EMBO/soda-nel huggingface dataset:
Distinct biogenesis pathways may have led to functional divergence of the human and Drosophila Arglu1 sisRNA (2022)
Figure 7

Figure 7. Cross-species binding of Arglu1 intron 2 to ARGLU1 protein and processing into sisRNAs.

A Confocal images of 125 nM d/hARGLU1-GFP protein added with 50 nM IVT RNA of either hArglu1 intron 2 WT (magenta) or dArglu1 intron 2 (red). White arrowheads indicate free IVT RNA dots that are not co-localized with the d/hARGLU1-GFP droplets. Scale bar: 10 μm.

B Quantification of non co-localized RNA dots from (A, white arrowheads). (n = 16 biological replicates).

C, D Quantification of total hARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates).

E, F Quantification of total dARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates).

G Top: Schematic showing exon2-intron2-exon3 (E2i2E3) of dArglu1 used for exogenous expression in MCF-7 cells and locations of primers used for RT-PCR (bottom). Bottom: RT-PCR of processed dArglu1 spliced isoforms and actin as control in dArglu1 E2i2E3 OE MCF-7 cells vs untransfected (WT) MCF-7 cells.

H hArglu1 gene locus showing location of primers used in (I-K).

I-K qPCR of hArglu1 sisRNA, mRNA and pre-mRNA levels in dArglu1 E2i2E3 OE MCF-7 cells vs untransfected (WT) MCF-7 cells. (n = 3 biological replicates).

L Top: Schematic showing exon2-intron2-exon3 (E2i2E3) of hArglu1 used for exogenous expression in S2 cells and locations of primers used for RT-PCR (bottom). Bottom: RT-PCR of processed hArglu1 spliced isoforms and actin5C as control in hArglu1 E2i2E3 OE S2 cells vs untransfected (WT) S2 cells.

M dArglu1 gene locus showing location of primers used in (N-P).

N-P qPCR of dArglu1 sisRNA, mRNA and pre-mRNA levels in hArglu1 E2i2E3 OE S2 cells vs untransfected (WT) S2 cells (n = 3 biological replicates).

Data information: In (B-F), Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. In (I-K, N-P), data are presented as mean ± SEM. ns=not significant, *P≤0.05, **P≤0.01 and ***P≤0.001 (Student's t-test).
Hippo kinases maintain polarity during directional cell migration in C. elegans (2016)
Figure 1

Figure 1. Hippo kinases promote AP migration via prevention of DV polarization.

(A) Model of Q cell position and migration direction in C. elegans L1 Larvae. Green arrow indicates that Q cells migrate along the AP body axis, whereas the dotted red arrows indicate that Q cells are potent to move towards the DV direction. (B) Schematics of the cst-1 and cst-2 gene models and DNA sequences of the cst-1 and/or cst-2 mutants that were generated via CRISPR-Cas9. Purple bar, Serine/Threonine kinase domain; blue bar, SARAH (Salvador-Rassf-Hippo homolog) domain. Arrow, sgRNA target site; green, target sequence; red, PAM sequence; blue, inserted nucleotide; dashed lines, deleted nucleotides. (C) Quantification of AQR position in adult WT, cst-1 and/or cst-2 mutant animals. The full length between the URXs and the end of the nematode tail is divided into 10 blocks, and the percent of AQR that stopped within each block is indicated. ***, P < 0.001 by Fisher’s exact test. N = 50 - 109. (D) Quantification scheme of the angle (α) between the longitudinal axis of a migrating cell and the AP body axis. (E - F) Fluorescence time-lapse images of F-actin, plasma membrane and histone during AQR/QR.ap migration in WT (E) or cst-1; cst-2 double mutant (F) animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrows, migration distance. F-actin is labeled with GFP-tagged with the actin-binding domain of Moesin (GFP::Moesin ABD), and the plasma membrane and Histone are labeled with mCherry-tagged with a myristoylation signal and a histone. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bars, 5 μm. (G - J) Quantification of the QR.ap migration angle (G & H), distance (I) and speed (J). Each line in (G & I) represents the measurement from one time-lapse movie. Error bars indicate SD. N = 10 - 17. Statistical significance is based on Student’s t-tests, **, P < 0.01; ***, P < 0.001.
Hippo kinases maintain polarity during directional cell migration in C. elegans (2016)
Figure 2

Figure 2. Ectopic DV polarization in mig-2 gain-of-function mutants and the phosphorylation of MIG-2S139 by Hippo kinases.

(A) Fluorescence time-lapse images of F-actin, plasma membrane and histone during QR.ap migration in mig-2(rh17) gain-of-function mutant animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrow, migration distance. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bar, 5 μm. (B - C) Quantification of the QR.ap migration angle (B) and distance (C). N = 10. (D) Mass spectrum of a MIG-2 peptide that was phosphorylated at Serine 139 in C. elegans. The peptide was identified from the GFP::MIG-2 immunoprecipitate. (E) In vitro phosphorylation assay of CST-1/2 to MIG-2. Bacterially expressed His-tagged MIG-2 or His-tagged MIG-2S139A were incubated with the GST-tagged kinase domain of GST-CST-1/2 (1-318 aa). The kinase may use ATP-γ-S as a phosphodonor to thiophosphorylate its substrate; thus, the phosphorylation reaction was performed with ATP-γ-S, and anti-thiophosphate ester antibody was used to detect substrate phosphorylation (Allen et al, 2005; Allen et al, 2007). The final reaction was probed via immunoblotting with antibodies as indicated. Thio, thiophosphate ester. (F) Sequence alignment of MIG-2. Serine 139 (red) is conserved among the Rac1 homologs across species. The accession version is as follows: C. elegans, CAB01691.1; Drosophila, CAC88352.1, AAA67041.1; Human, CAB53579.5; Rat, NP_599193.1; Mouse, AAH51053.1; Chicken, NP_990348.1, NP_001188381.1; Zebrafish, AAV85902.1; Xenopus, NP_001089332.1. (G & H) Three-dimensional ribbon (PDB 3TH5) representations of the human Rac1 polypeptide which indicate the position of the T135 (S139 in MIG-2) located between the last two α-helixes (Krauthammer et al, 2012).
Hippo kinases maintain polarity during directional cell migration in C. elegans (2016)
Figure 3

Figure 3. MIG-2S139 phosphorylation regulates actin assembly during cell migration.

(A) Quantification of AQR position in mig-2(mu28) null mutant animals with different rescue transgenes. N = 108 - 114. *, P < 0.05; ##, P < 0.01, phenotype enhanced. P values are based on Fisher’s exact tests. (B) Quantification of AQR position in WT and knock-in animals that harbor mig-2S139 point mutations. N = 100 - 111. ***, P < 0.001 by Fisher’s exact test. (C - F) Quantification of the QR.ap migration angle (C & D), distance (E) and speed (F) in mig-2S139A/E mutant animals. Each line in (C & E) represents the measurement from one movie of QR.ap migration. Error bars indicate SD. N = 12 -17. **, P < 0.01; ***, P < 0.001 by Student’s t tests. (G) Inverted fluorescence images (up) and schematics (down) of QR.ap in WT, cst-1; cst-2 double or mig-2 mutant animals. The plasma membrane and histones were labeled with mCherry. Scale bar, 5 μm. (H) Area ratio between the cell body and the nucleus in WT and mutant animals. Error bars indicate SD. N = 17 - 22. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s t tests. (I - K) Representative frames (upper) and quantification (lower) of F-actin enrichment at the QR.ap periphery in WT (I) and mig-2S139A/E mutant animals (J & K). The trace starts from the rear of the QR.ap and moves along the cell periphery to the leading edge (L) and back to the rear (R). F-actin was labeled with GFP::Moesin ABD; the plasma membrane and histone were marked with mCherry fluorescence. Green lines, F-actin fluorescence intensity; red lines, plasma membrane fluorescence intensity; dotted lines, cell periphery. Double-headed arrows indicate the F-actin enrichment region, in which the fluorescence intensity is 2-fold higher than the background. Scale bars, 5 μm. (L) Quantification of F-actin enrichment ratio in WT and mig-2 mutants. Error bars indicate SD. N = 28 - 68. **, P < 0.01; ***, P < 0.001 by Student’s t-tests.
Hippo kinases maintain polarity during directional cell migration in C. elegans (2016)
Figure 4

Figure 4. Hippo kinases are restricted in the cell body by MIG-2.

(A - C) Fluorescence images (upper) and quantifications (lower) of GFP-tagged CST-1 (A & B) or GFP-labeled F-actin (C) and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. The membrane protrusions in the white boxes are enlarged 3 times at the bottom. Yellow dashed lines indicate the cell periphery. White lines indicate the line to measure the fluorescence intensity. Green lines denote the CST-1 fluorescence intensity; red lines denote the plasma membrane fluorescence intensity; double-headed arrows denote the distance between the GFP signal and cell periphery. Scale bars, 5 μm. (D) Distance between GFP::CST-1 or GFP::Moesin ABD and the plasma membrane at the leading edge in WT and mig-2 null mutants. Error bars indicate SD. N = 9 - 14. ***, P < 0.001 by Student’s t test. (E - F) Fluorescence images (left) and quantifications (right) of GFP-tagged CST-1 and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. Blue lines, sites of measurements; dotted lines, cell periphery; Green lines; CST-1 fluorescence intensity; red lines, mCherry fluorescence intensity. Scale bars, 5 μm. (G) Quantification of the fluorescence intensity ratio of GFP::CST-1 and mCherry-membrane between the leading edge and the lagging edge of AQR in WT and mig-2 null mutant animals. Error bars indicate SD. N = 20. ***, P < 0.001 by Student’s t test. (H) Proposed model of Hippo-RhoG signaling negative feedback loop in the prevention of secondary fronts during directional cell migration.
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6 (2021)
Figure 1

Figure 1. CRTH2 is located in the endoplasmic reticulum (ER) and cell membrane in fibroblasts.

A, CRTH2 subcellular localization in GFP-fused CRTH2 expressing plasmid-transfected cells. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.

B, Western blot analysis of CRTH2 expression in cell membrane and ER in NIH 3T3 and HEK293T cells. Myc-tagged CRTH2 expressing plasmid was transfected into NIH 3T3 and HEK293T cells. Whole cell lysate, plasma, and ER fractions were obtained 48 h after transfection. Calnexin and ATP1B were used as markers for ER and cell membrane, respectively.

C, The effect of TGF-β1 (10 ng/mL) on the expression of caveolin-1 in NIH 3T3 cells.

D, Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF-β1 (10 ng/mL) treatment. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.

E, Quantification of CRTH2 colocalization with endoplasmic reticulum after TGF-β1 or vehicle treatment. *P < 0.05 vs vehicle (Mann-Whitney U test); n = 4 for all groups. Data are expressed as the mean ± standard error of the mean.
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6 (2021)
Figure 2

Figure 2. CRTH2 interacts with LARP6 and GRP78 in the ER.

A, Immunopurification and mass spectrometric analysis of CRTH2-containing protein complex in NIH 3T3 cells. Cellular extracts from NIH 3T3 cells stably expressing myc-CRTH2 protein, were immunopurified with anti-myc affinity beads and eluted with myc peptide. The eluates were used for SDS-PAGE and the gels were silver stained and analyzed using mass spectrometry.

B-D, Representative mass spectrogram of CRTH2 (B), LARP6 (C) and GRP78 (D) from CRTH2-containing protein complex in NIH 3T3 cells.

E-F, Co-immunoprecipitation analysis of association of CRTH2 with LARP6. HEK293T cells were co-transfected with Myc-CRTH2 and Flag-LARP6 expressing plasmids. Whole cell lysates from HEK293T cells were immunoprecipitated and then immunoblotted with the indicated antibodies.

G, Subcellular localization of CRTH2 and LARP6 in NIH 3T3 cells. NIH 3T3 cells were transfected with GFP-fused CRTH2 expressing plasmid, and fixed and immunostained with antibodies against the indicated proteins. Red arrow indicates colocalization of CRTH2 and LARP6 in the ER. Scale bar, 20 μm.

H-I, Co-IP analysis of association of CRTH2 with GPR78. HEK293T cells were co-transfected with Flag-CRTH2 and Myc-GPR78 expressing plasmids.

J, Colocalization of CRTH2, LARP6 and GRP78 in NIH 3T3 cells. White arrow indicates colocalization of CRTH2, LARP6 and GRP78. Scale bar, 20 μm.

K, Schematic diagram depicting different domains of CRTH2 involved in the interaction with LARP6 and GRP78 in the endoplasmic reticulum membrane.
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6 (2021)
Figure 3

Figure 3. CRTH2 deficiency increases collagen synthesis in fibroblasts through LARP6.

A, RNA immunoprecipitation and qRT-PCR (RIP-qPCR) analysis of collagen type 1, α1 (Col1a1), collagen type 1, α2 (Col1a2), and collagen type 3, α1 (Col3a1) mRNA in CRTH2-/- and wild type (WT) fibroblasts using LARP6 antibody. *P < 0.05 vs WT (two-tailed Student's t test); n = 6 for all groups.

B, Effect of the CRTH2 deficiency on the stability of Col1a1, Col1a2, and Col3a1 mRNA in primary mouse fibroblasts. Fibroblasts were treated with 5 μg/mL Actinomycin D (Act D). Total RNA was extracted immediately after addition of Act D at 0, 2, 4, 6, 8, 12, and 24 h. Col1a1, Col1a2, and Col3a1 mRNA expression was quantitated by qRT-PCR. Dotted lines indicate mRNA half-lives; *P < 0.05 vs WT (two-way ANOVA); n = 6 for all groups.

C, Effect of CRTH2 deficiency on Col1a1, Col1a2, and Col3a1 mRNA expression in primary mouse fibroblasts. GAPDH was used as housekeeping gene. *P < 0.05 vs WT (two-tailed Student's t test); n = 8 for all groups.

D, Effect of CRTH2 deficiency on Collagen (Col) I and III protein expression in primary mouse fibroblasts with and without TGF-β1 (10 ng/mL) treatment.

E, Knockdown efficiency of LARP6 si-RNA (Si-LARP6) detected by qRT-PCR in primary fibroblasts. *P < 0.05 vs scramble, (two-tailed Student's t test); n = 5 for all groups.

F, Effect of LARP6 knockdown on Col1a1, Col1a2 and Col3a1 mRNA stability in WT and CRTH2-/- fibroblasts. *P < 0.05 vs WT, (two-way ANOVA); n = 8 for all groups.

G-I, Effect of LARP6 knockdown on Col1a1, Col1a2, and Col3a1 mRNA expression in WT and CRTH2-/- fibroblasts. Fibroblasts were transfected with Si-LARP6 or scramble siRNA. After 48 h transfection, RNA was prepared for qRT-PCR analysis. GAPDH was used as housekeeping gene. *P < 0.05 vs WT, #P < 0.05 vs scramble (two-way ANOVA); n = 6 for all groups.

J, Effect of LARP6 knockdown on Collagen I and III protein expression in WT and CRTH2-/- fibroblasts.

Data information: All data are expressed as the mean ± standard error of the mean.
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6 (2021)
Figure 4

Figure 4. CRTH2 deficiency in fibroblasts exacerbates injury-induced organ fibrosis in mice.

A, Schematic diagram of the bleomycin-induced lung fibrosis model in mice.

B, Representative images of Masson′s trichrome staining of lung sections from CRTH2flox/floxCol1a2-CreERT (F-CRTH2 KO) and CRTH2flox/flox (Control) mice 14 days after bleomycin challenge. Blue indicates the collagen deposition regions. Scale bar, 100 μm.

C, Quantification of collagen content in mouse lung sections after bleomycin challenge. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each; bleomycin groups, n = 8 each.

D, Hydroxyproline content in lungs from control and F-CRTH2 KO mice treated with saline or bleomycin. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, bleomycin groups, n = 8 each.

E, Western blot analysis of collagen (Col) Ⅰ and Ⅲ expression in lung tissues from control and F-CRTH2 KO mice 14 days after bleomycin challenge.

F, Protocol for CCl4-induced liver fibrosis in mice.

G, Representative images of Masson′s trichrome staining of liver sections from control and F-CRTH2 KO mice 4 weeks after CCl4 challenge; Scale bar, 100 μm.

H, Quantification of collagen content in mouse liver sections after CCl4 challenge. *P < 0.05 vs control, #P < 0.05 vs mineral oil group (two-way ANOVA); mineral oil groups, n = 4 each; CCl4 groups, n = 8 each.

I, Hydroxyproline content in lungs from control and F-CRTH2 KO mice treated with CCl4. *P < 0.05 vs control, #P < 0.05 vs mineral oil group (two-way ANOVA); mineral oil groups, n = 4 each, CCl4 groups, n = 8 each.

J, Western blot analysis of Collagen Ⅰ and Ⅲ expression in liver tissues from control and F-CRTH2 KO mice 4 weeks after CCl4 challenge.

K, Schematic diagram of isoproterenol (ISO)-induced heart fibrosis in mice.

L, Representative images of Masson′s trichrome staining of heart sections from control and F-CRTH2 KO mice 21 days after ISO challenge; Scale bar, 50 μm.

M, Quantification of collagen content in mouse heart sections after ISO challenge. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); PBS groups, n = 3 each, ISO groups, n = 7-8.

N, Hydroxyproline content in lungs from control and F-CRTH2 KO mice treated with ISO. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each; ISO groups, n = 8 each.

O, Western blot analysis of Collagen Ⅰ and Ⅲ expression in heart tissues from control and F-CRTH2 KO mice 21 days after ISO challenge.

Data information: All data are expressed as the mean ± standard error of the mean.
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6 (2021)
Figure 5

Figure 5. LARP6 is required for deteriorated organ fibrosis in CRTH2-/- mice.

A, Representative images of Masson's trichrome staining of lung sections from F-LARP6 KO and F-LARP6/F-CRTH2 double KO (F-DKO) mice 14 days after bleomycin challenge; Scale bar, 100 μm.

B, Quantification of collagen content in mouse lung sections after bleomycin challenge. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, bleomycin groups, n = 7-8.

C, Hydroxyproline content in lungs from F-LARP6 KO and F-DKO mice treated with bleomycin. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, bleomycin groups, n = 8 each.

D, Western blot analysis of Collagen (Col) Ⅰ and Ⅲ expression in lung tissues from F-LARP6 KO and F-DKO mice 14 days after bleomycin challenge.

E, Representative images of Masson's trichrome staining of liver sections from F-LARP6 KO and F-DKO mice 4 weeks after CCl4 challenge; Scale bar, 100 μm.

F, Quantification of collagen content in mouse liver sections after CCl4 challenge. #P < 0.05 vs mineral oil group (two-way ANOVA); mineral oil groups, n = 4 each, CCl4 groups, n = 8 each.

G, Hydroxyproline content in livers from F-LARP6 KO and F-DKO mice treated with CCl4. #P < 0.05 vs mineral oil group (two-way ANOVA); saline groups, n = 4 each, CCl4 groups, n = 8 each.

H, Western blot analysis of Collagen Ⅰ and Ⅲ expression in liver tissues from F-LARP6 KO and F-DKO mice 4 weeks after CCl4 challenge.

I, Representative images of Masson's trichrome staining of heart sections from F-LARP6 KO and F-DKO mice 21 days after ISO challenge; Scale bar, 50 μm.

J, Quantification of collagen content in mouse heart sections after ISO challenge. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 3 each, ISO groups, n = 7 each.

K, Hydroxyproline content in hearts from F-LARP6 KO and F-DKO mice treated with ISO. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, ISO groups, n = 8 each.

L, Western blot analysis of Collagen Ⅰ and Ⅲ expression in heart tissues from F-LARP6 KO and F-DKO mice 21 days after ISO challenge.

M, Schematic diagram of the working model of CRTH2-mediated collagen homeostasis in fibroblasts.

Data information: All data are expressed as the mean ± standard error of the mean.
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6 (2021)
Figure 6

Figure 6. Bumetanide inhibits bleomycin-induced lung fibrosis in mice by targeting the LARP6 RRM domain.

A, Structure-based flow chart for the virtual screening of small molecules interacting with the LARP6-RRM domain.

B-C, Microscale thermophoresis (MST) analysis of the binding affinity of Bumetanide with MBP-LARP6 and MBP control protein.

D, Binding mode of the LARP6-RRM domain with Bumetanide. The protein is shown as cartoons and is green. Bumetanide and dominant residues are shown as sticks. Bumetanide is cyan and the dominant residues are green.

E-F, Effect of Bumetanide (2 μM) on direct interaction of CRTH2 N-terminal (CRTH2-2) with LARP6 RRM domain (LARP6-4) using GST and MBP pulldown assays.

G, Effect of Bumetanide on LARP6 binding to Col1a1, Col1a2, and Col3a1 mRNA in primary mouse fibroblasts. *P < 0.05 vs DMSO, (unpaired two-tailed t test); n = 6 for all groups.

H, Protocol for administration of Bumetanide to bleomycin-challenged mice. Bumetanide (2 mg/kg) was subcutaneously injected in mice daily for 14 days.

I, Representative Masson's trichrome staining of lung sections from Bumetanide-treated control and F-CRTH2 KO mice. Scale bars, 100 µm.

J, Quantification of collagen content in lung sections from Bumetanide-treated control (CRTH2flox/flox) and F-CRTH2 KO mice. *P < 0.05 vs control, #P < 0.05 vs Vehicle (two-way ANOVA); n = 7 for all groups.

K, Effect of Bumetanide treatment on pulmonary hydroxyproline levels in control and F-CRTH2 KO mice. *P < 0.05 vs control; #P < 0.05 vs Vehicle (two-way ANOVA); n = 7 for all groups.

L, Effect of Bumetanide treatment on Collagen (Col) I and III expression in lungs from control and F-CRTH2 KO mice.

Data information: All data are expressed as the mean ± standard error of the mean.
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6 (2021)
Figure 7

Figure 7. Bumetanide reduces TGF-β1-induced collagen biosynthesis in human fibroblasts.

A-B, Bumetanide interferes with the interaction of CRTH2 with LARP6 in MRC-5 human lung fibroblasts MRC-5. MRC-5 cells were co-transfected with myc-LARP6 and flag-CRTH2 expressing plasmids for 24 h, and then treated with Bumetanide (0.1, 2 μM) for an additional 24 h. Whole cell lysates from MRC-5 cells were immunoprecipitated and immunoblotted with the indicated antibodies.

C-E, Effect of Bumetanide on LARP6 binding to Col1a1, Col1a2, and Col3a1 mRNA in cultured MRC-5 cells. MRC-5 cells were treated with TGF-β1 and Bumetanide (2 μM) for 24 h. The cells were ultraviolet-cross-linked and RNA-bound proteins were immunoprecipitated with LARP6 antibody followed by RNA extraction for qRT-PCR. *P < 0.05 vs Vehicle, #P < 0.05 vs Control (two-way ANOVA); n = 6 for all groups.

F-H, Effect of Bumetanide on Col1a1, Col1a2 and Col3a1 mRNA expression in MRC-5 cells. GAPDH was used as housekeeping gene. *P < 0.05 vs vehicle, #P < 0.05 vs Control (two-way ANOVA); n = 6 for all groups.

I, Effect of Bumetanide on Collagen (Col) I and III protein expression in MRC-5 cells.

Data information: All data are expressed as the mean ± standard error of the mean.

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