF-1.
Figure Legend Snippet: Fig. 4. Binding of FGF-1 to free subunits of CK2. ( A ) MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated for 90 min with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing amounts of CK2β as indicated. The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. ( B ) The same conditions as described in (A), but MBP–CK2β was bound to CNBr-activated Sepharose and incubated with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing concentrations of FGF-1.

Techniques Used: Binding Assay, Incubation, In Vitro, SDS Page

Fig. 1. Identification of proteins binding selectively to FGF-1. ( A ) U2OS cells were incubated overnight (16 h) with [ 35 S]cysteine/methionine, then washed and lysed. The cell lysates were rotated for 1 h at 4°C with either MBP (lane 1), MBP–FGF-1 (lane 2) or MBP–IFN-γ (lane 4) covalently bound to Sepharose beads (∼20 µg of protein). The lysates were then transferred to another Eppendorf tube and subjected to a second round of precipitation for 1 h at 4°C with Sepharose-bound MBP–FGF-1. The Sepharose beads were washed, the proteins eluted with SDS sample buffer and the eluates were subjected to SDS–PAGE [12% (w/v) gel] and fluorography. An asterisk marks proteins that bound to MBP–FGF-1 after the lysate had first been incubated with MBP, and two asterisks mark proteins that bound to MBP–FGF-1 after the lysate had first been incubated with MBP–IFN-γ. Arrows point to proteins that bind FGF-1 specifically. Also shown are the positions of molecular weight standards. ( B ) The same conditions as in (A), but the rotations with fusion proteins were performed in the absence (lanes 2 and 4) or presence (lanes 1 and 3) of excess free FGF-1 (500 µg). Arrows mark proteins that bound FGF-1 specifically and which were competed out by excess FGF-1.
Figure Legend Snippet: Fig. 1. Identification of proteins binding selectively to FGF-1. ( A ) U2OS cells were incubated overnight (16 h) with [ 35 S]cysteine/methionine, then washed and lysed. The cell lysates were rotated for 1 h at 4°C with either MBP (lane 1), MBP–FGF-1 (lane 2) or MBP–IFN-γ (lane 4) covalently bound to Sepharose beads (∼20 µg of protein). The lysates were then transferred to another Eppendorf tube and subjected to a second round of precipitation for 1 h at 4°C with Sepharose-bound MBP–FGF-1. The Sepharose beads were washed, the proteins eluted with SDS sample buffer and the eluates were subjected to SDS–PAGE [12% (w/v) gel] and fluorography. An asterisk marks proteins that bound to MBP–FGF-1 after the lysate had first been incubated with MBP, and two asterisks mark proteins that bound to MBP–FGF-1 after the lysate had first been incubated with MBP–IFN-γ. Arrows point to proteins that bind FGF-1 specifically. Also shown are the positions of molecular weight standards. ( B ) The same conditions as in (A), but the rotations with fusion proteins were performed in the absence (lanes 2 and 4) or presence (lanes 1 and 3) of excess free FGF-1 (500 µg). Arrows mark proteins that bound FGF-1 specifically and which were competed out by excess FGF-1.

Techniques Used: Binding Assay, Incubation, SDS Page, Molecular Weight

Fig. 5. Sensitivity to salt of the binding between FGF-1 and CK2α and β. MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated in a 1:1 mixture of lysis buffer and PBS for 90 min at 4°C with in vitro translated [ 35 S]methionine-labelled CK2α (lanes 1–4) or CK2β (lanes 5–8). The samples were washed in the same buffer (lanes 1 and 5) or the same buffer supplemented with either 0.3 (lanes 2 and 6), 0.5 (lanes 3 and 7) or 1.0 M NaCl (lanes 4 and 8). The bound proteins were then eluted with SDS sample buffer and analysed by SDS–PAGE and fluorography.
Figure Legend Snippet: Fig. 5. Sensitivity to salt of the binding between FGF-1 and CK2α and β. MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated in a 1:1 mixture of lysis buffer and PBS for 90 min at 4°C with in vitro translated [ 35 S]methionine-labelled CK2α (lanes 1–4) or CK2β (lanes 5–8). The samples were washed in the same buffer (lanes 1 and 5) or the same buffer supplemented with either 0.3 (lanes 2 and 6), 0.5 (lanes 3 and 7) or 1.0 M NaCl (lanes 4 and 8). The bound proteins were then eluted with SDS sample buffer and analysed by SDS–PAGE and fluorography.

Techniques Used: Binding Assay, Incubation, Lysis, In Vitro, SDS Page

Fig. 2. ). U2OS cells were lysed and lysate was incubated for 1 h at 4°C with the indicated amount of immobilized MBP–FGF-1 or MBP. The bound proteins were separated by SDS–PAGE, transferred to a PVDF membrane and blotted with anti-CK2β antibodies. The arrow marks the position of CK2β.
Figure Legend Snippet: Fig. 2. ). U2OS cells were lysed and lysate was incubated for 1 h at 4°C with the indicated amount of immobilized MBP–FGF-1 or MBP. The bound proteins were separated by SDS–PAGE, transferred to a PVDF membrane and blotted with anti-CK2β antibodies. The arrow marks the position of CK2β.

Techniques Used: Incubation, SDS Page

Fig. 9. Phosphorylation of FGF-1 and FGF-2 by CK2 and the ability of the growth factors to stimulate autophosphorylation of CK2β. Purified rat liver CK2 was incubated for 20 min at 30°C either alone or with the indicated quantity of FGF-1 (lanes 2–4), FGF-2 (lanes 5–8) or FGF-1(K132E) (lane 9). The samples were then incubated for 2 h with either heparin–Sepharose (lanes 1–7 and 9) or anti-FGF-2 coupled to protein A–Sepharose (lane 8). The bound proteins were eluted with sample buffer, analysed by SDS–PAGE and exposed to a phosphoimager. It should be noted that CK2 binds to heparin–Sepharose independently of FGF.
Figure Legend Snippet: Fig. 9. Phosphorylation of FGF-1 and FGF-2 by CK2 and the ability of the growth factors to stimulate autophosphorylation of CK2β. Purified rat liver CK2 was incubated for 20 min at 30°C either alone or with the indicated quantity of FGF-1 (lanes 2–4), FGF-2 (lanes 5–8) or FGF-1(K132E) (lane 9). The samples were then incubated for 2 h with either heparin–Sepharose (lanes 1–7 and 9) or anti-FGF-2 coupled to protein A–Sepharose (lane 8). The bound proteins were eluted with sample buffer, analysed by SDS–PAGE and exposed to a phosphoimager. It should be noted that CK2 binds to heparin–Sepharose independently of FGF.

Techniques Used: Purification, Incubation, SDS Page

Fig. 6. Kinetics of binding between FGF-1 and CK2α or CK2β as measured by surface plasmon resonance. FGF-1 was injected over a sensorchip loaded with either GST–CK2α ( A ) or GST–CK2β ( B ). Sensorgrams obtained at the indicated concentrations from one representative series are shown. The calculated association ( K a ), dissociation ( K d ) and equilibrium ( K D ) constants (± SDs) based on four separate series are given.
Figure Legend Snippet: Fig. 6. Kinetics of binding between FGF-1 and CK2α or CK2β as measured by surface plasmon resonance. FGF-1 was injected over a sensorchip loaded with either GST–CK2α ( A ) or GST–CK2β ( B ). Sensorgrams obtained at the indicated concentrations from one representative series are shown. The calculated association ( K a ), dissociation ( K d ) and equilibrium ( K D ) constants (± SDs) based on four separate series are given.

Techniques Used: Binding Assay, SPR Assay, Injection

Fig. 3. Binding of FGF-1 to both CK2α and β. ( A ) U2OS cells were lysed and incubated for 1 h at 4°C with the indicated amounts of MBP–FGF-1 or MBP immobilized on Sepharose beads. The Sepharose beads were washed and the bound proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody (upper panel). Then the membrane was stripped and reprobed with an antibody against CK2α (lower panel). ( B ) MBP–FGF-1 (lanes 1–12) or MBP (lanes 13–15) bound to protein A–Sepharose beads was incubated for 2 h at 4°C with the indicated volumes of in vitro translated CK2β (lanes 2–4 and 13), CK2α (lanes 6–9 and 14) or CK2α′ (lanes 10–12 and 15). The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. For comparison, 1 µl of the in vitro translated proteins used in the precipitations was run in separate lanes (1, 5 and 9). ( C ) GST–CK2α (lanes 1, 4 and 7), GST–CK2β (lanes 2, 5 and 8) or GST (lanes 3, 6 and 9) bound to glutathione–Sepharose was incubated with MBP–FGF-1 (lanes 1–3), MBP–FGF-2 (lanes 4–6) or MBP (lanes 7–9) for 2 h at 4°C. After binding, the samples were washed and analysed by western blotting with an antibody against MBP.
Figure Legend Snippet: Fig. 3. Binding of FGF-1 to both CK2α and β. ( A ) U2OS cells were lysed and incubated for 1 h at 4°C with the indicated amounts of MBP–FGF-1 or MBP immobilized on Sepharose beads. The Sepharose beads were washed and the bound proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody (upper panel). Then the membrane was stripped and reprobed with an antibody against CK2α (lower panel). ( B ) MBP–FGF-1 (lanes 1–12) or MBP (lanes 13–15) bound to protein A–Sepharose beads was incubated for 2 h at 4°C with the indicated volumes of in vitro translated CK2β (lanes 2–4 and 13), CK2α (lanes 6–9 and 14) or CK2α′ (lanes 10–12 and 15). The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. For comparison, 1 µl of the in vitro translated proteins used in the precipitations was run in separate lanes (1, 5 and 9). ( C ) GST–CK2α (lanes 1, 4 and 7), GST–CK2β (lanes 2, 5 and 8) or GST (lanes 3, 6 and 9) bound to glutathione–Sepharose was incubated with MBP–FGF-1 (lanes 1–3), MBP–FGF-2 (lanes 4–6) or MBP (lanes 7–9) for 2 h at 4°C. After binding, the samples were washed and analysed by western blotting with an antibody against MBP.

Techniques Used: Binding Assay, Incubation, SDS Page, In Vitro, Western Blot

Fig. 10. Correlation between mitogenic potential and binding to CK2α. ( A ) NIH 3T3 cells were serum starved for 24 h before 10 U/ml heparin and different amounts of FGF-1, mutants of FGF-1, FGF-2 or the corresponding amount of MBP as in the fusion protein constructs were added. The cells were incubated for another 24 h, the last 6 h in the presence of [ 3 H]thymidine. The cell-associated, TCA-precipitable radioactivity was then measured. The data shown are from a representative experiment out of eight. ( B ) MBP–FGF-2 (lane 1), MBP–FGF-1 (lane 2) or MBP fused with the indicated FGF-1 mutants (lanes 3–10) were incubated for 2 h at 4°C with GST–CK2α bound to glutathione–Sepharose. The beads were washed and the bound proteins were analysed by western blotting with an antibody against MBP. Indicated above each lane is the mitogenicity of the corresponding mutant as a percentage of the mitogenicity of wild-type FGF-1, which is estimated from at least three independent DNA stimulation experiments. The amount of growth factor necessary to induce half-maximal stimulation of DNA synthesis was determined. The mitogenicity is taken as the reciprocal of the amount of growth factor needed and normalized to 100 for the mitogenic activity of wild-type FGF-1. ( C ) U2OS cells were lysed and incubated for 1 h at 4°C with Sepharose-bound MBP–FGF-1 or with the indicated mutant. The beads were washed, and adsorbed proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody. The mitogenicity was calculated as in (B). ( D ) NIH 3T3 cells were starved for 26 h and then 10 U/ml heparin and 5 ng/ml FGF-1, mutants of FGF-1, FGF-2 or MBP were added. The cells were stimulated for 10 min, washed, lysed with SDS sample buffer and analysed by western blotting with antibodies against phosphorylated p42/p44 MAP kinase (upper panel) and total p42/p44 MAP kinase (lower panel). The data shown are from a representative experiment.
Figure Legend Snippet: Fig. 10. Correlation between mitogenic potential and binding to CK2α. ( A ) NIH 3T3 cells were serum starved for 24 h before 10 U/ml heparin and different amounts of FGF-1, mutants of FGF-1, FGF-2 or the corresponding amount of MBP as in the fusion protein constructs were added. The cells were incubated for another 24 h, the last 6 h in the presence of [ 3 H]thymidine. The cell-associated, TCA-precipitable radioactivity was then measured. The data shown are from a representative experiment out of eight. ( B ) MBP–FGF-2 (lane 1), MBP–FGF-1 (lane 2) or MBP fused with the indicated FGF-1 mutants (lanes 3–10) were incubated for 2 h at 4°C with GST–CK2α bound to glutathione–Sepharose. The beads were washed and the bound proteins were analysed by western blotting with an antibody against MBP. Indicated above each lane is the mitogenicity of the corresponding mutant as a percentage of the mitogenicity of wild-type FGF-1, which is estimated from at least three independent DNA stimulation experiments. The amount of growth factor necessary to induce half-maximal stimulation of DNA synthesis was determined. The mitogenicity is taken as the reciprocal of the amount of growth factor needed and normalized to 100 for the mitogenic activity of wild-type FGF-1. ( C ) U2OS cells were lysed and incubated for 1 h at 4°C with Sepharose-bound MBP–FGF-1 or with the indicated mutant. The beads were washed, and adsorbed proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody. The mitogenicity was calculated as in (B). ( D ) NIH 3T3 cells were starved for 26 h and then 10 U/ml heparin and 5 ng/ml FGF-1, mutants of FGF-1, FGF-2 or MBP were added. The cells were stimulated for 10 min, washed, lysed with SDS sample buffer and analysed by western blotting with antibodies against phosphorylated p42/p44 MAP kinase (upper panel) and total p42/p44 MAP kinase (lower panel). The data shown are from a representative experiment.

Techniques Used: Binding Assay, Construct, Incubation, Radioactivity, Western Blot, Mutagenesis, DNA Synthesis, Activity Assay, SDS Page

40) Product Images from "Phenotypic Mutants of the Intracellular Actinomycete Rhodococcusequi Created by In Vivo Himar1 Transposon Mutagenesis"

Article Title: Phenotypic Mutants of the Intracellular Actinomycete Rhodococcusequi Created by In Vivo Himar1 Transposon Mutagenesis

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.8.2644-2652.2003

Schematic representation of in vivo transposition mutagenesis in R . equi . Plasmid pJA2.2, containing the Himar1 transposase, a ts origin of replication ( oriM ), and a Himar1 element marked with a hygromycin resistance gene and carrying an E . coli origin of replication ( oriE ), was electroporated into R . equi , and transformants were recovered on hygromycin plates at 30°C (permissive temperature) to allow for transposition (as indicated by the dashed arrow). Transformants were subsequently scraped from the plates and resuspended in medium, and aliquots were replated on hygromycin-containing agar. Plates were incubated at 42°C (nonpermissive temperature) to isolate transposon insertion mutants.
Figure Legend Snippet: Schematic representation of in vivo transposition mutagenesis in R . equi . Plasmid pJA2.2, containing the Himar1 transposase, a ts origin of replication ( oriM ), and a Himar1 element marked with a hygromycin resistance gene and carrying an E . coli origin of replication ( oriE ), was electroporated into R . equi , and transformants were recovered on hygromycin plates at 30°C (permissive temperature) to allow for transposition (as indicated by the dashed arrow). Transformants were subsequently scraped from the plates and resuspended in medium, and aliquots were replated on hygromycin-containing agar. Plates were incubated at 42°C (nonpermissive temperature) to isolate transposon insertion mutants.

Techniques Used: In Vivo, Mutagenesis, Plasmid Preparation, Incubation

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Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome
Article Snippet: .. Generation of DNMT1 plasmids The mammalian expression vector containing the long isoform of wild-type human DNMT1 with N-terminal GFP tag in pEGFP-C1 plasmid (Clontech) was obtained from Prof. Heinrich Leonhardt (Ludwig-Maximilians-University Biocentre, Munich). .. The pEGFP-C1-DNMT1 construct was transfected into competent DH5-alpha cells, and construct fidelity was assessed by sequencing plasmid DNA using primers designed to DNMT1 exons as described in the supplementary information (cDNMT1 sequencing primers).

Article Title: Molecular Cloning and Expression of MnGST-1 and MnGST-2 from Oriental River Prawn, Macrobrachium nipponense, in Response to Hypoxia and Reoxygenation
Article Snippet: .. Quantitative Real-Time PCR (QPCR) Analysis of MnGST-1 and MnGST-2 Expression The mRNA levels of MnGST-1 and MnGST-2 in different tissues and following different hypoxic treatments were measured by QPCR. cDNAs from different tissues and following different hypoxia treatments were synthesized from total DNA-free RNA (1 μg) using a Prime Script RT reagent kit (TaKaRa, Kusatsu, Japan) following the manufacturer’s instructions. .. Reactions were executed on a Bio-Rad iCycler i Q5 Real-Time PCR system (Bio-Rad, USA) using primers listed in .

Y2H Assay:

Article Title: GSK3β-mediated Ser156 phosphorylation modulates a BH3-like domain in BCL2L12 during TMZ-induced apoptosis and autophagy in glioma cells
Article Snippet: .. Cloning BCL2L12, BCL-XL, BCL2, BCL2 associated X (Bax), Beclin-1, induced myeloid leukemia cell differentiation protein MCL-1 (Mcl-1), and GSK3β were cloned into pACT2 and pAS2-1 vectors (Takara Bio Inc., Otsu, Japan) for yeast two-hybrid assay or into the pEGFP-C1 vector for overexpression. .. Polymerase chain reaction (PCR) technique was used to generate DNA fragments containing the desired gene sequences utilizing primers designed to contain the Xho I and Bam HI restriction enzyme recognition sites.

Sequencing:

Article Title: Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels
Article Snippet: .. Molecular Biology Human TREK1 (KCNK2, genbank accession number AAH69462.1 ) and TRAAK (KCNK4, NCBI Reference Sequence: NP_201567.1) were cloned into pIRES2-eGFP vector (Clontech). ..

DNA Fragmentation Assay:

Article Title: Cell-based chemical fingerprinting identifies telomeres and lamin A as modifiers of DNA damage response in cancer cells
Article Snippet: .. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay for detection of apoptotic cells was performed using the ApoAlert DNA Fragmentation Assay Kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. .. Quantitation of the sub-G1 fractions was performed by propidium iodide staining followed by flow cytometry, as described previously .

Purification:

Article Title: MiR‐486 promotes proliferation and suppresses apoptosis in myeloid cells by targeting Cebpa in vitro, et al. MiR‐486 promotes proliferation and suppresses apoptosis in myeloid cells by targeting Cebpa in vitro
Article Snippet: .. The wild type 3′‐UTR fragment of Cebpa was amplified from mouse genomic DNA using PrimeSTAR® HS DNA Polymerase (Catalog number: R010A; TakaRa, Tokyo, Japanese) and was purified from agarose gels using TIANgel Midi Purification Kit (Catalog number: DP‐209; TakaRa, Japanese). ..

Over Expression:

Article Title: GSK3β-mediated Ser156 phosphorylation modulates a BH3-like domain in BCL2L12 during TMZ-induced apoptosis and autophagy in glioma cells
Article Snippet: .. Cloning BCL2L12, BCL-XL, BCL2, BCL2 associated X (Bax), Beclin-1, induced myeloid leukemia cell differentiation protein MCL-1 (Mcl-1), and GSK3β were cloned into pACT2 and pAS2-1 vectors (Takara Bio Inc., Otsu, Japan) for yeast two-hybrid assay or into the pEGFP-C1 vector for overexpression. .. Polymerase chain reaction (PCR) technique was used to generate DNA fragments containing the desired gene sequences utilizing primers designed to contain the Xho I and Bam HI restriction enzyme recognition sites.

Plasmid Preparation:

Article Title: GSK3β-mediated Ser156 phosphorylation modulates a BH3-like domain in BCL2L12 during TMZ-induced apoptosis and autophagy in glioma cells
Article Snippet: .. Cloning BCL2L12, BCL-XL, BCL2, BCL2 associated X (Bax), Beclin-1, induced myeloid leukemia cell differentiation protein MCL-1 (Mcl-1), and GSK3β were cloned into pACT2 and pAS2-1 vectors (Takara Bio Inc., Otsu, Japan) for yeast two-hybrid assay or into the pEGFP-C1 vector for overexpression. .. Polymerase chain reaction (PCR) technique was used to generate DNA fragments containing the desired gene sequences utilizing primers designed to contain the Xho I and Bam HI restriction enzyme recognition sites.

Article Title: Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels
Article Snippet: .. Molecular Biology Human TREK1 (KCNK2, genbank accession number AAH69462.1 ) and TRAAK (KCNK4, NCBI Reference Sequence: NP_201567.1) were cloned into pIRES2-eGFP vector (Clontech). ..

Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome
Article Snippet: .. Generation of DNMT1 plasmids The mammalian expression vector containing the long isoform of wild-type human DNMT1 with N-terminal GFP tag in pEGFP-C1 plasmid (Clontech) was obtained from Prof. Heinrich Leonhardt (Ludwig-Maximilians-University Biocentre, Munich). .. The pEGFP-C1-DNMT1 construct was transfected into competent DH5-alpha cells, and construct fidelity was assessed by sequencing plasmid DNA using primers designed to DNMT1 exons as described in the supplementary information (cDNMT1 sequencing primers).

Article Title: Silkworm genetic sexing through W chromosome-linked, targeted gene integration
Article Snippet: .. The HAs (1,000 bp) flanking TALEN sites were amplified using genomic DNA as the template and cloned into pGEMT_HR5-IE1-DsRed2-SV40 to generate donor A. Donor B (pGEMT_L-Homo_HR5-IE1-EGFP-SV40_Nos-Cas9-SV40_R-Homo): An HR5-IE1-EGFP-SV40 cassette was subcloned into the pGEMT-simple vector (Takara) to generate pGEMT_HR5-IE1-EGFP-SV40. .. The same HAs as used in donor A were cloned into pGEMT_HR5-IE1-EGFP-SV40.

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  • 88
    TaKaRa recombinants genomic dna
    The detection of recombinant in the two transformations by PCR amplification. ( a ) The detection of recombinant in the first transformation by PCR amplification with primers Cm-F/R. M: Trans <t>DNA</t> Marker II. 1: Negative control, the PCR amplification band of Cm r gene with the genomic DNA of A. <t>limacinum</t> OUC88 as the template. 2: The PCR amplification band of Cm r gene with the genomic DNA of A. limacinum OUC_CG as the template. 3: Positive control, the PCR amplification band of Cm r gene with the plasmid pACYCDuet-1 as the template. B: Blank control; ( b ) The detection of recombinant in the second transformation by PCR amplification with primers 18S+-F/P pgk -R. M: Trans 15K DNA Marker. 1: The PCR amplification band with genomic DNA of A. limacinum OUC88 as the template; 2: The PCR amplification band (3655 bp) of 18S+- lox P- Cm r - lox P-P pgk with genomic DNA of A. limacinum OUC_CG as the template; 3: The PCR amplification band (2185 bp) of 18S+- lox P- lox P-P pgk with genomic DNA of A. limacinum OUC_EG as the template; B: Blank control.
    Recombinants Genomic Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa 16s rrna gene sequences dna extraction
    Phylogenetic tree derived from <t>16S</t> <t>rRNA</t> gene sequences, reconstructed using the maximum-likelihood method. The sequence of Trueperella pyogenes was used as an outgroup. Numbers at the branch points are percentages of 1,000 bootstrap replicates. Bar=0.02 substitutions per site.
    16s Rrna Gene Sequences Dna Extraction, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa residual dna
    Transcriptional changes in PD-1, PD-L1, PD-L2, PTEN, CTLA-4, LAG-3, and Foxp3 levels in postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy and PMWS-affected pigs, and total <t>RNA</t> was extracted from them. Residual <t>DNA</t> was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA. Results are expressed as relative fold changes of PD-1, PD-L1, PD-L2, PTEN CTLA-4, LAG-3, and Foxp3 mRNA using β-actin as an internal control. Values are shown as the mean±standard deviation ( SD ) from triplicate samples. Differences between groups were considered statistically significant at p
    Residual Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    TaKaRa dna double stranded breaks
    Radioresistance of cells under low-glucose and hypoxic conditions. ( a – e) With ( d ) or without ( a – c , e ) transient transfection using an empty vector or pcDNA4/p27 Kip1 , <t>HEK293/EGFP-53BP1-M</t> ( a , b , d , e ) and A549 ( c ) cells were cultured in medium containing a low (L: 0.45 g/l) or high (H: 4.5 g/l) concentration of glucose under normoxic (20% oxygen) or hypoxic (0.02% oxygen) conditions for 20 h, and treated with the indicated dose of X-radiation. ( a , d) The radiation-induced <t>DNA</t> double-stranded breaks (DNA DSBs) were then observed as EGFP-53BP1-M foci under a fluorescence microscope. The numbers of DNA DSB per nucleus in each group were quantified as the mean of 50 nuclei in 10 independent fields. Results are the mean±s.d. * P
    Dna Double Stranded Breaks, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The detection of recombinant in the two transformations by PCR amplification. ( a ) The detection of recombinant in the first transformation by PCR amplification with primers Cm-F/R. M: Trans DNA Marker II. 1: Negative control, the PCR amplification band of Cm r gene with the genomic DNA of A. limacinum OUC88 as the template. 2: The PCR amplification band of Cm r gene with the genomic DNA of A. limacinum OUC_CG as the template. 3: Positive control, the PCR amplification band of Cm r gene with the plasmid pACYCDuet-1 as the template. B: Blank control; ( b ) The detection of recombinant in the second transformation by PCR amplification with primers 18S+-F/P pgk -R. M: Trans 15K DNA Marker. 1: The PCR amplification band with genomic DNA of A. limacinum OUC88 as the template; 2: The PCR amplification band (3655 bp) of 18S+- lox P- Cm r - lox P-P pgk with genomic DNA of A. limacinum OUC_CG as the template; 3: The PCR amplification band (2185 bp) of 18S+- lox P- lox P-P pgk with genomic DNA of A. limacinum OUC_EG as the template; B: Blank control.

    Journal: Molecules

    Article Title: Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum

    doi: 10.3390/molecules200610110

    Figure Lengend Snippet: The detection of recombinant in the two transformations by PCR amplification. ( a ) The detection of recombinant in the first transformation by PCR amplification with primers Cm-F/R. M: Trans DNA Marker II. 1: Negative control, the PCR amplification band of Cm r gene with the genomic DNA of A. limacinum OUC88 as the template. 2: The PCR amplification band of Cm r gene with the genomic DNA of A. limacinum OUC_CG as the template. 3: Positive control, the PCR amplification band of Cm r gene with the plasmid pACYCDuet-1 as the template. B: Blank control; ( b ) The detection of recombinant in the second transformation by PCR amplification with primers 18S+-F/P pgk -R. M: Trans 15K DNA Marker. 1: The PCR amplification band with genomic DNA of A. limacinum OUC88 as the template; 2: The PCR amplification band (3655 bp) of 18S+- lox P- Cm r - lox P-P pgk with genomic DNA of A. limacinum OUC_CG as the template; 3: The PCR amplification band (2185 bp) of 18S+- lox P- lox P-P pgk with genomic DNA of A. limacinum OUC_EG as the template; B: Blank control.

    Article Snippet: Southern Blotting of the Recombinants Genomic DNA was extracted from A. limacinum OUC88 and the transformants respectively using a Yeast DNAiso Kit (D9082, Takara, Otsu, Japan).

    Techniques: Recombinant, Polymerase Chain Reaction, Amplification, Transformation Assay, Marker, Negative Control, Positive Control, Plasmid Preparation

    Southern blotting analysis of egfp in the genomic DNA of transformants ( a ) The results of total DNA digested by restriction enzymes. Molecular-size of markers (bp) is shown on the left. The genomic DNA of A. limacinum OUC88 (WT), A. limacinum OUC_CG and A. limacinum OUC_EG were double digested with either Bam HI/ Eco RI or Hin dIII/ Xba I; ( b ) The southern-blot hybridization results for the egfp gene. The number of fragment copies as deduced from the comparison of the hybridization bands are depicted for the transformants.

    Journal: Molecules

    Article Title: Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum

    doi: 10.3390/molecules200610110

    Figure Lengend Snippet: Southern blotting analysis of egfp in the genomic DNA of transformants ( a ) The results of total DNA digested by restriction enzymes. Molecular-size of markers (bp) is shown on the left. The genomic DNA of A. limacinum OUC88 (WT), A. limacinum OUC_CG and A. limacinum OUC_EG were double digested with either Bam HI/ Eco RI or Hin dIII/ Xba I; ( b ) The southern-blot hybridization results for the egfp gene. The number of fragment copies as deduced from the comparison of the hybridization bands are depicted for the transformants.

    Article Snippet: Southern Blotting of the Recombinants Genomic DNA was extracted from A. limacinum OUC88 and the transformants respectively using a Yeast DNAiso Kit (D9082, Takara, Otsu, Japan).

    Techniques: Southern Blot, Hybridization

    Phylogenetic tree derived from 16S rRNA gene sequences, reconstructed using the maximum-likelihood method. The sequence of Trueperella pyogenes was used as an outgroup. Numbers at the branch points are percentages of 1,000 bootstrap replicates. Bar=0.02 substitutions per site.

    Journal: The Journal of Veterinary Medical Science

    Article Title: Actinomyces denticolens as a causative agent of actinomycosis in animals

    doi: 10.1292/jvms.18-0207

    Figure Lengend Snippet: Phylogenetic tree derived from 16S rRNA gene sequences, reconstructed using the maximum-likelihood method. The sequence of Trueperella pyogenes was used as an outgroup. Numbers at the branch points are percentages of 1,000 bootstrap replicates. Bar=0.02 substitutions per site.

    Article Snippet: Analysis of 16S rRNA gene sequences DNA extraction of 4 Higuchi strains, Actinomyces denticolens DSM 20671T and strain Chiba 101 were carried out using GenTEL with high recovery (TaKaRa Bio, Kusatsu, Japan) according to the manufacturer’s protocol.

    Techniques: Derivative Assay, Sequencing

    Transcriptional changes in PD-1, PD-L1, PD-L2, PTEN, CTLA-4, LAG-3, and Foxp3 levels in postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy and PMWS-affected pigs, and total RNA was extracted from them. Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA. Results are expressed as relative fold changes of PD-1, PD-L1, PD-L2, PTEN CTLA-4, LAG-3, and Foxp3 mRNA using β-actin as an internal control. Values are shown as the mean±standard deviation ( SD ) from triplicate samples. Differences between groups were considered statistically significant at p

    Journal: Viral Immunology

    Article Title: Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome

    doi: 10.1089/vim.2014.0097

    Figure Lengend Snippet: Transcriptional changes in PD-1, PD-L1, PD-L2, PTEN, CTLA-4, LAG-3, and Foxp3 levels in postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy and PMWS-affected pigs, and total RNA was extracted from them. Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA. Results are expressed as relative fold changes of PD-1, PD-L1, PD-L2, PTEN CTLA-4, LAG-3, and Foxp3 mRNA using β-actin as an internal control. Values are shown as the mean±standard deviation ( SD ) from triplicate samples. Differences between groups were considered statistically significant at p

    Article Snippet: Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA with a PrimeScript RT Reagent Kit with gDNA eraser (perfect real-time; Takara, code: RR047A) according to the manufacturer's protocol.

    Techniques: Isolation, Synthesized, Standard Deviation

    Transcriptional changes in IL-10, IL-2, and IFN-γ levels in PMWS-affected pigs. PBMCs were isolated from healthy and PMWS-affected pigs, and total RNA was extracted from them. Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA. Results are expressed as relative fold changes of IL-10, IL-2, and IFN-γ mRNA using β-actin as an internal control. Values are shown as the mean± SD from triplicate samples. Differences between groups were considered statistically significant at p

    Journal: Viral Immunology

    Article Title: Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome

    doi: 10.1089/vim.2014.0097

    Figure Lengend Snippet: Transcriptional changes in IL-10, IL-2, and IFN-γ levels in PMWS-affected pigs. PBMCs were isolated from healthy and PMWS-affected pigs, and total RNA was extracted from them. Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA. Results are expressed as relative fold changes of IL-10, IL-2, and IFN-γ mRNA using β-actin as an internal control. Values are shown as the mean± SD from triplicate samples. Differences between groups were considered statistically significant at p

    Article Snippet: Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA with a PrimeScript RT Reagent Kit with gDNA eraser (perfect real-time; Takara, code: RR047A) according to the manufacturer's protocol.

    Techniques: Isolation, Synthesized

    Radioresistance of cells under low-glucose and hypoxic conditions. ( a – e) With ( d ) or without ( a – c , e ) transient transfection using an empty vector or pcDNA4/p27 Kip1 , HEK293/EGFP-53BP1-M ( a , b , d , e ) and A549 ( c ) cells were cultured in medium containing a low (L: 0.45 g/l) or high (H: 4.5 g/l) concentration of glucose under normoxic (20% oxygen) or hypoxic (0.02% oxygen) conditions for 20 h, and treated with the indicated dose of X-radiation. ( a , d) The radiation-induced DNA double-stranded breaks (DNA DSBs) were then observed as EGFP-53BP1-M foci under a fluorescence microscope. The numbers of DNA DSB per nucleus in each group were quantified as the mean of 50 nuclei in 10 independent fields. Results are the mean±s.d. * P

    Journal: Oncogene

    Article Title: Involvement of decreased hypoxia-inducible factor 1 activity and resultant G1-S cell cycle transition in radioresistance of perinecrotic tumor cells

    doi: 10.1038/onc.2012.223

    Figure Lengend Snippet: Radioresistance of cells under low-glucose and hypoxic conditions. ( a – e) With ( d ) or without ( a – c , e ) transient transfection using an empty vector or pcDNA4/p27 Kip1 , HEK293/EGFP-53BP1-M ( a , b , d , e ) and A549 ( c ) cells were cultured in medium containing a low (L: 0.45 g/l) or high (H: 4.5 g/l) concentration of glucose under normoxic (20% oxygen) or hypoxic (0.02% oxygen) conditions for 20 h, and treated with the indicated dose of X-radiation. ( a , d) The radiation-induced DNA double-stranded breaks (DNA DSBs) were then observed as EGFP-53BP1-M foci under a fluorescence microscope. The numbers of DNA DSB per nucleus in each group were quantified as the mean of 50 nuclei in 10 independent fields. Results are the mean±s.d. * P

    Article Snippet: To construct the plasmid pEGFP-53BP1-M, the coding sequence of the M domain of the p53-binding protein 1 (53BP1 ) gene, which comprises residues 1220–1703 and is responsible for the recruitment of 53BP1 to DNA double-stranded breaks, was amplified by PCR and inserted between the Xho I-Hind III sites of pEGFP-C1 (Clontech, Palo Alto, CA, USA).

    Techniques: Transfection, Plasmid Preparation, Cell Culture, Concentration Assay, Fluorescence, Microscopy