Journal: bioRxiv

Article Title: The cold-inducible RNA-binding protein RBM3 stabilises viral mRNA at the cooler temperatures of the upper respiratory tract

doi: 10.1101/2024.07.25.605104

Figure Lengend Snippet: (A) Cold-responsiveness of a panel of cell lines and primary nasal epithelial cells (PNECs) as determined by the induction of RBM3 expression by qPCR over 48 h and normalised to 37°C levels. (B) Generation of a lentiviral RBM3 overexpression (RBM3+) A549 cell line alongside a control empty vector line (EMP). (C) Titres of WSN at 72 hpi after an MOI 0.01 infection in EMP or RBM3+ A549 cells. (D) RBPmap predicted RBM3 binding motifs for each WSN positive strand segment, separated by predicted confidence of each motif. (E) Western blot showing input and pulldown samples for CLIP-qPCR, blotting for Actin and overexpressed RBM3-Flag. (F) qPCR quantification of the RNA extracted from CLIP samples demonstrated a significant increase in viral NP mRNA levels with minimal differences in HA or Actin transcripts, when comparing RBM3 to no antibody control pulldowns.

Article Snippet: Cells were submerged in modified Promocell Airway Epithelial Cell Growth Medium which was replaced on the apical and basal side every 2 days until the cells reached confluency.

Techniques: Expressing, Over Expression, Control, Plasmid Preparation, Infection, Binding Assay, Western Blot

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: bioRxiv

Article Title: Stepwise developmental mimicry generates proximal-biased kidney organoids

doi: 10.1101/2024.06.28.601028

Figure Lengend Snippet: a . Live imaging of HNF4A-YFP (Vanslambrouck et al., 2019 JASN ) kidney organoids on differentiation day 18 with Alexa 647-conjugated dextran uptake. Quantification of individual uptake events displayed in lower left, and insets detail HNF4A-YFP and dextran distinction. Scale bars: whole organoid-500 microns, insets-100 microns. b . Whole-mount immunofluorescence of day 18 control and proximal-biased kidney organoids following dextran uptake assay. Boxed region is magnified and split by individual channels. Scale bars: 10 microns. c . Whole-mount immunofluorescence of uninjured and 5 µM cisplatin-injured kidney organoids on differentiation day 21, split by control and proximal-biased conditions. Boxed area magnifies HNF4A + proximal organoid nephron regions. Scale bars: 10 microns. d . Timeline schematic of kidney organoid injury strategy. e . Quantification of relative protein signal from HAVCR1 + regions in control and proximal-biased kidney organoid nephrons on day 21 following injury. Data are averaged from n = 3 independent and consistent imaging panels. SEM error bars are shown. Statistical significance is determined using Student’s t test. f . Quantification of HNF4A + proximal organoid nephron segments with HAVCR1 expression for each of the four conditions from (c). Positive segments are counted from a 20X widefield panel of 4 images per organoid, from n = 2 biological replicate organoids for each condition. SEM error bars are shown. Statistical significance is determined using Student’s t test. g . Whole-mount immunofluorescence of uninjured and 5 µM cisplatin-injured kidney organoid nephrons on day 21. Dashed white line indicates an uninterrupted segment through the lumen of the organoid tubule. Scale bars: 10 microns. h . Quantification of continuous lumen segments from (h). Data are quantified from n = 4 organoids per condition, and SEM error bars are shown. Statistical significance is determined using Student’s t test. i . Whole-mount immunofluorescence of uninjured and 5 µM cisplatin-injured proximal-biased kidney organoid nephrons on day 21. Yellow arrows denote cells of the HNF4A + organoid nephron tubule with low HNF4A protein expression and positive expression of γH2AX. White arrows indicate cells with high HNF4A, and low γH2AX detection. Split channels separate HNF4A and γH2AX for cisplatin-injured condition. Scale bars: 10 microns. j . Whole-mount immunofluorescence of uninjured and 5 µM cisplatin-injured proximal-biased kidney organoid nephrons on day 21. White arrowheads indicate interstitial cells surrounding the tubules that are positive for SOX9. Scale bars: 10 microns. k-l . Quantification of HNF4A and SOX9 signal intensities from (j) for uninjured (k) and +cisplatin (l) samples. Intensities were quantified from individual cells of technical and biological duplicates (4 organoids per condition) and normalized using min-max normalization.

Article Snippet: Primary antibodies used in this study were: WT1 (abcam, ab89901, 1:1000), JAG1 (R&D Systems, AF599, 1:300), HNF1B (Thermo Fisher Scientific, MA5-24605, 1:500), HNF4A (R&D Systems, MAB4605, 1:200), CDH1 (BD Biosciences, 610181, 1:300), ZO-1 (Thermo Fisher Scientific, 33-9100, 1:200), PAX2 (R&D Systems, AF3364, 1:50), SIX1 (Cell Signaling Technology, 12891S, 1:300), HES1 (Cell Signaling Technology, 11988, 1:300), POU3F3 (Novus Biologicals, NBP1-49872, 1:500), HNF4G (Thermo Fisher Scientific, PA5-82189, 1:200), LRP2 (My Bio Source, MBS690201, 1:500), HAVCR1 (R&D Systems, AF1750, 1:200), γH2AX (Cell Signaling Technology, 2577), LAMB1 (Santa Cruz Biotechnology, sc-33709, 1:250), ATP1A1 (Abcam, ab7671, 1:200), and SOX9 (Abcam, ab185230, 1:300).

Techniques: Imaging, Immunofluorescence, Control, Expressing

Journal: bioRxiv

Article Title: Ubiquitin Ligase ITCH Regulates Life Cycle of SARS-CoV-2 Virus

doi: 10.1101/2024.12.04.624804

Figure Lengend Snippet: (A) Culture medium from vT2-WT and vT2-ITCH-KO cells expressing HA-tagged ubiquitin (Ub) and Flag-2×Strep tagged E was harvested for Strep AP. A decrease of the extracellular E protein induced by ITCH ablation was visualized by immunoblot (left) and dot blot analysis (right). (B) Lysates from HEK293 cells expressing Flag-tagged E with ITCH or ITCH-CS were subjected to Flag IP, followed by immunoblotting for autophagosome cargo receptors. E specifically precipitated p62 and ITCH promoted their interaction. (C-E) HEK293 cells were transfected with Flag-tagged E with ITCH or ITCH-CS. 24 h later, cells were analyzed by immunofluorescence with Flag, ITCH and p62 or LC3B or LAMP1 antibodies. ITCH enhanced the colocalization between E and p62 (C) or LC3B (D), while no change in the colocalization between E and LAMP1 (E) was noted. Scale bar, 10 μm. (F) Culture media and denatured lysates from control (CTRL) and p62 knock down (#1, #2) HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged E were subjected to Flag IP (with incorporation of a washing step with urea before elution for culture media samples), followed by immunoblotting or dot blot analysis. p62 depletion resulted in the accumulation of intracellular E (both unmodified and ubiquitinated), while decreasing the level of extracellular E (n=3). (G, H) vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 h were subjected to immunofluorescence analysis with E and p62 or LC3B antibodies. ITCH-ablation decreased the colocalization between E and p62 (G) or LC3B (H). Scale bar, 10 μm. (I) A model of the function of ITCH in promoting autophagosome-mediated SARS-CoV-2 virion egress. ITCH-dependent ubiquitin modification enhances E binding with S and M binding with non-ubiquitinated E, resulting in the increase in virion formation and p62-dependent autophagosome targeting for release.

Article Snippet: The following primary antibodies were used: Flag (Sigma, F1804); Flag (Sigma, F3165); Flag (Cell Signaling Technology, 14793S); GAPDH (Invitrogen, MA5-27912); β-tubulin (Cell Signaling Technology, 2128S);s (BioLegend, 688102); Strep (Invitrogen, MA5-17283); CBD (New England BioLabs, E8034S); ubiquitin (Cell Signaling Technology, 58395S); K63-linkage-specific antibody (Enzo Life Sciences, BML-PW0600-0100); K48-linkage-specific antibody (Cell Signaling Technology, 8081S); Spike (Proteintech, 28867-1-AP); M (Proteintech, 28882-1-AP); E (Proteintech, 28904-1-AP); ITCH (Santa Cruz, sc-28367); ITCH (Novus Biologicals, NB100-68142); p62 (Cell Signaling Technology, 88588S and 7695S); GM130 (Proteintech, 11308-1-AP); LAMP1 (Cell Signaling Technology, 9091S); OPTN (Cayman Chemical, 100002); LC3 (Proteintech,14600-1-AP); LC3B (Cell Signaling Technology, 3868S); SARS-CoV-2 Membrane protein (Cell Signaling Technology, 15333S); SARS-CoV-2 Envelope protein (Cell Signaling Technology, 74698S); furin (Proteintech, 18413-1-AP); Cathepsin L (Proteintech, 10938-1-AP); NDP52 (Proteintech, 12229-1-AP); NBR1 (Proteintech, 16004-1-AP); FAM134B (Proteintech, 21537-1-AP); RTN3 (Proteintech, 12055-2-AP) and NIX (Proteintech, 12986-1-AP).

Techniques: Expressing, Western Blot, Dot Blot, Transfection, Immunofluorescence, Control, Knockdown, Infection, Modification, Binding Assay

Journal: bioRxiv

Article Title: Ubiquitin Ligase ITCH Regulates Life Cycle of SARS-CoV-2 Virus

doi: 10.1101/2024.12.04.624804

Figure Lengend Snippet: (A) Culture medium from vT2-WT and vT2-ITCH-KO cells expressing HA-tagged ubiquitin (Ub) and Flag-2×Strep tagged E was harvested for Strep AP. A decrease of the extracellular E protein induced by ITCH ablation was visualized by immunoblot (left) and dot blot analysis (right). (B) Lysates from HEK293 cells expressing Flag-tagged E with ITCH or ITCH-CS were subjected to Flag IP, followed by immunoblotting for autophagosome cargo receptors. E specifically precipitated p62 and ITCH promoted their interaction. (C-E) HEK293 cells were transfected with Flag-tagged E with ITCH or ITCH-CS. 24 h later, cells were analyzed by immunofluorescence with Flag, ITCH and p62 or LC3B or LAMP1 antibodies. ITCH enhanced the colocalization between E and p62 (C) or LC3B (D), while no change in the colocalization between E and LAMP1 (E) was noted. Scale bar, 10 μm. (F) Culture media and denatured lysates from control (CTRL) and p62 knock down (#1, #2) HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged E were subjected to Flag IP (with incorporation of a washing step with urea before elution for culture media samples), followed by immunoblotting or dot blot analysis. p62 depletion resulted in the accumulation of intracellular E (both unmodified and ubiquitinated), while decreasing the level of extracellular E (n=3). (G, H) vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 h were subjected to immunofluorescence analysis with E and p62 or LC3B antibodies. ITCH-ablation decreased the colocalization between E and p62 (G) or LC3B (H). Scale bar, 10 μm. (I) A model of the function of ITCH in promoting autophagosome-mediated SARS-CoV-2 virion egress. ITCH-dependent ubiquitin modification enhances E binding with S and M binding with non-ubiquitinated E, resulting in the increase in virion formation and p62-dependent autophagosome targeting for release.

Article Snippet: The following primary antibodies were used: Flag (Sigma, F1804); Flag (Sigma, F3165); Flag (Cell Signaling Technology, 14793S); GAPDH (Invitrogen, MA5-27912); β-tubulin (Cell Signaling Technology, 2128S);s (BioLegend, 688102); Strep (Invitrogen, MA5-17283); CBD (New England BioLabs, E8034S); ubiquitin (Cell Signaling Technology, 58395S); K63-linkage-specific antibody (Enzo Life Sciences, BML-PW0600-0100); K48-linkage-specific antibody (Cell Signaling Technology, 8081S); Spike (Proteintech, 28867-1-AP); M (Proteintech, 28882-1-AP); E (Proteintech, 28904-1-AP); ITCH (Santa Cruz, sc-28367); ITCH (Novus Biologicals, NB100-68142); p62 (Cell Signaling Technology, 88588S and 7695S); GM130 (Proteintech, 11308-1-AP); LAMP1 (Cell Signaling Technology, 9091S); OPTN (Cayman Chemical, 100002); LC3 (Proteintech,14600-1-AP); LC3B (Cell Signaling Technology, 3868S); SARS-CoV-2 Membrane protein (Cell Signaling Technology, 15333S); SARS-CoV-2 Envelope protein (Cell Signaling Technology, 74698S); furin (Proteintech, 18413-1-AP); Cathepsin L (Proteintech, 10938-1-AP); NDP52 (Proteintech, 12229-1-AP); NBR1 (Proteintech, 16004-1-AP); FAM134B (Proteintech, 21537-1-AP); RTN3 (Proteintech, 12055-2-AP) and NIX (Proteintech, 12986-1-AP).

Techniques: Expressing, Western Blot, Dot Blot, Transfection, Immunofluorescence, Control, Knockdown, Infection, Modification, Binding Assay