EMBO/soda-nel huggingface dataset:
ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors (2016)
Figure 7

Figure 7. ZEB1 knock-down sensitizes naive melanoma cells to BRAFi and induces cell death in BRAFi-resistant melanoma cells

(A) Western blot analyses of ZEB1 and P-ERK in shRNA-control or shRNA-ZEB1-expressing A375 cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control. (B) Soft agar colony formation assay in A375 cells in the presence or absence of PLX4032 (150 nM). Scale bar = 100 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student’s t-test). (C) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or shRNA-ZEB1-expressing cells (n=3, Student’s t-test). (D) RPMI7951, A375-R and SKMEL5-R were infected with a retrovirus encoding a constitutive shRNA-ZEB1. Short-term cultures of GOKA and ESP cells, derived from vemurafenib-resistant patients, were infected with a lentivirus encoding an IPTG-inducible shRNA-ZEB1. Western blot analyses showing efficient ZEB1 knock-down in the different models, ±IPTG (200µM), ±PLX4032 (3µM) as indicated. Induction of cell death was assessed by PARP cleavage. GAPDH or actin were used as loading control. (E) Clonogenic assays in the presence of 3 µM PLX4032, and with or without IPTG (200 µM) as indicated. The graphs represent the mean number of colonies in 3 independent experiments (Student’s t-test). (F) Western blot analyses of ZEB1, p75 and quantitative PCR analyses of MITF in shRNA-Control or shRNA-ZEB1 ESP vemurafenib-resistant patient-derived short-term culture cells. Actin was used as a loading control. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student’s t-test). (G) 2,5 x 106 shRNA-Control or shRNA-ZEB1 ESP vemurafenib-resistant cells were injected subcutaneously in nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by providing mice with IPTG (10 mM) in their drinking water and orally administering vemurafenib (50 mg/kg) daily for 4 weeks. The mean tumor volume for 5 mice is represented (± SEM). (Student’s t-test). (H) Upper part: Western blot analyses of ZEB1 in shRNA-Control (1 to 4) or shRNA-ZEB1 (5 to 8) ESP xenograft tumors, showing efficient ZEB1 knock-down directly in the tumors, after IPTG ±PLX4032 treatment in vivo. Lower part: Representative pictures of ZEB1 immunostaining in shRNA-Control or shRNA-ZEB1 tumors, after IPTG treatment in vivo. Scale bar = 40µm.
Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma (2022)
Figure 1

Figure 1 - CCN4 is associated with reduced overall survival of patients diagnosed with primary melanoma and a shift in immune contexture.

(A) Kaplan-Meier estimate of overall survival of patients diagnosed with primary melanoma stratified by CCN4 transcript abundance, with patients at risk tabulated below graph, as similarly shown in Deng et al. (2019). Original data obtained from SKCM arm of TCGA and stratified based on CCN4 mRNA expression (CCN4 high/positive > 1 FPKM: blue, CCN4 low/negative < 1 FPKM: red). P-value calculated using the Peto & Peto modified Gehan-Wilcoxon test.

(B) The proportion of CCN4 positive melanoma cells obtained from patients with melanoma obtained prior to treatment (o, n = 15) and from patients that did not respond to immune checkpoint blockade (x, n = 10). Values shown as the proportion of CCN4 positive melanoma cells of the sample ± SE of the sample proportion, given a binomial distribution. A binomial test assessed significance between the proportion of CCN4 positive cells in the sample relative to a null proportion of 1% or less CCN4 positive melanoma cells. Samples with significantly enriched CCN4 positive cells are indicated in red.

(C) Immune contexture in corresponding primary SKCM tissue samples estimated from bulk RNAseq data using CIBERSORTx deconvolution. Columns ordered from low (left) to high (right) CCN4 expression. Rows hierarchically clustered based on a Euclidian distance metric in R (Ward.D). A non-parametric Mann-Whitney U test assessed significance of difference in immune subset signature between CCN4 high/positive and low/negative groups. The log10 p-values were color coded.
Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma (2022)
Figure 2

Figure 2 - CCN4 knockout suppressed melanoma tumor growth in immunocompetent mice.

(A) CCN4 secretion after CRISPR/Cas9 knockout in B16F0 and YUMM1.7 cell lines. Cell culture media conditioned for 48 hours by the indicated cell line were tested by ELISA (n.d. = not detected). Three biological replicates represented as mean ± SEM.

(B-E) B16F0 knockout created with Homology-Directed Repair approach with controls for puromycin selection (B16F0-Ctr) created with pBabe-puro retrovirus. YUMM1.7 knockout created with double nickase approach with KO1 and KO2 indicating two different clones. Wildtype (WT: red) B16F0 (B,C) and YUMM1.7 (D,E) cells and CCN4 knockout variants (KO1: blue, KO2: black) were subcutaneously injected into (B,D) C57BL/6 immunocompetent and (C,E) NSG immunocompromised mice. Tumor volumes measured by caliper as a function of time after tumor challenge, with n expressed as the number of mice with tumors over the number of mice injected. The tumor growth profiles for every mouse in the different cohorts is provided in Dataset EV1.

(F-H) The tumor growth model used to interpret the log-linear tumor growth curves (F). Posterior distributions in the growth rate constant of tumors in NSG mice (G) and in the log-ratio of the net growth rate constants of tumors in NSG relative to C57BL/6 mice (H). Statistical differences in these distributions were assessed using a Chi-squared test summarized in text.

(I) In a time-matched experiment, average tumor weights of both B16F0 and YUMM1.7 were significantly decreased following CCN4 knockout (KO1). Results summarized as mean ± SEM.

(J) Significant increases in the number of CD45+ cells were observed in both B16F0 and YUMM1.7 cell lines following CCN4 knockout (KO1). Results summarized as mean ± SEM.

Data information: In I and J, statistical significance was assessed using a Student's t-test with *: 0.01<p<0.05, **: 0.001<p<0.01, ***: p<0.001 and results representative of at least six biological replicates.
Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma (2022)
Figure 3

Figure 3 - CCN4 knockout increased CTL and NK effector cells and decreased MDSC in the tumor microenvironment.

(A) Representative flow cytometry data of the frequency of tumor-infiltrating CD4+ and CD8+ T cells, NK effector cells, tumor-associated neutrophils (TAN) and MDSC from the live CD45+ compartment in WT and CCN4 knockout (KO1) YUMM1.7 tumors. Gating strategies are illustrated in Appendix Figures S4 and S5. In the right panel, the contour lines enclose 90% of TANs (red line) and MDSC (blue) that were determined based on gating shown in Appendix Figure S5 but here backgated along the GR1/CD11b axes.

(B-I) Graphs summarizing the change in frequency of immune cells in the YUMM1.7 tumor microenvironment following CCN4 knockout (n=6 /group). (B) CD3+, (C) CD4+, (D) CD8+, (E) NK cells, (F) CD11c+ TAMs, (G) TANs, (H) DCs, and (I) MDSC. Statistical significance was assessed using a Student's t-test using six biological replicates with results annotated with *: 0.01<p<0.05, **: 0.001<p<0.01, and ***: p<0.001.. Results summarized as mean ± SEM.

(J) CCN4 secretion from CCN4-inducible cells and associated control cell lines in conditioned media with or without 0.5 μg/ml doxycycline, as measured by ELISA (n.d. = not detected). Results from three biological replicates summarized as mean ± SEM.

(K) Kaplan-Meier summary of the fraction tumor free in a 2x2 factor experimental design (n = 5 / group), where Tet-on vector control (red) versus inducible mCCN4 vector (black) and in the presence (dotted lines) or absence (solid lines) of doxycycline were the two factors.

(L) Summary of a Cox multivariate proportional hazards analysis where x-axis of the dot-and-whisker plot corresponds to the mean hazard ratio and 95% confidence interval for the indicated variable.

(M) Representative flow cytometry data of the frequency of tumor-infiltrating MDSC (GR1+ CD11c−/lo : dotted box) from the live CD45+ CD11b+ compartment isolated from CCN4-induced rescue (ID mCCN4 + Dox) and CCN4 knockout (YM1.7-KO1) YUMM1.7 tumors.

(N) Frequency of MDSC for similarly sized tumors generated from CCN4-induced rescue (gray triangle), CCN4 knockout (blue square), and WT YUMM1.7 cells (red circles). Pairwise differences were assessed using a two-sided homoscedastic (pooled variance) Student's t-test.
Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma (2022)
Figure 4

Figure 4 - CCN4 expanded MDSC in the spleens of tumor bearing mice.

(A-C) Tumor volume of YUMM1.7-WT and CCN4 KO (KO1) compared when size-matched and time-matched on day 28 after tumor challenge. MDSC in the spleens of tumor bearing mice expressed as percentage (B) or weight-basis (C) observed in size-matched versus time-matched experimental designs (n = 5 for size-matched, 4 for time-matched experiments). Statistical significance was assessed using a Student's t-test with results annotated with *: 0.01<p<0.05, **: 0.001<p<0.01, and ***: p<0.001. Results summarized as mean ± SEM.

(D) Representative flow cytometry data of CD11b+/−/GR-1+/− cells found in WT, KO1, and tumor free (TF) groups.

(E-H) Graphs summarizing G-MDSCs (E,G) and M-MDSCs (F,H) in the spleens of time-matched YUMM1.7-WT and CCN4 KO (KO1) tumor bearing mice (n = 4/group) expressed as percentage of CD45+ (E,F) and total number per gram spleen (G,H). Statistical significance was assessed using a Student's t-test with results annotated with *: 0.01<p<0.05, **: 0.001<p<0.01, and ***: p<0.001. Results summarized as mean ± SEM.

(I) Representative flow cytometry data of CD11b+/Ly6Chi/low/Ly6G+/− cells found in WT, KO1, and TF groups.

(J,K) Suppression of naïve CD8+ T cell proliferation by (J) freshly isolated G-MDSCs and (K) total splenocytes from WT, KO1, and TF groups (n = 3 biological replicates/group) on a per cell basis. ANOVA with post-hoc Tukey tests were used to assess statistical significance, where different letter (a, b, or c) denotes statistically different groups.

(L) Cell trace profiles of live CD8+ T cells contained within CD45+ cells extracted from WT (red) and KO1 (blue) tumors in a time-matched experiment (TME) and stimulated for three days in vitro with αCD3/αCD28-loaded beads (representative of n = 3 biological replicates/group), with unstimulated cells as a staining control (gray).

Data information: In panels A-C and E-H, dotted line represents average value found in tumor free mice.
Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma (2022)
Figure 5

Figure 5 - Knockout of CCN4 down-regulated CCL2 and CXCL1 expression and decreased glycolysis and glycolytic capacity in YUMM1.7 melanoma cells.

(A) Cytokine, chemokine, and growth factor expression by YUMM1.7-WT and CCN4-KO cells in vitro assayed using R&D Systems' Mouse XL Cytokine Array. Orange dots represent results for specific cytokine probes while blue dots represent positive and negative controls. Dotted lines enclose a null distribution estimated from positive and negative controls.

(B-G) ELISA results for CCL2 and CXCL1 secretion after 36 h ex vivo culture (n = 3 biological replicates/group). CCL2 assayed in (B) tumor-conditioned medium (TCM), (D) CD45− medium, and (F) serum. CXCL1 assayed in (C) TCM, (E) CD45− medium, and (G) serum.

(H-I) Live-dead staining of CD45+ cells isolated from WT and CCN4-KO tumors. Statistical significance was assessed using a Student's t-test with results annotated with ***: p<0.001. Results of biological replicates summarized as mean ± SEM.

(J-K) Analysis of the extracellular acidification rate (ECAR) associated with (J) glycolysis and (K) glycolytic capacity assayed by Seahorse Analyzer in WT and CCN4-KO tumors (n ≥ 12 biological replicates/group) after 36 hours ex vivo. Statistical significance was assessed using a Student's t-test with results annotated with ***: p<0.001. Results of biological replicates summarized as mean ± SEM.

(L) Lactate production by isolated CD45− cells after 36 h ex vivo culture (n = 4 biological replicates/group). Statistical significance was assessed using a Student's t-test with results annotated with ***: p<0.001. Results of biological replicates summarized as mean ± SEM.
Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma (2022)
Figure 6

Figure 6 - CCN4 directly inhibited CD8+ T cell function.

(A) ELISpot for IFNγ release by CD8+ T cells using parental YUMM1.7 and CCN4-KO YUMM1.7 (KO1) cells as targets and different amount of in vivo activated CD8+ T cells. Statistical significance was assessed using a Student's t-test with results annotated with ***: p<0.001. Results of three biological replicates summarized as mean ± SEM.

(B) ELISpot for IFNγ release by in vivo activated CD8+ T cells with CCN4-inducible cells as targets in the presence or absence of 0.5 mg/ml doxycycline. Statistical significance was assessed using a Student's t-test with results annotated with ***: p<0.001. Results of biological replicates summarized as mean ± SEM.

(C) CD8+ T cells isolated from the spleens of C57BL/6 mice that rejected YUMM1.7 tumors were assayed by in vitro ELISpot using variants of the YUMM1.7 cell line as targets (WT YUMM1.7 (Ym1.7) - yellow, CCN4-KO YUMM1.7 (Ym1.7-KO1)- light green, CCN4-KO YUMM1.7 with a blank inducible expression vector (Ym1.7-KO1-IDvector) - dark green and blue, CCN4-KO YUMM1.7 with a CCN4 inducible expression vector (Ym1.7-KO1-IDmCCN4) - purple and red). Representative images shown under indicated condition with scale bar indicating 1 mm. Variants containing the inducible expression vector were also cultured in the absence (dark green and purple) or presence of 0.5 μg/ml doxycycline (blue and red). CD8+ T cells expressing IFNγ and TNFα were quantified following 24 hour co-culture (bar graph). Results shown as mean ± S.D. for three biological replicates. Statistical significance was assessed using a Student's t-test with results annotated with ***: p<0.001 and n.s.: p>0.05.
Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma (2022)
Figure 7

Figure 7 - CCN4 knockout further promoted the anti-tumor effect of immune checkpoint blockade therapy.

(A) Average tumor volumes of mice bearing YUMM1.7-WT (squares and triangles) or CCN4-KO (KO1, circles and inverted triangles) tumors (n = 4 mice/group). Groups were treated with either αPD1 (triangles and inverted triangles) or isotype control (squares and circles) antibodies when the tumors reached 100 mm3 (dotted line). Statistical significance at each time point was assessed using a Student's t-test with results annotated with *: 0.01<p<0.05, **: 0.001<p<0.01, and ***: p<0.001. Results summarized as mean ± SEM.

(B) Expression of H-2Kb (top panel) and PD-L1 (bottom panel) were assayed by flow cytometry in WT (red curves) and CCN4-KO (KO1 - blue curves) YUMM1.7 cells with (dotted curves) and without (solid curves) preconditioning with IFNγ. Unstained cells were used as a negative control (shaded curve).

(C-F) CD45− fraction isolated from WT and CCN4-KO YUMM1.7 tumors in a time-matched experiment were assayed for H-2Kb and PD-L1 expression by flow cytometry. Contour curves enclose 90% (dotted curve) and 50% (solid curves) of CD45− events obtained from WT (red) and CCN4-KO (blue) YUMM1.7 tumors. CD8+ T cells expressing PD1 within the tumor (D) and spleen (E) were assayed by flow cytometry in mice bearing WT and CCN4-KO YUMM1.7 tumors (F). Results representative of three biological replicates and summarized as mean ± SD. Statistical significance was assessed using a Student's t-test.

Expanded View Figure Legends
LONP1 Targets HMGCS2 to Protect Mitochondrial Function and Attenuate Chronic Kidney Disease (2022)
Figure 1

Figure 1. LONP1 is down-regulated in the kidneys of CKD patients and mice and proximal tubular-specific overexpression of Lonp1 alleviates renal injury and mitochondrial dysfunction in UUO model.

A Immunohistochemical analysis of LONP1 expression in CKD children with mild (n=11), moderate (n=10), or severe fibrosis (n=9). N=4 in Normal group. Scale bar: 50μm.

B Immunohistochemical semi-quantitative IOD analysis of LONP1.

C Pearson correlation analysis of LONP1 and atrophy and fibrosis score (AFS) in renal biopsy specimens.

D Western blot analysis for the expression of LONP1 in UUO models at different time points.

E Densitometric analysis for the expression of LONP1 (n=3, biological replicates).

F Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm.

G Sirius red staining in WT and cKI mice after UUO. Scale bar: 50μm.

H Fibrotic area statistics of Sirius red staining in WT and cKI mice after UUO (n=6 in WT+Sham group, n=8 in cKI+Sham or WT+UUO group, n=7 in cKI+UUO group, biological replicates).

I qRT-PCR analysis of FN1, Collagen I and Collagen III in WT and cKI mice after UUO (n=7 in WT+Sham group, n=8 in the other three groups, biological replicates).

J, K Western blot and densitometric analysis for the expression of FN1, Collagen I, Collagen III and TGF-β1 (monomer) in WT and cKI mice after UUO (n=3 or 4, biological replicates).

L qRT-PCR analysis of mtDNA expression in WT and cKI mice after UUO (n=6 or 7, biological replicates).

M Succinate dehydrogenase (SDH) staining in WT and cKI mice after UUO. Scale bar: 50μm.

N Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKI mice after UUO. Scale bar: 500nm.

Data information: In (B, E), data are presented as mean ± SEM. Student's t-test. In (H-L), data are presented as mean ± SEM. One-way ANOVA.
LONP1 Targets HMGCS2 to Protect Mitochondrial Function and Attenuate Chronic Kidney Disease (2022)
Figure 2

Figure 2. Proximal tubular-specific deletion of Lonp1 aggravates renal injury and mitochondrial dysfunction in UUO model.

A Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm.

B Sirius red staining in WT and cKO mice after UUO. Scale bar: 50μm.

C Fibrotic area statistics of Sirius red staining in WT and cKO mice after UUO. N=5 in Sham groups. N=7 in UUO groups, biological replicates.

D, E Western blot and densitometric analysis for the expression of FN1, Collagen I, Collagen III and α-SMA in WT and cKO mice after UUO (n=3, biological replicates).

F qRT-PCR analysis of FN1, Collagen I, Collagen III and α-SMA in WT and cKO mice after UUO (n=7 or 5 in Sham groups, n=9 in UUO groups, biological replicates).

G qRT-PCR analysis of mtDNA expression in WT and cKO mice after UUO (n=7 or 5 in Sham groups, n=7 in UUO groups, biological replicates).

H Mitochondrial respiratory chain complex I enzymatic activity in WT and cKO mice after UUO (n=4, biological replicates).

I Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKO mice after UUO. Scale bar: 500nm.

Data information: Data are presented as mean ± SEM. One-way ANOVA.
LONP1 Targets HMGCS2 to Protect Mitochondrial Function and Attenuate Chronic Kidney Disease (2022)
Figure 3

Figure 3. Proximal tubular-specific overexpression or deletion of Lonp1 mitigated or aggravated renal injury and mitochondrial dysfunction in 5/6Nx model.

A Analysis of blood urea nitrogen (BUN) after 5/6Nx model in WT and cKI mice (n=6 in cKI+Sham group, n=5 in the other three groups, biological replicates).

B Systolic blood pressure was measured by the computerized tail-cuff system (n=6 in cKI+Sham group, n=5 in the other three groups, biological replicates).

C Masson's trichrome staining of WT and cKI mice after 5/6Nx. Scale bar: 100μm.

D qRT-PCR analysis of FN1, Collagen I and Collagen III in WT and cKI mice after 5/6Nx (n=6 in cKI+Sham group, n=5 in the other three groups, biological replicates)

E Analysis of blood urea nitrogen (BUN) after 5/6Nx model in WT and cKO mice (n=5 in WT+Sham group, n=6 in the other three groups, biological replicates).

F Systolic blood pressure in WT and cKO mice (n=5 in WT+Sham or cKO+5/6Nx group, n=8 in cKO+Sham or WT+5/6Nx group, biological replicates).

G Masson's trichrome staining of WT and cKO mice after 5/6Nx. Scale bar: 100μm.

H qRT-PCR analysis of FN1, Collagen I, Collagen III and α-SMA in WT and cKO mice after 5/6Nx (n=5 in WT+Sham group, n=7 in cKO+Sham group, n=6 in 5/6Nx groups, biological replicates).

I, J Western blot and densitometric analysis for the expression of Collagen I and α-SMA in WT and cKO mice after 5/6Nx (n=4, biological replicates).

K Mitochondrial respiratory chain complex I enzymatic activity in WT and cKO mice after 5/6Nx (n=4-8, biological replicates).

L Transmission electron microscopy images of the mitochondria in tubular cells of WT and cKO mice after 5/6Nx. Scale bar: 500nm.

Data information: Data are presented as mean ± SEM. One-way ANOVA.
LONP1 Targets HMGCS2 to Protect Mitochondrial Function and Attenuate Chronic Kidney Disease (2022)
Figure 4

Figure 4. LONP1 attenuated TGF-β1-induced mitochondrial dysfunction and fibrotic response in HK2 cells.

A Quantification of mitochondrial ROS production (n=4, biological replicates).

B, C Measurement of oxygen consumption rate (OCR) using an XF96 Extracellular Flux Analyzer. OSR, spare respiratory capacity (n=8-10 in each group, biological replicates).

D qRT-PCR analysis of FN1, Collagen I, Collagen III, Collagen IV, α-SMA and Vimentin (n=3, biological replicates).

E, F Western blot and densitometric analysis for the expression of Collagen I, α-SMA, and Vimentin (n=3, two independent experiments were carried out).

G Quantification of mitochondrial ROS production (n=4, biological replicates).

H, I Measurement of OCR and OSR using an XF96 Extracellular Flux Analyzer (n=8 or 10 in each group, biological replicates).

J qRT-PCR analysis of FN1, Collagen I, Collagen III, Collagen IV, α-SMA and Vimentin (n=3, biological replicates).

K, L Western blot and densitometric analysis for the expression of Collagen I, α-SMA, and Vimentin (n=3, two independent experiments were carried out).

Data information: In figure A-F, HK2 cells were transfected with vector or Lonp1 plasmid, and then treated with TGF-β1 (10ng/mL) for 24h. In figure G-L, HK2 cells were transfected with vector or Lonp1 shRNA, and then treated with TGF-β1 (10ng/mL) for 24h. Data are presented as mean ± SEM. One-way ANOVA.
LONP1 Targets HMGCS2 to Protect Mitochondrial Function and Attenuate Chronic Kidney Disease (2022)
Figure 5

Figure 5. HMGCS2 may be the degradation substrate of LONP1.

A The proximal renal tubules of Lonp1 cKO mice were extracted and detected by mass spectrometry (n=3, biological replicates).

B Subcellular localization of up-regulated proteins.

C Western blot validation of the up-regulated proteins in mitochondria (n=2, biological replicates).

D CoIP analysis of interaction between LONP1 and HMGCS2.

E GST pull down analysis of interaction between LONP1 and HMGCS2.

F Enzymatic hydrolysis of LONP1 to HMGCS2.

G Immunofluorescence co-localization analysis of LONP1, HMGCS2 and mitochondria in mPTC (up, scale bar: 20μm) and human kidney tissue (down, scale bar: 200μm).