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Extracellular [Pi] and inhibitors of Pi transport affect PIT-2V functions. (A to C) Control experiments performed with 1 mM [Pi] in the absence of drug. (A) Increased Pi uptake in CHO-PIT-2V cells (CRAV) compared to parental CHO cells (CHO). (B) Flow cytometry revealed amphotropic envelopes bound to CHO-PIT-2V cells (solid line and no asterisks) but not to parental CHO cells (broken line). Asterisks indicate signals without viral envelopes. (C) Exposure to an amphotropic lacZ retrovirus vector induced β-galactosidase-positive foci on CHO-PIT-2V cells but not parental CHO cells (data not shown). Equal numbers were scored with various concentrations (x axis) of NaCl (closed circles) or NaHCO2 (open circles). (D to F) CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were switched to medium containing the indicated [Pi] (x axis) for 30 min at 37°C. (D) NaH232PO4 uptake was affected by changing the Na2HPO4 concentration (closed circles; sulfate, 0.4 mM) but not the Na2SO4 concentration (open circles; phosphate, 0 mM). (E) Equivalent binding of amphotropic envelopes at 0 mM (solid line) and 40 mM (broken line) [Pi]. (F) Infection efficiency for CHO-PIT-2V cells (closed circles; 100%, 97 ± 9 FFUs) or CEAR 13 cells (closed squares; 100%, 265 ± 21 FFUs) with an amphotropic vector and different [Pi]. In contrast, infection of CHO-PIT-2V cells with a VSV-G pseudotype (open circles; 100%, 90 ± 7 FFUs) or of CEAR 13 cells with an ecotropic pseudotype (open squares; 100%, 179 ± 5 FFUs) was unaffected. (G to I) To CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were added the indicated concentrations of PCMB (squares) or <t>PCMBS</t> (circles) for 30 min at 37°C. (G) Drugs inhibited Na2HPO4 uptake. (H) Unmodified binding of amphotropic envelopes (solid line, no drug; broken line, 200 μM PCMB; dotted line, 200 μM PCMBS). (I) Drugs inhibited infection with an amphotropic (Ampho) vector (closed symbols; 100%, 125 ± 12 FFUs) but not with a VSV-G pseudotype (open symbols; 100%, 95 ± 17 FFUs). Data are means ± standard deviations for triplicate experiments.
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Extracellular [Pi] and inhibitors of Pi transport affect PIT-2V functions. (A to C) Control experiments performed with 1 mM [Pi] in the absence of drug. (A) Increased Pi uptake in CHO-PIT-2V cells (CRAV) compared to parental CHO cells (CHO). (B) Flow cytometry revealed amphotropic envelopes bound to CHO-PIT-2V cells (solid line and no asterisks) but not to parental CHO cells (broken line). Asterisks indicate signals without viral envelopes. (C) Exposure to an amphotropic lacZ retrovirus vector induced β-galactosidase-positive foci on CHO-PIT-2V cells but not parental CHO cells (data not shown). Equal numbers were scored with various concentrations (x axis) of NaCl (closed circles) or NaHCO2 (open circles). (D to F) CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were switched to medium containing the indicated [Pi] (x axis) for 30 min at 37°C. (D) NaH232PO4 uptake was affected by changing the Na2HPO4 concentration (closed circles; sulfate, 0.4 mM) but not the Na2SO4 concentration (open circles; phosphate, 0 mM). (E) Equivalent binding of amphotropic envelopes at 0 mM (solid line) and 40 mM (broken line) [Pi]. (F) Infection efficiency for CHO-PIT-2V cells (closed circles; 100%, 97 ± 9 FFUs) or CEAR 13 cells (closed squares; 100%, 265 ± 21 FFUs) with an amphotropic vector and different [Pi]. In contrast, infection of CHO-PIT-2V cells with a VSV-G pseudotype (open circles; 100%, 90 ± 7 FFUs) or of CEAR 13 cells with an ecotropic pseudotype (open squares; 100%, 179 ± 5 FFUs) was unaffected. (G to I) To CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were added the indicated concentrations of PCMB (squares) or <t>PCMBS</t> (circles) for 30 min at 37°C. (G) Drugs inhibited Na2HPO4 uptake. (H) Unmodified binding of amphotropic envelopes (solid line, no drug; broken line, 200 μM PCMB; dotted line, 200 μM PCMBS). (I) Drugs inhibited infection with an amphotropic (Ampho) vector (closed symbols; 100%, 125 ± 12 FFUs) but not with a VSV-G pseudotype (open symbols; 100%, 95 ± 17 FFUs). Data are means ± standard deviations for triplicate experiments.
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Extracellular [Pi] and inhibitors of Pi transport affect PIT-2V functions. (A to C) Control experiments performed with 1 mM [Pi] in the absence of drug. (A) Increased Pi uptake in CHO-PIT-2V cells (CRAV) compared to parental CHO cells (CHO). (B) Flow cytometry revealed amphotropic envelopes bound to CHO-PIT-2V cells (solid line and no asterisks) but not to parental CHO cells (broken line). Asterisks indicate signals without viral envelopes. (C) Exposure to an amphotropic lacZ retrovirus vector induced β-galactosidase-positive foci on CHO-PIT-2V cells but not parental CHO cells (data not shown). Equal numbers were scored with various concentrations (x axis) of NaCl (closed circles) or NaHCO2 (open circles). (D to F) CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were switched to medium containing the indicated [Pi] (x axis) for 30 min at 37°C. (D) NaH232PO4 uptake was affected by changing the Na2HPO4 concentration (closed circles; sulfate, 0.4 mM) but not the Na2SO4 concentration (open circles; phosphate, 0 mM). (E) Equivalent binding of amphotropic envelopes at 0 mM (solid line) and 40 mM (broken line) [Pi]. (F) Infection efficiency for CHO-PIT-2V cells (closed circles; 100%, 97 ± 9 FFUs) or CEAR 13 cells (closed squares; 100%, 265 ± 21 FFUs) with an amphotropic vector and different [Pi]. In contrast, infection of CHO-PIT-2V cells with a VSV-G pseudotype (open circles; 100%, 90 ± 7 FFUs) or of CEAR 13 cells with an ecotropic pseudotype (open squares; 100%, 179 ± 5 FFUs) was unaffected. (G to I) To CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were added the indicated concentrations of PCMB (squares) or <t>PCMBS</t> (circles) for 30 min at 37°C. (G) Drugs inhibited Na2HPO4 uptake. (H) Unmodified binding of amphotropic envelopes (solid line, no drug; broken line, 200 μM PCMB; dotted line, 200 μM PCMBS). (I) Drugs inhibited infection with an amphotropic (Ampho) vector (closed symbols; 100%, 125 ± 12 FFUs) but not with a VSV-G pseudotype (open symbols; 100%, 95 ± 17 FFUs). Data are means ± standard deviations for triplicate experiments.
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Extracellular [Pi] and inhibitors of Pi transport affect PIT-2V functions. (A to C) Control experiments performed with 1 mM [Pi] in the absence of drug. (A) Increased Pi uptake in CHO-PIT-2V cells (CRAV) compared to parental CHO cells (CHO). (B) Flow cytometry revealed amphotropic envelopes bound to CHO-PIT-2V cells (solid line and no asterisks) but not to parental CHO cells (broken line). Asterisks indicate signals without viral envelopes. (C) Exposure to an amphotropic lacZ retrovirus vector induced β-galactosidase-positive foci on CHO-PIT-2V cells but not parental CHO cells (data not shown). Equal numbers were scored with various concentrations (x axis) of NaCl (closed circles) or NaHCO2 (open circles). (D to F) CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were switched to medium containing the indicated [Pi] (x axis) for 30 min at 37°C. (D) NaH232PO4 uptake was affected by changing the Na2HPO4 concentration (closed circles; sulfate, 0.4 mM) but not the Na2SO4 concentration (open circles; phosphate, 0 mM). (E) Equivalent binding of amphotropic envelopes at 0 mM (solid line) and 40 mM (broken line) [Pi]. (F) Infection efficiency for CHO-PIT-2V cells (closed circles; 100%, 97 ± 9 FFUs) or CEAR 13 cells (closed squares; 100%, 265 ± 21 FFUs) with an amphotropic vector and different [Pi]. In contrast, infection of CHO-PIT-2V cells with a VSV-G pseudotype (open circles; 100%, 90 ± 7 FFUs) or of CEAR 13 cells with an ecotropic pseudotype (open squares; 100%, 179 ± 5 FFUs) was unaffected. (G to I) To CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were added the indicated concentrations of PCMB (squares) or <t>PCMBS</t> (circles) for 30 min at 37°C. (G) Drugs inhibited Na2HPO4 uptake. (H) Unmodified binding of amphotropic envelopes (solid line, no drug; broken line, 200 μM PCMB; dotted line, 200 μM PCMBS). (I) Drugs inhibited infection with an amphotropic (Ampho) vector (closed symbols; 100%, 125 ± 12 FFUs) but not with a VSV-G pseudotype (open symbols; 100%, 95 ± 17 FFUs). Data are means ± standard deviations for triplicate experiments.
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Image Search Results


Extracellular [Pi] and inhibitors of Pi transport affect PIT-2V functions. (A to C) Control experiments performed with 1 mM [Pi] in the absence of drug. (A) Increased Pi uptake in CHO-PIT-2V cells (CRAV) compared to parental CHO cells (CHO). (B) Flow cytometry revealed amphotropic envelopes bound to CHO-PIT-2V cells (solid line and no asterisks) but not to parental CHO cells (broken line). Asterisks indicate signals without viral envelopes. (C) Exposure to an amphotropic lacZ retrovirus vector induced β-galactosidase-positive foci on CHO-PIT-2V cells but not parental CHO cells (data not shown). Equal numbers were scored with various concentrations (x axis) of NaCl (closed circles) or NaHCO2 (open circles). (D to F) CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were switched to medium containing the indicated [Pi] (x axis) for 30 min at 37°C. (D) NaH232PO4 uptake was affected by changing the Na2HPO4 concentration (closed circles; sulfate, 0.4 mM) but not the Na2SO4 concentration (open circles; phosphate, 0 mM). (E) Equivalent binding of amphotropic envelopes at 0 mM (solid line) and 40 mM (broken line) [Pi]. (F) Infection efficiency for CHO-PIT-2V cells (closed circles; 100%, 97 ± 9 FFUs) or CEAR 13 cells (closed squares; 100%, 265 ± 21 FFUs) with an amphotropic vector and different [Pi]. In contrast, infection of CHO-PIT-2V cells with a VSV-G pseudotype (open circles; 100%, 90 ± 7 FFUs) or of CEAR 13 cells with an ecotropic pseudotype (open squares; 100%, 179 ± 5 FFUs) was unaffected. (G to I) To CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were added the indicated concentrations of PCMB (squares) or PCMBS (circles) for 30 min at 37°C. (G) Drugs inhibited Na2HPO4 uptake. (H) Unmodified binding of amphotropic envelopes (solid line, no drug; broken line, 200 μM PCMB; dotted line, 200 μM PCMBS). (I) Drugs inhibited infection with an amphotropic (Ampho) vector (closed symbols; 100%, 125 ± 12 FFUs) but not with a VSV-G pseudotype (open symbols; 100%, 95 ± 17 FFUs). Data are means ± standard deviations for triplicate experiments.

Journal:

Article Title: Modulation of Phosphate Uptake and Amphotropic Murine Leukemia Virus Entry by Posttranslational Modifications of PIT-2

doi:

Figure Lengend Snippet: Extracellular [Pi] and inhibitors of Pi transport affect PIT-2V functions. (A to C) Control experiments performed with 1 mM [Pi] in the absence of drug. (A) Increased Pi uptake in CHO-PIT-2V cells (CRAV) compared to parental CHO cells (CHO). (B) Flow cytometry revealed amphotropic envelopes bound to CHO-PIT-2V cells (solid line and no asterisks) but not to parental CHO cells (broken line). Asterisks indicate signals without viral envelopes. (C) Exposure to an amphotropic lacZ retrovirus vector induced β-galactosidase-positive foci on CHO-PIT-2V cells but not parental CHO cells (data not shown). Equal numbers were scored with various concentrations (x axis) of NaCl (closed circles) or NaHCO2 (open circles). (D to F) CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were switched to medium containing the indicated [Pi] (x axis) for 30 min at 37°C. (D) NaH232PO4 uptake was affected by changing the Na2HPO4 concentration (closed circles; sulfate, 0.4 mM) but not the Na2SO4 concentration (open circles; phosphate, 0 mM). (E) Equivalent binding of amphotropic envelopes at 0 mM (solid line) and 40 mM (broken line) [Pi]. (F) Infection efficiency for CHO-PIT-2V cells (closed circles; 100%, 97 ± 9 FFUs) or CEAR 13 cells (closed squares; 100%, 265 ± 21 FFUs) with an amphotropic vector and different [Pi]. In contrast, infection of CHO-PIT-2V cells with a VSV-G pseudotype (open circles; 100%, 90 ± 7 FFUs) or of CEAR 13 cells with an ecotropic pseudotype (open squares; 100%, 179 ± 5 FFUs) was unaffected. (G to I) To CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were added the indicated concentrations of PCMB (squares) or PCMBS (circles) for 30 min at 37°C. (G) Drugs inhibited Na2HPO4 uptake. (H) Unmodified binding of amphotropic envelopes (solid line, no drug; broken line, 200 μM PCMB; dotted line, 200 μM PCMBS). (I) Drugs inhibited infection with an amphotropic (Ampho) vector (closed symbols; 100%, 125 ± 12 FFUs) but not with a VSV-G pseudotype (open symbols; 100%, 95 ± 17 FFUs). Data are means ± standard deviations for triplicate experiments.

Article Snippet: Cytochalasin D, lysophosphatidic acid (LPA), 4-chloromercuribenzoic acid (PCMB), and 4-(chloromercuri)benzene-sulfonic acid (PCMBS) were from Sigma-Aldrich (St. Louis, Mo.).

Techniques: Flow Cytometry, Plasmid Preparation, Concentration Assay, Binding Assay, Infection

Effects of extracellular [Pi], PCMB, PCMBS, and LPA on the actin network. CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were switched to the indicated conditions for 30 min, stained with phalloidin-FITC, and examined by fluorescence microscopy. Bar, 5 μm.

Journal:

Article Title: Modulation of Phosphate Uptake and Amphotropic Murine Leukemia Virus Entry by Posttranslational Modifications of PIT-2

doi:

Figure Lengend Snippet: Effects of extracellular [Pi], PCMB, PCMBS, and LPA on the actin network. CHO-PIT-2V cells maintained at physiological [Pi] (1 mM) were switched to the indicated conditions for 30 min, stained with phalloidin-FITC, and examined by fluorescence microscopy. Bar, 5 μm.

Article Snippet: Cytochalasin D, lysophosphatidic acid (LPA), 4-chloromercuribenzoic acid (PCMB), and 4-(chloromercuri)benzene-sulfonic acid (PCMBS) were from Sigma-Aldrich (St. Louis, Mo.).

Techniques: Staining, Fluorescence, Microscopy