Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Sar1 membrane association is efficiently detected using the Split mNeonGreen system. (A) Schematic representation of the SAIYAN system. The membrane-spanning regions of TANGO1S and HA-tag fused to 10 of the 11 strands of mNG (mNG 1–10 ) were expressed in cells. In addition, Sar1A constructs with a FLAG-tag and a glycine linker fused to the 11th strand of mNG (mNG 11 ) were also expressed. Upon Sar1A activation, mNG 1–10 and mNG 11 combined to form the complete mNG proteins, inducing mNG signals. (B) HA-mNG 1–10 cells transfected with the indicated Sar1A constructs were fixed and stained with anti-Sec16-C and anti-FLAG antibodies. Scale bar = 10 µm. (C) Quantification of mNG intensity from B (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Membrane, Construct, FLAG-tag, Activation Assay, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Validation of SAIYAN technology. (A) Doxycycline-inducible HeLa cells expressing the membrane-spanning region of TANGO1S and HA-tag fused to 10 of the 11 strands of mNG (HA-mNG 1–10 cells) were either non-transfected or transfected with Sar1A constructs with a FLAG tag and a glycine linker fused to the 11th strand of mNG (Sar1A-FLAG-mNG 11 ). The cells were fixed and stained with anti-HA and anti-PDI antibodies. Scale bar = 10 µm. (B) HA-mNG 1–10 cells, treated with or without doxycycline, were transfected with Sar1A-FLAG-mNG 11 . The cells were fixed and stained with anti-HA and anti-FLAG antibodies. Scale bar = 10 µm.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Expressing, Membrane, Transfection, Construct, FLAG-tag, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Production of Sar1A/SAIYAN cells. (A) Doxycycline-inducible stable cell lines expressing the membrane-spanning regions of TANGO1S and HA-tag fused to 10 of the 11 strands of mNG were established using a lentiviral system and G418 selection (HA-mNG 1–10 cells). Stable cells were subsequently electroporated with Cas9 protein, sgRNA, and ssDNA to facilitate the knock-in of FLAG-mNG 11 into the Sar1A locus of the genome. Cells were treated with doxycycline for 24 h and further sorted via FACS to isolate single cells exhibiting mNG signals into 96-well plates. The expanded cell population was then collected and subjected to genomic sequencing. Positive clones were identified and selected for further analysis (Sar1A/SAIYAN cells). (B) Sar1A/SAIYAN (HeLa) cells, either treated or non-treated with doxycycline, were fixed and stained with anti-HA and anti-PDI antibodies. Boxed areas in the middle panels are shown at high magnification in the bottom panels. Scale bars: 10 µm (main), 5 µm (magnification). (C) Sar1A/SAIYAN (HeLa) cells, either treated or non-treated with doxycycline, were fixed and stained with anti-HA and anti-FLAG antibodies. Boxed areas in the middle panels are shown at high magnification in the bottom panels. Scale bar:10 µm (main), 5 μm (magnification). (D) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were fixed and stained with an anti-Sec16-C antibody. Scale bar = 10 µm. (E) Quantification of mNG intensity from D (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells. (F) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sar1A, and anti-GAPDH antibodies. (G) Sar1A/SAIYAN (HeLa) cells, either treated or non-treated with doxycycline, were fractionated via centrifugation. The lysates, the supernatants, and the pellets were subjected to SDS-PAGE, followed by western blotting with anti-FLAG, Sar1A, HA, ERK1, and calnexin antibodies. Source data are available for this figure: .

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Stable Transfection, Expressing, Membrane, Selection, Knock-In, Genomic Sequencing, Clone Assay, Staining, Transfection, SDS Page, Western Blot, Centrifugation

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Sar1A/SAIYAN (HeLa) cells proliferate and secrete normally. (A) HeLa and Sar1A/SAIYAN (HeLa) cells were treated with or without doxycycline for 24 h, and cell viability was measured and normalized using untreated HeLa cells as control. Error bars represent the means ± SEM. n = 4. (B) Sar1A/SAIYAN (HeLa) cells, treated with or without doxycycline, were fixed and stained with anti-Sec16-C and anti-GM130 antibodies. Scale bars = 10 µm. (C) Sar1A/SAIYAN (HeLa) cells treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry. RUSH chase was started with biotin addition, and live imaging was performed. Scale bars = 10 μm.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Control, Staining, Transfection, Imaging

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: ER exit site organization is required for the efficient activation of Sar1A. (A, C, E, and G) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were fixed and stained with anti-Sec16-C antibodies. Scale bar = 10 µm. (B, D, F, and H) Quantification of mNG signals from A, C, E, and G, respectively (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Activation Assay, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Western blotting analysis of Sar1A/SAIYAN (HeLa) cells on , , and . (A) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec12, and anti-GAPDH antibodies. (B) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-cTAGE5 CC1, anti-Sec12, and anti-GAPDH antibodies. (C) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-TANGO1 CC1, and anti-GAPDH antibodies. (D) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec16-N, and anti-GAPDH antibodies. (E) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec23A (11D8), and anti-GAPDH antibodies. (F) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec31A (rabbit), and anti-GAPDH antibodies. (G) Sar1A/SAIYAN (HeLa) cells were stably expressed using mCherry-tagged Sec23A constructs as indicated. Cells were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec23A (11D8), anti-Sec31A (rabbit), and anti-GAPDH antibodies. Source data are available for this figure: .

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Western Blot, Transfection, SDS Page, Stable Transfection, Construct

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Sec23A and Sec31A depletion exerts opposite effects on the activation of Sar1A in living cells. (A and C) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were fixed and stained with anti-Sec16-C antibodies. Scale bar = 10 µm. (B and D) Quantification of mNG signals from A and C, respectively (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells. (E) Sar1A/SAIYAN (HeLa) cells transfected with the indicated plasmids were fixed and processed for microscopic analysis. Scale bar = 10 µm. (F) Quantification of mNG signals from E (A.U.). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Activation Assay, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Each CLSD mutant of Sec23A exhibits different properties on Sar1 activation. (A) Sar1A/SAIYAN (HeLa) cells stably expressing mCherry-tagged Sec23A constructs, as indicated, were fixed and processed for microscopic analysis. Scale bar = 10 µm. (B) Quantification of mNG signals from A (A.U.). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Mutagenesis, Activation Assay, Stable Transfection, Expressing, Construct

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: DPD treatment accumulates collagen I within the ER of Sar1A/SAIYAN (BJ-5ta) cells. Sar1A/SAIYAN (BJ-5ta) cells were treated with DMSO or 0.5 mM DPD and incubated for 16 h. Cells were fixed and stained with an anti-collagen I antibody. Scale bar = 10 µm.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Incubation, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Activated Sar1 prevails in the ERGIC region of Sar1A/SAIYAN (BJ-5ta) cells. (A–O) Sar1A/SAIYAN (BJ-5ta) cells were fixed and stained with anti-Sec16-C (A), anti-ERGIC53 (B), anti-Sec23 (5H2) (C), anti-Sec24B (D), anti-Sec24D (E), anti-p125A (F), anti-TANGO1-CT (G), anti-Sec12 (H), anti-TFG (I), anti-Sec13 (J), anti-Sec31A (mouse) (K), anti-β-COP (L), anti-GM130 (M), anti-PDI (N), and anti-Rab1A (O) antibodies. Images were captured using Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (A–L) Right; top: Magnification of the indicated regions is on the left. Right; bottom: Magnification of the indicated regions on the upper. (P) Pearson’s correlation coefficient was used to quantify the degree of colocalization. n = 5. Cyan; outer COPII coats, blue; inner COPII coats, purple; ER exit site resident proteins, red; ERGIC proteins, orange; COPI protein, magenta; ER and Golgi proteins. Error bars represent the mean 95% CI.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Triple staining of Sar1A/SAIYAN (BJ-5ta) and parental BJ-5ta reveals the organization of the ER-Golgi interface of collagen-secreting cells. (A–C) Sar1A/SAIYAN (BJ-5ta) cells were fixed and stained with anti-Sec16-C and anti-ERGIC53 (A), anti-Sec23 (5H2), and anti-ERGIC53 (B), and anti-Rab1A and anti-ERGIC53 (C) antibodies. Images were captured using the Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (D) BJ-5ta cells were fixed and stained with anti-Sec16-C, anti-Sec23 (5H2), and anti-ERGIC53 antibodies. Images were captured using the Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (A–D) (right; top) Magnification of the indicated regions is on the left. (right; bottom) Double staining of the magnified region on the top.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Staining, Double Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Quantification of Pearson’s correlation coefficient to quantify the degree of colocalization in Sar1A/SAIYAN (HeLa) cells. Sar1A/SAIYAN (HeLa) cells were fixed and stained with anti-Sec16-C, anti-ERGIC53, anti-Sec23, anti-Sec24B, anti-Sec24D, anti-p125A, anti-TANGO1-CT, anti-Sec12, anti-TFG, anti-Sec13, anti-Sec31A (mouse), anti-β-COP, anti-GM130, anti-PDI, and anti-Rab1A antibodies. Images were captured using the Airyscan2. n = 5. Cyan; outer COPII coats, blue; inner COPII coats, purple; endoplasmic reticulum (ER) exit site resident proteins, red; ERGIC proteins, orange; COPI protein, magenta; ER and Golgi proteins. Error bars represent the mean 95% CI.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Reticular pattern of activated Sar1 signals diminished with DPD treatment in Sar1A/SAIYAN (BJ-5ta) cells. (A) Sar1A/SAIYAN (BJ-5ta) cells were treated with DMSO or 0.5 mM DPD and incubated for 16 h. Cells were fixed and stained with anti-Sec16-C antibodies. Images were captured using the Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (B) Pearson’s correlation coefficient was quantified to assess the degree of colocalization. n = 5. Error bars represent the mean 95% CI.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Incubation, Staining

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: The expression profile of cytokines between NFs and CAFs. ELISA of αSMA levels in cell lysates of primary NFs and paired CAFs (six pairs). (B, C) NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs cultured with the CM from KYSE30, KYSE410, KYSE510, or primary ESCC cells for 3 days. After the tumor CM was removed, NFs (a mixture of pairs 1, 2, and 3) and adipose‐derived MSCs were incubated with fresh RPMI1640 medium for 2 days, and fluorescent staining of αSMA in NFs, or adipose‐derived MSCs alone or these cells incubated with the CM from indicated ESCC cells. Scale bar, 20 μm as indicated (B). ELISA of αSMA levels in cell lysates of indicated stromal cells (C). Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. (D) The differential secreting status of cytokines in primary NF versus CAF from the same ESCC patient (pair 2), as assayed by cytokine antibody array. (E) ELISA assay showing the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from six paired primary NFs and CAFs. (F) The experimental condition of (F) was consistent with that of (B). ELISA assay showing the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs alone or incubated with the CM from indicated ESCC cells. Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. (G) Transcriptional factor activity assay of NF‐κB p65 activity in the nucleus of primary NFs and paired CAFs (six pairs). (H) The experimental condition of (H) was consistent with that of (B). NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs cultured with the CM from indicated ESCC cells. NF‐κB p65 activity was examined using transcriptional factor activity assay. Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. ** p < 0.01;*** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars represent mean ± SD of three independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Incubation, Staining, Positive Control, Ab Array, Activity Assay, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: Intratumoral NOX5 is critically contributed to the activation of NFs into CAFs. NFs (a mixture of pairs 1, 2, and 3) were incubated with the CM from KYSE30, KYSE410, KYSE510, and primary ESCC cells harbored control or NOX5 shRNA for 3 days. Then, fibroblasts were cultured with fresh RPMI1640 medium for 2 days. The intracellular expression of αSMA was evaluated using immunoblotting (B) and confocal assay (C). The secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from fibroblasts was assessed using ELISA assay (D). Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. (B, C) Stably silencing NOX5 in the indicated ESCC cell lines and primary ESCC cells analyzed by immunoblotting. GAPDH was used as the internal control (B, upper panel). Immunoblotting (B, lower panel) or confocal (C) of αSMA levels in NFs or fibroblasts derived from NFs incubated with the CM from indicated ESCC cells harbored control shRNA or NOX5 shRNA. Scale bar, 20 μm as indicated. (D) ELISA assay showing the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from NFs or fibroblasts derived from NFs incubated with the CM from indicated ESCC cells harbored control shRNA or NOX5 shRNA. # represents the statistical significance of NFs alone versus NFs treated with the CM from indicated ESCC cells harbored control shRNA. * represents the statistical significance of NFs treated with the CM from indicated ESCC cells harbored control shRNA versus NFs treated with the CM from indicated ESCC cells harbored NOX5 shRNA. ## p < 0.01; ### p < 0.001; ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of three independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Incubation, shRNA, Cell Culture, Expressing, Western Blot, Confocal Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Stable Transfection, Derivative Assay, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: Intratumoral NOX5 is critically contributed to the activation of adipose‐derived MSCs into CAFs. The experimental condition of this figure was consistent with that of Figure 3. (A, B) Immunoblotting (A) or confocal (B) of αSMA levels in adipose‐derived MSCs, or fibroblasts derived from adipose‐derived MSCs incubated with the CM from KYSE410, KYSE30, KYSE510, or primary ESCC cells harbored control shRNA or NOX5 shRNA. Scale bar, 20 μm as indicated. (C) ELISA assay showing the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from adipose‐derived MSCs and fibroblasts derived from adipose‐derived MSCs incubated with the CM from indicated ESCC cells harbored control shRNA or NOX5 shRNA. Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. # represents the statistical significance of adipose‐derived MSCs alone versus adipose‐derived MSCs incubated with the CM from indicated ESCC cells harbored control shRNA. *represents the statistical significance of adipose‐derived MSCs incubated with the CM from indicated ESCC cells harbored control shRNA versus adipose‐derived MSCs incubated with the CM from indicated ESCC cells harbored NOX5 shRNA. ### p < 0.001; ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of three independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Derivative Assay, Western Blot, Incubation, shRNA, Enzyme-linked Immunosorbent Assay, Positive Control, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: NOX5 Y476/478 sites are critical for NOX5‐mediated CAFs activation. The experimental condition of this figure was consistent with that of Figure 3. (A) Transfection of NOX5 Y476/478F plasmid into KYSE30 and KYSE410 cells. The transfection efficiency was evaluated using immunoblotting. GAPDH was used as the loading control. (B) The concentration of TNF‐α and IL‐1β in CM from KYSE30 and KYSE410 cells harbored control vector or NOX5 Y476/478F plasmid, was assayed by ELISA. (C) Confocal analysis of αSMA levels in NFs (a mixture of pairs 1, 2, and 3), adipose‐derived MSCs incubated with the CM from indicated ESCC cells harbored control vector or NOX5 Y476/478F plasmid. Scale bar, 20 μm as indicated. (D) ELISA assay showing the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from NFs (a mixture of pairs 1, 2, and 3) and adipose‐derived MSCs incubated with the CM from KYSE30 and KYSE410 cells harbored control vector or NOX5 Y476/478F plasmid. ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of three independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Incubation, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: NOX5‐induced TNF‐α or IL‐1β mediates the activation of NFs and adipose‐derived MSCs into CAFs. Control or NOX5‐overexpressing KYSE30 and KYSE410 cells were treated with inhibitors of ROS/Src/NF‐κB pathway, and the secretion of TNF‐α and IL‐1β was evaluated using ELISA assay (B). NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs were incubated with recombinant human TNF‐α or IL‐1β protein, or CM from KYSE30 and KYSE410 cells alone or in the presence of TNF‐α or IL‐1β Ab for 3 days. Then, fibroblasts were cultured with fresh RPMI1640 medium for 2 days. The intracellular expression of αSMA was evaluated using immunoblotting and confocal assay (C). The secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from fibroblasts was assessed using ELISA assay (D). Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. (B) Control vector or NOX5‐overexpressing KYSE30 (black bar) and KYSE410 (gray bar) cells were pretreated with ROS scavenger NAC (2 mM, pretreated with 90 min), or treated with H 2 O 2 scavenger‐PEG‐catalase (400 units/ml), 100 nM dasatinib, 1 μM PP2, 5 μM JSH‐23, 5 μM SC75741, or control solvent. The concentration of TNF‐α and IL‐1β in CM from indicated ESCC cells was assayed by ELISA. # represents the statistical significance of indicated treatments versus control cells. * represents the statistical significance of indicated treatments versus NOX5‐overexpressing cells. # p < 0.05; ## p < 0.01; ### p < 0.001; *** p < 0.001; two‐tailed unpaired Student's t ‐test. (C, D) NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs were treated with recombinant TNF‐α or IL‐1β protein (10 ng/ml), or the CM from KYSE30 or KYSE410 cells in the presence or absence of the TNF‐α or IL‐1β Ab (10 μg/ml) for 3 days. Then, culture media were removed and added fresh RPMI1640 medium to these fibroblasts for 2 days. (C) Immunoblotting (left panel) or confocal assay (right panel) evaluating the expression of αSMA in NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs. Scale bar, 20 μm as indicated. (D) ELISA assay was used to detect the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from indicated stromal cells. Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of three independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Cell Culture, Expressing, Western Blot, Confocal Assay, Positive Control, Plasmid Preparation, Concentration Assay, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: Activated CAFs assist ESCC malignant progression in vitro . NFs (a mixture of pairs 1, 2, and 3) were incubated with the CM from KYSE30 or KYSE410 cells for 3 days. Then, the tumor CM was removed, the fresh RPMI1640 medium was added to fibroblasts for 2 days to generate CM for MTS assay, and the activated CAFs were applied to Transwell invasion assay. KYSE30 or KYSE410 harbored control shRNA or NOX5 shRNA cells were incubated with the CM from corresponding parental cells‐activated CAFs. The growth ability was evaluated using MTS assay (B). CAFs regulated the invasion of ESCC cells was assessed by Transwell invasion assay. The activated CAFs primed by KYSE30 or KYSE410 cells were plated in the lower chamber. KYSE30 or KYSE410 harbored control shRNA or NOX5 shRNA cells (corresponding to their respective parental cells‐activated CAFs) were placed in the upper chamber (C). For evaluation of CAFs‐induced HLECs migration, NFs (a mixture of pairs 1, 2, and 3) were incubated with the CM from KYSE30 or KYSE410 harbored control shRNA or NOX5 shRNA cells for 3 days. Then, the tumor CM was removed, the fresh RPMI1640 medium was added to fibroblasts for 2 days, and these fibroblasts were used for subsequent assay. Migration of HLECs was evaluated using Transwell migration assay. Fibroblasts primed by KYSE30 or KYSE410 harbored control shRNA or NOX5 shRNA cells were plated in the lower chamber. HLECs were seeded in the upper chamber (D). (B) Growth rates of KYSE30 or KYSE410 cells harbored control shRNA incubated with the CM from corresponding parental cells‐activated CAFs alone or in the presence of IL‐6, IL‐7, IL‐8, CCL5, or TGF‐β1 Ab (10 μg/ml), or KYSE30 or KYSE410 cells harbored NOX5 shRNA alone or incubated with CM from their corresponding parental ESCC cells for 4 days. Cell growth was assayed by MTS assay. (C) Boyden chamber assay for KYSE30 or KYSE410 cells harbored control shRNA plated on the upper cell culture inserts with their corresponding parental ESCC cells‐activated CAFs in lower chambers alone or in the presence of IL‐6, IL‐7, IL‐8, CCL5, or TGF‐β1 Ab (10 μg/ml), or KYSE30 or KYSE410 cells harbored NOX5 shRNA plated on the upper cell culture inserts with or without their corresponding parental ESCC cells‐primed CAFs in lower chambers. (D) CytoSelect 96‐well cell migration assay for HLECs plated on upper cell culture inserts with NFs (a mixture of pairs 1, 2, and 3), NFs (a mixture of pairs 1, 2, and 3)‐activated CAFs (primed by KYSE30 or KYSE410 cells harbored control shRNA) alone or in the presence of IL‐6, IL‐7, IL‐8, CCL5, or TGF‐β1 Ab (10 μg/ml), or NFs (a mixture of pairs 1, 2, and 3) primed by the CM from KYSE30 or KYSE410 cells harbored NOX5 shRNA in lower chambers. n.s. no significant difference; * p < 0.05; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of five independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: In Vitro, Incubation, MTS Assay, Transwell Invasion Assay, shRNA, Migration, Subsequent Assay, Transwell Migration Assay, Boyden Chamber Assay, Cell Culture, Cell Migration Assay, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: CAFs promote the malignancy of ESCC tumors in vivo . Tumor volume was measured at day 33. Mice‐bearing control or NOX5 shRNA KYSE30 tumors with NFs (a mixture of pairs 1, 2, and 3)‐activated CAFs (primed by KYSE30 cells) were treated with control solvent or several Abs, including IL‐6, IL‐7, IL‐8, or TGF‐β1 Ab (10 μg/mouse twice times per week, i.v.). (B) The expression of Ki‐67, CD31, or LYVE1 in indicated KYSE30 tumor tissues was evaluated using IHC assay. n.s. no significant difference; ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of five independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: In Vivo, shRNA, Expressing, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: CAFs promote the malignancy of ESCC tumors in vivo . A popliteal lymph node metastasis model was established in mice ( n = 5 biologically independent mouse/group) by inoculating the foot pads with KYSE30 cells and NFs (a mixture of pairs 1, 2, and 3)‐activated CAFs (primed by KYSE30 cells). The popliteal lymph nodes were enucleated and analyzed 5 weeks after inoculation, and the volumes of popliteal lymph nodes were shown. * p < 0.05; ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars represent mean ± SD of five independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: In Vivo, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: Expression and internalization analyses of FcγRs. (a) Histogram overlays show expression of FcγRs (FcγRI: CD64, FcγRIIB: CD32, FcγRIII: CD16, and FcγRIV: 9E9) on splenic single lin − MHCII + CD11c + CD8 + DCs, CD11c + CD8 − DCs, and pDCs (see gating strategy in Fig. S1 a) from C57BL/6 (black) and FcγRI −/− , FcγRIIB −/− , FcγRIII −/− , and FcγRIV −/− mice (gray). This experiment was repeated three times with similar results. (b) Internalization of αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, αFcγRIIB/III-Ova, or isotype-Ova into the indicated splenic cell populations. Splenic single-cell suspensions were incubated in media containing the antibodies labeled with an oligo containing an Atto647N fluorochrome for 10, 30, 60, or 120 min at 37°C or kept on ice (0-min time point). Extracellular fluorescence was quenched by incubation with a complementary oligo containing a specific BBQ650 quencher. Cells were gated on single CD19 + B cells, CD11c + CD8 + DCs, CD11c + CD8 − DCs, CD11c low PDCA-1 + B220 + pDCs, Ly6C high inflammatory and Ly6C low resident monocytes, T cells, and NK cells as described in Fig. S1 b. The presented experiments were repeated three times with two samples each. All data points ± SD are shown in the graph. MFI, mean fluorescence intensity.

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Expressing, Incubation, Labeling, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: In vivo proliferation of CD4 + and CD8 + T cells by antigen delivery to FcγRs in the steady state. (a and b) MACS-purified, CFSE-labeled, congenic 10 6 CD8 + OT-I T cells (a) or 2 × 10 6 CD4 + OT-II T cells (b) were i.v. transferred into C57BL/6 mice. 16 h later, recipients were i.p. injected with doses of 10, 3, 1, 0.3, 0.1, and 0 µg isotype-Ova, αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, or αFcγRIIB/III-Ova targeting antibodies in PBS. 3 d after targeting antibody injection, in vivo T cell proliferation in dependency of the injected antibody amount was measured by CFSE-dilution analysis via flow cytometry of Vα2 + CD45.1 + CD8 + (a) and Vα2 + CD45.1 + CD4 + (b) splenic T cells. All experiments were repeated at least three times with similar results.

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: In Vivo, Purification, Labeling, Injection, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: T cell proliferation induced by FcγR targeting is ITAM independent, and long-term proliferative responses require additional co-stimulatory signals in vivo. (a) Injection of antigen-targeting antibodies does not change activation status of DCs in vivo. 10 µg αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, αFcγRIIB/III-Ova, or isotype-Ova, was i.p. injected into C57BL/6 mice. As positive control, a mixture of each 25 µg poly(I:C) and αCD40 or 10 9 HKLM was used. PBS was i.p. injected as negative control. 12 h later, splenic single lin − (CD3 − CD19 − NKp46 − ) MHCII + CD11c + CD8 + DCs, CD11c + CD8 − DCs, and CD11c low PDCA1 + pDCs were analyzed by flow cytometry for the activation markers CD80, CD86, and CD69. Data were analyzed using DIVA and FlowJo Software. This experiment was repeated more than five times with similar results and was used as quality control in the production of antigen-targeting antibodies. (b–e) MACS-purified, CFSE-labeled, congenic 10 6 CD8 + OT-I T cells (b and d) or 2 × 10 6 CD4 + OT-II T cells (c and e) were i.v. transferred into C57BL/6 mice (b–e) or NOTAM mice (b and c). 16 h later, recipients were i.p. injected with 3 µg of the targeting antibodies in PBS (b and c) or together with 25 µg αCD40 antibody and 12.5 µg poly(I:C) (pIC) or HKLM (d and e). (b and c) 3 d after targeting antibody injection, in vivo T cell proliferation was measured by CFSE-dilution analysis via flow cytometry of Vα2 + CD45.1 + CD8 + (b) and Vα2 + CD45.1 + CD4 + (c) splenic T cells. The graph shows the proliferation indices of all mice analyzed (n.s., nonsignificant; *, P < 0.05). (d and e) T cell proliferation was analyzed by cell numbers of gated Vα2 + CD45.1 + CD8 + (d) or Vα2 + CD45.1 + CD4 + (e) splenic T cells 3 d and 9 d later, when PBS, αCD40 + poly(I:C), or HKLM was coinjected. Graphs show the relative cell number expansion compared with the isotype control. (a) These experiments were repeated at least three times with similar results. (b–e) The data were generated within three independent experiments, and all data points ± SD are presented (n.s., nonsignificant; *, P < 0.05; Mann–Whitney U test).

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: In Vivo, Injection, Activation Assay, Positive Control, Negative Control, Flow Cytometry, Software, Control, Purification, Labeling, Generated, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: Induction of T cell responses by FcγR targeting is dependent on the presence of FcγRs. (a–h) C57BL/6, FcγRIIB −/− (a–d), or FcγRIV −/− (e–h) mice were i.v. injected with congenic10 6 CD45.1 + CD8 + OT-I (a, b, e, and f) or 2 × 10 6 CD45.1 + CD4 + OT-II (c, d, g, and h) MACS-purified, CFSE-labeled T cells. 3 µg of the antigen-targeting antibodies αDEC205-Ova, αDCIR2-Ova, and αFcγRIV-Ova or 10 µg αFcγRIIB-Ova or isotype-Ova was i.p. injected 16 h after T cell transfer. T cell proliferation was analyzed by CFSE dilution in gated Vα2 + CD45.1 + CD8 + OT-I (a, b, e, and f) or Vα2 + CD45.1 + CD4 + OT-II (c, d, g, and h) T cells 72 h later. (a, c, e, and g) Shown are exemplary overlay histograms (gray: C57BL/6, black: [a and c] FcγRIIB −/− , [e and g] FcγRIV −/− ) of at least three independent experiments with similar results. (b, d, f, and h) Scatter plots represent the proliferation indices of all mice analyzed ± SD (gray circles: C57BL/6, black squares: [b and d] FcγRIIB −/− , [f and h] FcγRIV −/− ). (n.s., nonsignificant; **, P < 0.01; ***, P < 0.001 by Mann–Whitney U test.)

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Injection, Purification, Labeling, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: Antigen targeting to FcγRIIB and FcγRIV needs receptor expression on cDCs . (a–h) Mice were i.v. injected with MACS-purified, CFSE-labeled 10 6 CD45.1 + CD8 + OT-I (a, c, e, and g) or 2 × 10 6 CD45.1 + CD4 + OT-II (b, d, f, and h) T cells. 3 µg αDEC205-Ova, αDCIR2-Ova, or αFcγRIV-Ova or 10 µg αFcγRIIB-Ova or isotype-Ova control antibodies was i.p. injected 16 h after T cell transfer. T cell proliferation was evaluated 72 h later. Shown is the CFSE dilution of Vα2 + CD45.1 + CD8 + OT-I (a, c, e, and g) or Vα2 + CD45.1 + CD4 + OT-II (b, d, f, and h) gated congenic T cells. (a and b) C57BL/6 mice and CD11c-DTR mice were treated i.p. with PBS or DT 8 h before T cell transfer. (c and d) T cell transfer into FcγRIV fl/fl , LysM-Cre × FcγRIV fl/fl , CD11c-Cre × FcγRIV fl/fl mice. (e and f) C57BL/6 mice were treated three times with 200 µg αPDCA-1 antibody 64 h, 40 h, and 16 h before T cell transfer. (g and h) T cell transfer into C57BL/6 or µMT −/− mice. All experiments were repeated at least three times with similar results.

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Expressing, Injection, Purification, Labeling, Control

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: Differential antigen presentation to CD8 + and CD4 + T cells induced by FcγRIIB and FcγRIV targeting to CD11c + CD8 + or CD11c + CD8 − DCs. 10 µg αDEC205-Ova, αDCIR2-Ova, or αFcγRIV-Ova or 30 µg αFcγRIIB-Ova or isotype-Ova was i.p. injected into C57BL/6 mice. 12 h later, splenocytes were sorted into CD11c + CD8 + DCs, CD11c + CD8 − DCs, pDCs, B cells, and Ly6C high and Ly6C low monocytes. (a and b) Antigen-presenting cells were co-cultured in different numbers with 10 5 MACS-enriched CD8 + OT-I T cells (a) or CD4 + OT-II T cells (b). Proliferation was evaluated by addition of 3 H-thymidine 16 h (a) or 40 h (b) after start of the co-culture. Incorporation of 3 H-thymidine was measured 24 h later. This experiment was repeated at least three times, and all data points ± SD are shown in the graph.

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Immunopeptidomics, Injection, Cell Culture, Co-Culture Assay

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: Induction of effector T cell responses in naive mice mediated by antigen delivery through FcγRs. (a–d) C57BL/6 mice were i.p. injected with 10 µg of the targeting antibodies αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, αFcγRIIB/III-Ova, or isotype-Ova together with 50 µg αCD40 antibody and 25 µg poly(I:C) after injection of PBS or 200 µg αFcγRIV w/o Ova. 14 d later, splenocytes were in vitro restimulated for 12 h with freshly isolated CD11c-positive MACS-enriched DCs loaded with a peptide pool of Ova. Intracellular IFNγ (a and c) and IL-2 (b and d) production was analyzed by flow cytometry. The graphs represent the number of cytokine-positive TCRβ + NKp46 − CD19 − CD8 + CD4 − (a and b) and TCRβ + NKp46 − CD19 − CD8 − CD4 + (c and d) T cells. This experiment was performed twice with at least five mice, and all data points were used for the analysis. Shown is the median ± interquartile range (n.s., nonsignificant; **, P < 0.01; ***, P < 0.001; Mann–Whitney U test). (e and f) C57BL/6 mice were immunized with 3 µg αDEC205-Ova, αDCIR2-Ova, αFcγRIV-Ova, or αFcγRIIB/III-Ova or 10 µg αFcγRIIB or isotype-Ova in combination with 50 µg αCD40 and 25 µg poly(I:C) (e) or 0.03, 0.10, 0.3, 1, or 3 µg αDEC205-Ova, αDCIR2-Ova, αFcγRIV-Ova, or αFcγRIIB/III-Ova or 10 µg αFcγRIIB or isotype-Ova in combination with 50 µg αCD40 and 25 µg poly(I:C) (f). 8 d later, mice were challenged i.v. with a cocktail of freshly isolated CD45.1 + splenocytes labeled with different concentrations of CFSE and/or cell trace violet and loaded with 2.4, 40, 160, or 625 nM SIINFEKL peptide (unloaded cells were used as injection control and control for specificity of the lysis). This allowed for the simultaneous analysis of target cell lysis loaded with different amounts of SIINFEKL within one mouse. 16 h later, splenocytes were analyzed for the presence of the transferred CD45.1 + cells. The data points shown in this graph have been generated within three independent experiments. Each dot represents the degree of lysis observed for splenocytes loaded with a specific amount of peptide (median ± interquartile range in e and one line for each single mouse in f). (e) This experiment was performed three times with two mice per group, and all data points are shown in the graphs. (f) This experiment was performed three times with three to five mice per group, and all data points are shown in the graphs.

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Injection, In Vitro, Isolation, Flow Cytometry, MANN-WHITNEY, Labeling, Control, Lysis, Generated

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, PCR analysis of the input HIV-1 gRNA stocks and viral variants obtained after the indicated days of passaging. The upper panel shows primary PCR data and the lower panel the position of the primer binding sites and the fragments obtained. b, Schematic structure of the HIV-1 genome and modifications at its 3′end to insert the U6-gRNA-scaffold expression cassette. Duplicated T-rich regions, poly-purine tract (PPT) and LTR sequences are highlighted in red. The arrow indicates the major recombination event. Mutations introduced to minimize recombination and the predicted Nef amino acid sequence are indicated. Numbers refer to nucleotide positions in the NL4-3 nef gene. c, PCR analysis was performed as in panel A but the optimized HIV-1 NL4-3 gRNA construct containing the changes shown in panel B were used for passaging. d, HEK293T cells were transfected with the parental HIV-1 NL4-3 construct, the original or optimized derivative containing the U6-gRNA-scaffold cassette or the gRNA library targeting 511 potential antiviral factors. Infectious virus yield was measured using the TZM-bl reporter cell infectivity assay and values were normalized to the infectious virus yield obtained for the parental NL4-3 construct (100%). Bars represent the mean of three independent experiments ±SD, Unpaired T-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. e, Representative Western blot of Nef and GAPDH expression levels in HEK293T cells transfected with the indicated HIV-1 NL4-3 constructs. f, NGS results showing the coverage of the HIV-1 NL4-3 gRNA libraries.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Passaging, Binding Assay, Expressing, Sequencing, Construct, Transfection, Virus, Infection, Western Blot

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, Infected cells, infectious virus yields and p24 antigen production in CEM-M7 cells infected with the HIV-1 TC-NL4-3-CRF-gRNA library. Cells were infected and the virus passaged as indicated in . Every five days, the cell cultures were analyzed for the proportion of productively infected (p24+) cells by flow cytometry (left), infectious virus and p24 in the supernatant by TZM-bl infection assay (middle) and p24 antigen ELISA (right), respectively. b, Correlation between the enrichment of known RFs at the 15 and 20 day time-points and in the presence or absence of IFN-β in CEM-M7 Cas9 cells. c, Absolute counts of the indicated HIV-1 gRNA constructs during passage in CEM-M7 Cas9 cells. d, Correlation between MAGeCK score obtained in independent experiments in CEM-M7 vs SupT1 CCR5 high Cas9 cells.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Infection, Virus, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Construct

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, Expression of cellular factors targeted by gRNAs selected during passage of HIV-1 in CEM-M7 and SupT1 CCR5 high Cas9 cells with or without the indicated IFNs (1000U/ml). Whole-cell lysates were immunoblotted and stained with antibodies against the indicated proteins. b, Percentage of eGFP positive cells indicating infected CEM-M7 Cas9 cells at 4 days post infection electroporated with either the NT or GRN gRNA and infected with WT NL4-3. Bars represent the mean of infected cells at 2dpi relative to the control (100%) of three independent experiments, ±SEM, *p<0.05, Student’s t-test Welch’s correction, ** p<0.001, ***p<0.0001 In the lower panel a representative WB showing PGRN KO efficiency. c, HEK293T cells were cotransfected with increasing amounts of GRN expression construct and proviral mutants of NL4-3 or CH077 lacking the accessory genes. Each point represents the average of three independent experiments ±SEM. In the lower panel a representative WB indicating expression of Env, p55, p24 and PGRN in virus supernatants or cell lysates. d, HEK293T cells were cotransfected with a luciferase reporter constructs under the control of the HIV-1 LTR and expression constructs for GRN in presence and absence of NL4-3 Tat or a vector control. Bars represent the mean of three independent experiments ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. e, HEK293T cells were cotransfected with different amount of GRN expression plasmid with either NL4-3_eGFP, CH077_eGFP or CH058_eGFP. Early infection was measured with Flow cytometry and bars represent the mean fluorescence intensities (MFI) of eGFP in the eGFP+/GRN+ population relative to vector control (100%). Bars represent the mean of three independent experiments ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. f, Representative WB and quantification of PGRN KO.in primary CD4 + T cells. Bars represent the mean of three independent experiments ±SD, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. g, CD4 + T cells from 3 to 6 donors were electroporated with either the GRN gRNA or the NT control, infected with the indicated WT HIV-1 strains and infectious virus yields measured from 2 to 6dpi by TZM-bl infection assays. Values represent the mean of three to six experiments normalized to the NT control (100%) ±SEM, Two-way anova Sidak’s multiple comparison, *p<0.05, ** p<0.001, ***p<0.0001.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Expressing, Staining, Infection, Construct, Virus, Luciferase, Plasmid Preparation, Flow Cytometry, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, HEK293T cells were cotransfected with increasing amount of either CIITA, CC2D1B or CEACAM3 expression constructs with and indicated proviral constructs. Values represent the mean of three independent experiments ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. b-d, CD4 + T cells from 3 to 4 donors were electroporated with either the gRNA targeting CIITA , CC2D1B , CEACAM3 or the NT control, infected with the indicated WT HIV-1 strains and infectious virus yields measured from 2 to 6 dpi by TZM-bl infection assays. Values represent the mean of two to six experiments normalized to the NT control (100%) ±SEM, Two-way anova Sidak’s multiple comparison *p<0.05, ** p<0.001, ***p<0.0001. Examples from primary data are shown in . e, Percentage of p24 antigen in the supernatants of CD4 + T cells from three to four donors electroporated with either the gRNA targeting CC2D1B or NT control at 3 days post infection with VSV-G pseudo-typed Δ env NL4-3 or CH077. Bars represent the mean of the infectious viral yield at two days post-infection relative to the control (100%) of three to four independent experiments, ±SEM, Student’s t-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001 In the lower panel a representative WB showing CC2D1B KO efficiency.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Expressing, Construct, Infection, Virus, Comparison

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, HEK293T cells were cotransfected with different amount of CIITA expression plasmid with with either NL4-3_eGFP, CH077_eGFP or CH058_eGFP. Early infection was measured with Flow cytometry and bars represent the mean fluorescence intensities (MFI) of eGFP in the eGFP+/AF647+ population relative to vector control (100%). Bars represent the mean of two independent experiments ±SD. b, Representative Western blot showing Env, p24 and CC2D1B in virus containing supernatants or cell lysates of HEN293Ts cotransfected with increasing amounts of CC2D1B and the indicated proviral constructs. c, d, Quantification the WB in panel (b) of the p24 release (c) and Env processing (d). Values represent the mean of three independent experiments ±SD, Unpaired T-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. e, Representative western blot and quantification of CIITA, CC2D1B and CEACAM3 expression upon KO.in primary CD4 + T cells. Bars represent the mean of three independent experiments ±SD, Unpaired T-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. f, CD4 + T cells were electroporated with gRNA targeting CIITA or the NT control, infected with the indicated WT HIV-1 strains and infectious virus yields measured from 2 to 6 dpi by TZM-bl infection assays. Shown are representative results.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Expressing, Plasmid Preparation, Infection, Flow Cytometry, Fluorescence, Western Blot, Virus, Construct

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, Schematic structure of modifications at 3′end to insert the U6-gRNA-scaffold expression cassette. Duplicated T-rich regions, poly-purine tract (PPT) and LTR sequences are highlighted in red. b, Nef and GAPDH expression levels in HEK293T cells transfected with the HIV-1 NL4-3 constructs. c, Infection rate of parental HIV-1 CH077 construct, the original or optimized containing the U6-gRNA-scaffold cassette or the gRNA library. Bars represent the mean of four independent experiments ±SEM, Unpaired T-test Welch’s correction, *p<0.05, ** p<0.001, ***p<0.0001. d, Coverage of the HIV-1 NL4-3 gRNA libraries. e, CEM-M7 Cas9 cells infected with the HIV-1 TC-CH077-CRF-gRNA library. the cell cultures were analyzed by flow cytometry (left), infectious virus in the supernatant by TZM-bl infection assay (right), respectively.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Expressing, Transfection, Construct, Infection, Flow Cytometry, Virus

Journal: bioRxiv

Article Title: Traitor-virus-guided discovery of novel antiviral factors

doi: 10.1101/2023.11.03.565501

Figure Lengend Snippet: a, Infectious virus yields in CEM-M7 Cas9 cells infected with the TV-NL4-3-CRF-gRNA WT or Δ nef library. Cells were infected and the virus passaged as indicated in . Every five days, the cell cultures were analysed for infectious virus in the supernatant by TZM-bl infection. b, Correlation between the enrichment of genes at the 10, 15 and 20 day time-points between the WT and Δ nef kinetics. c, Read counts relative to input virus from the MAGeCK analysis showing the enrichment of gRNAs targeting GRN , CIITA , CC2D1B and IRF3 in Δ nef and WT kinetics.

Article Snippet: TZM-bl cells were provided and authenticated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. TZM-bl are derived from HeLa cells, which were isolated from a 30-year-old female.

Techniques: Virus, Infection

Journal: bioRxiv

Article Title: RGG peptide induces the disassembly of disease-relevant FUS and TDP43 condensates

doi: 10.1101/2025.03.19.643735

Figure Lengend Snippet: (A) Graph representing the distribution of FUS-P525L protein in nucleus and cytoplasm. The fluorescent intensities of the nucleus and cytoplasm were calculated from the experiment as performed in , and the ratio is plotted here. A student-paired t-test was used to calculate the significance value (n=6). (B) Incucyte images (real-time cell death analysis) representing the cellular uptake of propidium iodide (PI) in different conditions in HeLa cells. Scale bar=100um. (C) Graph representing the number of propidium iodide (PI) positive cells (dead cells) from the experiment as performed in B. The time on the x-axis reflects the time-point after transfection in hours. The significance was calculated using 2-way ANOVA with multiple comparisons (n=4). Error bars in all graphs represent mean ±SEM, and the same color points in A depict the data from a single experimental set. *, **, ***, and **** denote p-value≤0.05, ≤0.01, ≤0.001, and ≤0.0001, respectively.

Article Snippet: The cell death analysis was performed using the IncuCyte S3 live-cell analysis instrument (Sartorius), and the change in the number of PI-positive cells (dead cells) in different conditions was plotted in the graph.

Techniques: Transfection

Journal: bioRxiv

Article Title: RGG peptide induces the disassembly of disease-relevant FUS and TDP43 condensates

doi: 10.1101/2025.03.19.643735

Figure Lengend Snippet: Incucyte images representing the cellular uptake of propidium iodide (PI) in different conditions in HeLa cells. Scale bar=100um. The images are part of and .

Article Snippet: The cell death analysis was performed using the IncuCyte S3 live-cell analysis instrument (Sartorius), and the change in the number of PI-positive cells (dead cells) in different conditions was plotted in the graph.

Techniques: