EMBO/soda-nel huggingface dataset:
Optogenetic control of YAP cellular localisation and function (2022)
Figure 3

Figure 3. Validation of optofYap in zebrafish.

(A) Imaging and light activation protocol in zebrafish. Shield-stage embryos were subjected to activation protocol as described in Fig. 1A and imaged at the animal pole.

(B) Example images of embryos expressing optofYap (mCherry-tagged) in the EVL and deep cells kept in the dark or subjected to the activation protocol at 6 hpf. Scale bars, 10 μm. (B') Fold-change in nuclear localisation of mCherry-optofYap (n=20 cells from 3 independent experiments). Box plots represent median and 25th to 75th percentiles. Whiskers show minimum and maximum points. ***P < 10-3 (paired t-test).

(C) The N/C ratio measured using mCherry signal in zebrafish embryos injected with optofYap tracked over 10 min of activation protocol and 10 min recovery in the dark (see also Fig. EV3A-B). (n=10 cells from 2 independent experiments, red line=average). Vertical dashed line represents time when 488 nm stimulation ceased.

(D) qPCR of Yap target genes. Embryos were kept in the dark until 10 hpf and then subjected to pulsed light activation for 16 h. Gene expression level was normalized to the housekeeping gene, rpl13. Horizontal bars represent mean and 95% confidence interval from 4 biological replicates for control and 8 biological replicates for injected samples, *P < 0.05, **P < 10-2, ***P < 10-3 (unpaired t-test).
Cancer cell adaptation to hypoxia involves a HIF-GPRC5A-YAP axis (2018)
Figure 1

Figure 1. Hypoxia induces GPRC5A directly via HIFs

A. SILAC-based proteomics data identify known (red) & novel (green) hypoxia-induced proteins in SW620 cells. One sample T-test was performed.

B. Western blotting confirmed GPRC5A as a hypoxia-induced protein in SILAC lysates.

C. Validation of GPRC5A western blot data using siRNA. *Non-specific band of ~60kDa not depleted by GPRC5A siRNA.

D. Confocal microscopy showing plasma membrane GPRC5A expression in hypoxic SW620 cells (scale bars: 75um).

E. Western blotting showing GPRC5A upregulation by hypoxia in a panel of colorectal tumour cell lines.

F. Basal & hypoxia-induced GPRC5A protein expression was decreased by HIF-1/2α depletion.

G. Depletion of HIF-1β decreased GPRC5A protein upregulation in hypoxia.

H. Hypoxia mimetic DMOG induced HIF-1/2α, CA9 & GPRC5A protein expression. Dual HIF-1/2α depletion reduced GPRC5A induction by DMOG.

I. qRT-PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).

J. qRT-PCR demonstrating that HIF-1/2α depletion decreased GPRC5A induction during hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).

K. ChIP/PCR analyses identify HIF-1α a binding to the GPRC5A promoter region containing a putative optimal HRE (error bars ± SD).

Data information: Levels adjustments were made to images in Adobe Photoshop post-acquisition for clarity (equal changes applied to the entire image). Representative examples of n=3 independent experiments are shown.
Cancer cell adaptation to hypoxia involves a HIF-GPRC5A-YAP axis (2018)
Figure 2

Figure 2. GPRC5A is hypoxia/HIF induced in vivo

A-B. Expression of CA9 & GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced CA9 & GPRC5A expression with siRNA confirms antibody specificity (scale bars: 200um).

C. IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia (strangulated colon). GPRC5A is co-expressed with CA9 in the colonic epithelial cells (scale bars: 50um).

D. Quantitative RT-PCR analysis of mouse intestinal tissue. Gene expression was normalised to housekeeping gene Tbp. Raw data from three independent experiments (n=3 mice) are shown (error bars ± SEM).

E. Tg[fli1:eGFP; vhl-/-] & Tg[fli1:eGFP] zebrafish embryos (5 days post-fertilisation) demonstrate excessive angiogenesis & increased expression of HIF target genes (scale bars: 100um).

F. gprc5ba was induced in vhl mutant zebrafish embryos & fli1:eGFP zebrafish embryos exposed to 5% O2 (vs normoxia) for 24 hours (RT-PCR).

G-H. Bioinformatic analysis of transcriptomics dataset GSE24551. Gene set analyses reveals GPRC5A mRNA strongly correlated with HIF/hypoxia gene signatures. GSEA datasets used were Semenza_HIF1_Targets (M12299) Broad_Hallmark_Hypoxia (M5891). Analysis was performed using R2 (http://r2.amc.nl).

I. Kaplan-Meier curve following analysis of transcriptomics dataset GSE24551. Event-free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA. Analysis was performed using R2 (http://r2.amc.nl).

Data information: Levels adjustments were made to images in Adobe Photoshop post-acquisition for clarity (equal changes applied to the entire image). Representative examples of n=3 independent experiments are shown.
Cancer cell adaptation to hypoxia involves a HIF-GPRC5A-YAP axis (2018)
Figure 3

Figure 3. GPRC5A promotes hypoxic cancer cell survival

A. GPRC5A depletion markedly increases caspase-3 activation/PARP cleavage during hypoxia.

B. Three independent siRNA sequences targeting GPRC5A induce caspase-3 activation/PARP cleavage during hypoxia.

C. GPRC5A depletion reduces hypoxic cell growth/survival. Crystal violet cell assays show reduced cell growth/survival in GPRC5A depleted cells during hypoxia [n=3].

D. Expression of an siRNA-resistant GPRC5A cDNA rescues hypoxic GPRC5A-depleted cells from apoptosis. Upper: doxycycline-induced expression GPRC5Asi1R rescues increases caspase-3/PARP cleavage induced by GPRC5A depletion in hypoxia. Lower: generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations.

E. Caspase inhibitor QVD prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.

F. GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe/dead cell apoptosis assay and flow cytometry [n=3 independent experiments].

Data information: Levels adjustments were made to images in Adobe Photoshop post-acquisition for clarity (equal changes applied to the entire image). Representative examples of n=3 independent experiments shown; data are presented as ± SEM. One-way ANOVA with Tukey's multiple comparisons test was carried out in C and F.
Cancer cell adaptation to hypoxia involves a HIF-GPRC5A-YAP axis (2018)
Figure 4

Figure 4. GPRC5A promotes hypoxic cell survival via a novel HIF-GPRC5A-YAP axis

A. Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells.

B. Hypoxia-induced nuclear localisation of YAP was attenuated in GPRC5A depleted cells.

C. Hypoxia activated TEAD activity (8x GTIIC-luc reporter) but this was reduced by GPRC5A depletion. A representative triplicate experiment is shown (n=3).

D. Hypoxia reduced LATS activity and expression but this was prevented by GPRC5A depletion.

E. Constitutively active RhoA (G14V) expression restored YAP stabilisation (Ser397 dephosphorylation) by hypoxia in GPRC5A depleted cells.

F. The YAP/TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay (n=3 independent experiments).

G. YAP knockdown was sufficient to induce caspase-3 activation/PARP cleavage in hypoxia and was not further enhanced by GPRC5A depletion.

H. Crystal violet assays show that YAP was required downstream of GPRC5A to promote cell survival. GPRC5A depleted cells were rescued by expression of an siRNA-resistant GPRC5A cDNA (GPRC5Asi1R) but this was prevented by co-depletion of YAP (n=3 independent experiments).

I. Expression of an siRNA-resistant GPRC5A rescued the critical phenotypes of GPRC5A depletion. GPRC5Asi1R expression prevented PARP cleavage in hypoxia as well as restoring hypoxia-induced YAP stabilisation (Ser397 dephosphorylation); these phenotypes were reversed by YAP depletion.

J. GPRC5A depletion attenuated hypoxia-induced BCL-XL expression.

K. Constitutively active YAP (S127A) expression induced BCL-XL expression and prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.

L. GPRC5Asi1R expression restored BCL-XL expression and prevents the appearance of cleaved caspase-3 induced by GPRC5A depletion in hypoxia.

Data information: Levels adjustments were made to images in Adobe Photoshop post-acquisition for clarity (equal changes applied to the entire image). Representative examples of n=3 independent experiments shown; data are presented as ± SEM. One-way ANOVA with Tukey's multiple comparisons test was carried out in F and H.
5-Fluorouracil Efficacy Requires Anti-tumor Immunity Triggered by Cancer-cell-intrinsic STING (2021)
Figure 1

Figure 1. Efficient 5-FU-induced reduction of tumor burden in immunocompetent host is dependent on cancer-cell-intrinsic STING.

A. Schematic illustration of the syngeneic tumor model in which MC38 colon cancer cells were injected subcutaneously. 5-FU (25 mg/kg per dose) was administered intraperitoneally from day 6 post cancer cell injection, with one dose every two days. Tumor and spleen were harvested at 2 weeks.

B. Tumors formed with WT MC38 cells were treated with PBS or 5-FU. Tumor volumes were quantified at the indicated days. N=5.

C. STING-KO or sgRNA control (Ctrl) MC38 cells were treated in vitro with the indicated concentrations of 5-FU. Cell viability were determined using the CellTiter-Glo assay after two days, with normalized luminescence levels shown. N=3.

D-F. Mice were injected with Ctrl or STING-KO MC38 cells, and treated with PBS or 5-FU. (D) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. (E) Tumor volumes were quantified at the indicated days post cancer cell injection. N=4 to N=5 as shown in (D). (F) Tumor and spleen weights at the endpoint for (E), with each dot representing a mouse.

G-I. WT MC38 cells were injected into STING+/+ or STING-/- mice, and treated with 5-FU or PBS following the schematics in (A). (G) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. (H) Tumor volumes were quantified at the indicated days post cancer cell injection. N=5. (I) Tumor and spleen weights at the endpoint for (H), with each dot representing a mouse.

Data information: For all panels, error bars stand for s.d., and center values represent mean. Two-tailed unpaired student's t-test was used. ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001; ∗∗∗∗: p < 0.0001; ns: not significant. Data in (B, D-F) are representative of at least four independent experiments. Data in (C, G-I) are representative of two experiments.
5-Fluorouracil Efficacy Requires Anti-tumor Immunity Triggered by Cancer-cell-intrinsic STING (2021)
Figure 2

Figure 2. Cancer-cell-intrinsic STING is required for the response of YUMM1.7 melanoma to 5-FU but not DTIC in vivo.

A. Schematic illustration of the tumor model in which YUMM1.7 melanoma cells were injected subcutaneously into syngeneic wildtype C57BL/6 mice. 5-FU (25 mg/kg) or DTIC (10mg/kg) were administered intraperitoneally from day 6 post cancer cell injection, with one dose every two days. Tumor and spleen were harvested at day 16.

B. Western blot for Ctrl and STING KO YUMM1.7 cells, assayed with STING and Gapdh antibodies. C. STING-KO or sgRNA control (Ctrl) YUMM1.7 cells were treated in vitro with the indicated concentrations of DTIC. Cell viability were determined using the CellTiter-Glo assay after two days, with relative cell viability shown. N=3.

D. For cells in (C), proliferation assays were performed with cell viability assayed on indicated days after plating. Relative luminescence in arbitrary units (AU) is shown. N=3.

E-G. YUMM1.7 Ctrl or STING KO cells were injected into mice. 5-FU and DTIC responses were followed according to the schematics in (A). (E) Representative images of tumors and spleens. Image panels were cropped from the same picture. (F) Mean tumor volumes of Ctrl or STING KO YUMM1.7 with DMSO, DTIC or 5-FU treatment were followed during the experiment. N=3 to N=5, as shown in (E). (G) Tumor (left panel) and spleen (right panel) weights were determined at the endpoint for the experiment followed in (F). Each dot represents data from a mouse.

Data information: For all panels, error bars represent s.d., and center values represent mean. Two-tailed unpaired student's t-test was used. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns: not significant. Data are representative of two independent experiments.
5-Fluorouracil Efficacy Requires Anti-tumor Immunity Triggered by Cancer-cell-intrinsic STING (2021)
Figure 3

Figure 3. Cancer-cell-intrinsic type I IFNs are required for efficient 5-FU-induced reduction of tumor burden.

A. Control (Ctrl) or STING-KO MC38 cells were treated in vitro with STING agonist cGAMP (left) for 6 hours or 5-FU (right) or relevant vehicle controls for 24 hours. The expression of indicated genes was determined by qRT-PCR. N=3.

B. Ctrl or STING-KO CT26 cells were treated with 5-FU or vehicle control for 24 hours. The RNA expression levels of indicated genes were determined by qRT-PCR. N=3.

C. Ctrl or STING-KO YUMM1.7 cells were treated with (left panel) vehicle control (DMSO) or DTIC, or (right panel) PBS or 5-FU for 24 hours. The expression of indicated genes was determined by qRT-PCR. N=3.

D. Western blot analysis was performed with Ifnα and Gapdh antibodies, for control (Ctrl) and Ifna1-Ifnb1-double-KO (Ifn-DKO) MC38 cells. Two independent KO clones are shown.

E. MC38 Ctrl or Ifn-DKO cells were treated with (left panel) control or the STING-agonist DMXAA for 4 hours, or (right panel) control or 5-FU for 16 hours. Ifnβ levels were determined in harvested culture media by ELISA. N=2.

F-H. Mice were injected with Ctrl MC38 cells or Ifn-DKO cells and treated with 5-FU or PBS. (F) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. (G) Tumor volumes were quantified at the indicated days post cancer cell injection. N=5. (H) Tumor and spleen weights at the endpoint for (G), with each dot representing a mouse.

Data information: For all panels, error bars stand for s.d., and center values represent mean. Two-tailed unpaired student's t-test was used. ∗∗: p < 0.01; ∗∗∗: p < 0.001; ∗∗∗∗: p < 0.0001; ns: not significant. Data are representative of two or more independent experiments.
5-Fluorouracil Efficacy Requires Anti-tumor Immunity Triggered by Cancer-cell-intrinsic STING (2021)
Figure 4

Figure 4. Cancer-cell-intrinsic cGAS is required for efficient 5-FU-induced reduction of tumor burden.

A. Western blot for control sgRNA (Ctrl) and cGAS-KO MC38 cells, assayed with cGAS and Gapdh antibodies.

B-D. Mice were injected with Ctrl or cGAS-KO MC38 cells and treated with PBS or 5-FU. (B) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. (C) Tumor volumes were quantified at the indicated days post cancer cell injection. N=4 to N=5, as shown in (B). (D) Tumor and spleen weights at the endpoint for (C), with each dot representing a mouse.

Data information: For all panels, error bars stand for s.d., and center values represent mean. Two-tailed unpaired student's t-test was used. ∗: p < 0.05; ∗∗∗∗: p < 0.0001; ns: not significant. Data are representative of two or more independent experiments.
5-Fluorouracil Efficacy Requires Anti-tumor Immunity Triggered by Cancer-cell-intrinsic STING (2021)
Figure 5

Figure 5. Efficient 5-FU-induced tumor inhibition depends on IFN-sensing by bone-marrow-derived cells.

A. Western blot for Ctrl and Ifnar1-KO MC38 cells, analyzed with Ifnar1 and Gapdh antibodies.

B. Ctrl or Ifnar1-KO MC38 cells were treated with PBS or recombinant Ifnβ for 4 hours. The RNA expression levels of indicated ISGs were analyzed using qRT-PCR. N=3.

C-E. Mice were injected with control (Ctrl) or Ifnar1-KO MC38 cells, and treated with 5-FU or PBS. (C) Pictures of tumors and spleens from a representative experiment. (D) Tumor volumes were quantified at the indicated days post cancer cell injection. N=5. (E) Tumor and spleen weights at the endpoint for (D), with each dot representing a mouse. Ctrl tumor data in (C-E) are the same as those in Figure 4B-4D.

F-I. Schematics (F) of the experiment to test the function of Ifnar1 in bone marrow (BM) derived cells. WT C57BL/6 mice were transplanted with either Ifnar1+/+ or Ifnar1-/- BM cells. Recipient mice were allowed to recover followed by the injection of WT MC38 cells, before treatment with 5-FU or PBS. (G) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. (H) Tumor volumes were quantified at the indicated days post cancer cell injection. N=4 to N=5, as shown in (G). (I) Tumor and spleen weights at the endpoint for (H), with each dot representing a mouse.

Data information: For all panels, error bars stand for s.d., and center values represent mean. Two-tailed unpaired student's t-test was used. ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001; ∗∗∗∗: p < 0.0001; ns: not significant. Data are representative of two independent experiments.
5-Fluorouracil Efficacy Requires Anti-tumor Immunity Triggered by Cancer-cell-intrinsic STING (2021)
Figure 6

Figure 6. 5-FU treatment alters intratumoral immune cell populations

Mice were injected with control (Ctrl) or STING-KO MC38 cells and treated with PBS or 5-FU. Tumors were harvested 2 weeks after cancer cell injection and intratumoral immune cells were examined by flow cytometry.

A. The counts of CD45+ cells per gram of tumor.

B. The percentages of CD45+ cells among forward scatter (FSC) and side scatter (SSC) gated live cell population.

C. The counts of CD3+, CD3-NK1.1+, CD3-CD19+, CD11b+ and CD11c+CD103+ cells per gram of tumor were quantified.

D. The percentages of CD3+, CD3-NK1.1+, CD3-CD19+, CD11b+ and CD11c+CD103+ cells among CD45+ cells were quantified.

E. The counts of CD4+ and CD8+ T cells per gram of tumor.

F. The percentages of CD4+ and CD8+ cells among CD3+ T cells were quantified.

Data information: For all panels, N=5 from a representative experiment. Each dot represents one mouse. Error bars stand for s.d., and center values represent mean. Two-tailed unpaired student's t-test was used. ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001; ∗∗∗∗: p < 0.0001; ns: not significant. Data are representative of two independent experiments.
5-Fluorouracil Efficacy Requires Anti-tumor Immunity Triggered by Cancer-cell-intrinsic STING (2021)
Figure 7

Figure 7. T cells are required for efficient 5-FU-induced tumor inhibition.

A-D. Schematics (A) of the experiment to examine the effect of T cell depletion on 5-FU response, during which anti-CD4/anti-CD8 antibodies (Ab), control IgG Ab, or anti-NK1.1 Ab were administrated on day 5 and day 9. (B) Pictures of tumors and spleens. Image panels were cropped from the same picture. N=10. (C) Tumor volumes were quantified at the indicated days post cancer cell injection. N=10. (D) Tumor and spleen weights at the endpoint for (C), with each dot representing a mouse.

Data information: For all panels, error bars stand for s.d., and center values represent mean. Two-tailed unpaired student's t-test was used. ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001; ∗∗∗∗: p < 0.0001; ns: not significant. Data were pooled from two parallel experiments.
5-Fluorouracil Efficacy Requires Anti-tumor Immunity Triggered by Cancer-cell-intrinsic STING (2021)
Figure 8

Figure 8. STING-KO tumors require higher concentrations of 5-FU for inhibition in vivo.

Mice were injected with control (Ctrl) or STING-KO MC38 cells, and subjected to treatments, with each dose consisting of 25 mg/kg, 50 mg/kg or 75 mg/kg 5-FU, or PBS.

A. Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture.

B. Tumor volumes were quantified at the indicated days post cancer cell injection. N=5.

C. Tumor and spleen weights at the endpoint for (B), with each dot representing a mouse.

Data information: For all panels, error bars stand for s.d., and center values represent mean. Two-tailed unpaired student's t-test was used. ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001; ns: not significant. Data are representative of two independent experiments. Data are representative of two independent experiments.
5-Fluorouracil Efficacy Requires Anti-tumor Immunity Triggered by Cancer-cell-intrinsic STING (2021)
Figure 9

Figure 9. Association between STING expression and clinical responses of human colon cancer patients, and a model of our findings

A. STING protein expression of 58 colon adenocarcinoma specimens within the TCGA COAD collection were obtained from the CPTAC project website. Samples were partitioned into STING-Low (non-detectable STING protein, N=15) and STING-Hi (detectable STING protein, N=43) groups. Kaplan Meier curve is shown with P value indicated.

B. STING RNA expression from the same 58 specimens were partitioned into STING-Low (bottom 15 samples) and STING-Hi (top 43 samples) groups. Kaplan Meier curve is shown with P value indicated.

C. Stage II and Stage III patients from the TCGA COAD dataset were partitioned into STING-Hi (top 1/3 of samples, N=87) and STING-Low (bottom 1/3 of samples, N=87). Kaplan Meier curve is shown with P value indicated.

D. From a published colorectal cancer chemotherapy response study, STING RNA expression in responders and non-responders were compared, with each dot representing a tumor.

E. A model summarizing our findings.

Data information: For panels A-D, error bars represent s.d., and center values represent mean. Log rank test was used to analyze survival data. Unpaired two-tailed student's t-test was used for comparing responders and non-responders. ∗: p < 0.05; ns: not significant.
MiR-342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death (2021)
Figure 1

Figure 1 MiR-342-3p associates with TB susceptibility

(A) Expression changes of pri-, pre- and mature miR-342 transcripts were analyzed by semiquantitative PCR in C3H or B6 BMDMs after Mtb infection. Representative blots from n = 3 biological replicates are shown.

(B, C) The relative expressions of miR-342-3p and miR-342-5p were analyzed by northern blotting (B, representative blots from n = 3 biological replicates are shown) or quantitative real-time PCR (C, data are shown as the mean ± s.e.m. of n = 3 biological replicates) in Mtb-stimulated C3H and B6 BMDMs.

(D-G) Cell death mechanisms of C3H BMDMs transfected with miR-342-3p mimic (D), or B6 BMDMs transfected with miR-342-3p inhibitor (F), followed by Mtb infection for 36 hours. Cell viabilities of C3H BMDMs transfected with miR-342-3p mimic (E), or B6 BMDMs transfected with miR-342-3p inhibitor (G), followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.

(H, I) Mtb growth rates of C3H BMDMs transfected with miR-342-3p mimic (H), or B6 BMDMs transfected with miR-342-3p inhibitor (I) after Mtb infection. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.

(J) Expression levels of miR-342-3p in PBMCs from healthy controls (n = 27) or TB patients (n = 34) were detected by qRT-PCR. Data are shown as the medians ± interquartile ranges.

Data information: ANOVA followed by Bonferroni post hoc test (C-I) and Mann-Whitney U test (J) were used for data analysis. *, p < 0.05, **, p < 0.01. Abbreviation: NC, negative control.