EMBO/soda-nel huggingface dataset:
KUP9 maintains root meristem activity by regulating K+ and auxin homeostasis in response to low K (2020)
Figure 9

Figure 9. KUP9 regulates intracellular K+ and auxin homoeostasis in the root tip.

(A) K+ levels of various materials using the fluorescent dye Asante Potassium Green-2 (AM). Scale bar, 50 μm.

(B) Quantification of K+ concentration under LK and HK conditions. Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.

(C) to (E) The contents of free IAA (C), IA-Asp (D), and IA-Glu (E) in the root tips of wild type and the kup9-1 mutant. Data are means ± SE (n = 3, biological replicates; each replicate contains 1000-1200 individual plants). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represents control.

(F) Phenotype of the kup9-1/ProKUP9:PIN8 transgenic lines. Seeds were germinated and grown on LK or HK medium for 7 d. Scale bar, 1 cm.

(G) Primary root length of the plants tested in (F). Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance.

(H) QC division rates of the indicated plants. The statistical method is described in Materials and Methods. Data are means ± SE (n = 50-60, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance
The SUMO Protease SENP3 regulates Mitochondrial Autophagy mediated by Fis1 (2021)
Figure 1

Figure 1. Iron chelation induces protein deSUMO-2/3-ylation coinciding with SENP3 stabilisation.

Treatment of HeLa cells with DFP (1mM for 24 h) reduces the conjugation of SUMO-2/3 (n=5, biological replicates; **, p<0.01; paired t-test).

Treatment of HeLa cells with DFP (1mM for 24 h) does not change global SUMO-1-ylation (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).

Treatment of HeLa cells with DFP (1mM for 24 h) leads to increased levels of SENP3 (n=8, biological replicates; **p<0.01; paired t-test).

Treatment of HeLa cells with DFP (1mM for 24 h) does not change levels of SENP5 (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).

Treatment of HeLa cells with DFP (1mM for 24 h) leads to decreased levels of SENP3 mRNA (n=3, biological replicates; *p<0.05; unpaired t-test).

Treatment of DFP (1mM for 24 h)-treated HeLa cells with 50μM MG132 for the final 6 h prevents further increase in SENP3 levels induced by DFP.

DFP treatment increases SENP3 stability. DFP (1mM for 24 h)-treated HeLa cells were treated with cycloheximide (CHX; 50μg/ml) for the indicated durations in a chase experiment. Lysate samples were separated on different lanes in the same gel and immunoblotted as indicated.

Data information: In (A)~(E) values are presented as mean ± SEM and are normalised to the control value.
The SUMO Protease SENP3 regulates Mitochondrial Autophagy mediated by Fis1 (2021)
Figure 2

Figure 2. Iron chelation reduces levels of both CHIP and CHIP mRNA.

Treatment of HeLa cells with DFP (1mM for 24 h) decreases levels of CHIP (n=8, biological replicates; ***p<0.001; paired t-test).

Treatment of HeLa cells with DFP (1mM for 24 h) leads to decreased levels of CHIP mRNA (n=3, biological replicates; *p<0.05; unpaired t-test).

Treatment of DFP (1mM for 24 h)-treated HeLa cells with 50μM MG132 for the final 6 h does not recover reduced levels of CHIP induced by DFP. Histogram shows relative levels of cellular CHIP upon four different conditions (n=3, biological replicates; ****p<0.0001; *** p<0.001; repeated measures analysis of variance followed by Tukey's multiple comparisons test).

CHIP knockdown increases levels of SENP3 but not SENP5 in HeLa cells. Nsi or CHIPi (Nsi, non-specific siRNA; CHIPi, CHIP siRNA; concentration, 20 nM) was transfected into HeLa cells for 48 h. Lysate samples were blotted as indicated.

CHIP knockdown prevents the DFP-induced increase in SENP3 levels. Nsi, SENP3i or CHIPi (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; CHIPi, CHIP siRNA; siRNA concentration, 20nM) was transfected into HeLa cells for 48 h. Two days post-transfection the cells were treated with DFP for a further 24 h.

Data information: In (A, C, D, E) whole cell lysate samples were blotted as indicated. In (A)~(C) values are presented as mean ± SEM and are normalised to the control value.
The SUMO Protease SENP3 regulates Mitochondrial Autophagy mediated by Fis1 (2021)
Figure 3

Figure 3. SENP3 is required for mitophagy induced by DFP.

SENP3 knockdown abolishes the DFP-induced increase in LC3-II in HeLa cells. Nsi or SENP3i (I) was transfected into HeLa cells. Two days post-transfection the cells were treated with DFP for a further 24 h. Whole cell lysate samples were blotted as indicated (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; siRNA concentration, 20nM; DFP, 1mM for 24 h; values normalized to the control value; n=3, biological replicates; ***, p<0.001; N.S., not significant, p>0.05; paired t-test).

Replacement of endogenous SENP3 with GFP-SENP3R WT, but not GFP-SENP3R C532A, restores DFP-induced LC3-II levels. GFP-SENP3R WT or SENP3R C532A was expressed in HeLa cells after knockdown of endogenous SENP3 using SENP3i (II). The cells were treated with DFP (1mM for 24 h) in the presence of CQ (50μM; for final 12 h of DFP treatment). Whole cell lysate samples were blotted as indicated.

Establishment of HeLa cells stably expressing mito-Keima (Live image of cells grown in a T75 flask; Scale bar, 100 μm).

Nsi or SENP3i (II) was transfected into HeLa cells with stable expression of mito-Keima grown on 6-well plates. Two days post-transfection the cells were treated with DFP (1mM; 24 h). Cells transfected with Nsi and treated with CQ (50μM; 24 h) in the absence or presence of DFP (1mM; 24 h) were included as a negative control for mitophagy induction. The images show overlaid fluorescence and transmitted light images of unfixed, live cells obtained using an EVOS-fl inverted LED fluorescence microscope (Red: mitophagic puncta; Scale bar, 100 μm). Histogram shows the percentages of mitophagic cells under 6 different conditions (n=3, biological replicates; ****, p<0.0001; Ordinary one-way ANOVA followed by Tukey's multiple comparisons test).

SENP3 knockdown abolishes DFP-induced mitophagic autolysosomes. Nsi or SENP3i (I) together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), and were analyzed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Magenta: SENP3; Scale bar, 10 μm; n=28 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test).

Data information: In (A), (D) and (E) values are presented as mean ± SEM.
The SUMO Protease SENP3 regulates Mitochondrial Autophagy mediated by Fis1 (2021)
Figure 4

Figure 4. Fis1 is required for DFP-induced mitophagy.

Fis1 knockdown abolishes DFP-induced LC3-II. Fis1 siRNA was introduced into HeLa cells for 48 h, and the cells were treated with DFP for further 24 h (Nsi, non-specific siRNA; Fis1i, Fis1 siRNA; siRNA concentration, 20nM; DFP, 1mM). Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=3, biological replicates; **, p<0.01; paired t-test).

Fis1 knockout abolishes DFP-induced LC3-II. Fis1+/+ or Fis1-/- cells were treated with DFP for 24 h. Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=6, biological replicates; *, p<0.05; paired t-test).

Expressing exogenous Fis1 restores LC3-II induction by DFP in Fis1 KO cells. WT or Fis1 KO HeLa cells transfected with pcDNA3 or Flag-Fis1 for 48 h were treated with DFP for a further 24 h. Whole cell lysate samples were blotted as indicated.

Fis1 knockdown prevents DFP-induced mitophagic autolysosomes. Nsi or Fis1i together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), which were analysed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Blue: Fis1; Scale bar, 10 μm; n=25 cells per condition from three individual experiments; *** p<0.001; unpaired-t test).

Data information: In (A), (B) and (D) values are presented as mean ± SEM.
The SUMO Protease SENP3 regulates Mitochondrial Autophagy mediated by Fis1 (2021)
Figure 5

Figure 5. SENP3 mediates deSUMOlyation of Fis1.

Fis1 is a bona fide target for SUMOylation. Fis1 is a SUMO target in mammalian cells. Flag-Fis1 together with pcDNA3 or His-SUMO-2 were transfected into HEK293 cells expressing Ubc9 for 48 h. His-pulldown (His-PD) and lysate samples were detected by immunoblotting for Flag or β-actin.

Endogenous Fis1 is modified by His-SUMO-2 stably expressed in HEK293 cells. His-pulldown (His-PD) and lysate samples were blotted as indicated.

Flag-Fis1 is deSUMOylated by SENP3. SENP3 knockdown enhances Fis1 SUMOylation. Nsi or SENP3i was transfected into HEK293 cells expressing Flag-Fis1, His-SUMO-2 and Ubc9 for 48 h. His-pulldown and lysate samples were detected by immunoblotting for Flag, SENP3 or β-actin. Values are presented as mean ± SEM (n=3, biological replicates; * p<0.05; paired-t test).

SENP3 knockdown increases endogenous Fis1 SUMO-2/3-ylation in HeLa cells. SUMO-2/3 conjugates were immunoprecipitated (IP) from whole cell lysates prepared from HeLa cells using SUMO-2/3 affinity beads. IP and lysate samples were blotted as indicated.

Amino acids including lysine 149 at the C-terminus of Fis1 are required for Fis1 SUMOylation. Flag-Fis1, Flag-Fis1 K149R mutant or Flag-Fis1 Δ149-152 mutant together with His-SUMO-2 were transfected into HEK293 cells expressing Ubc9 for 48 h. His-pulldown and lysate samples were blotted as indicated.

Alignment (the right panel) shows amino acid sequences of the C-terminal tail of Fis1 from the indicated organisms: human (h), mouse (m), rat (r) and S. cerevisiae (y); red letters mark the lysine residue required for Fis1 SUMOylation. Schematic (the left panel) illustrates SUMO conjugation at the C-terminal tail of Fis1 (N, N-terminus; TM, Transmembrane domain; C, C-terminus).
The SUMO Protease SENP3 regulates Mitochondrial Autophagy mediated by Fis1 (2021)
Figure 6

Figure 6. SUMOylation influences Fis1 subcellular distribution

The SUMOylation status of Fis1 influences its mitochondrial localization. Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG was expressed in HeLa cells for 48 h. Whole cell lysates and cytosolic and mitochondrial fractions were prepared and blotted as indicated (the upper panel). Histograms (the right panel) show normalized levels of Flag-Fis1 WT or Flag-Fis1 K149R mutant associated with the cytosolic or mitochondrial fraction (Value are presented as mean ± SEM and are normalized to the control value; n=7, biological replicates; *, p<0.05; paired t-test).

The SUMOylation status of Fis1 may influence its association with the Endoplasmic Reticulum (ER). Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG along with GFP-Cb5 as an ER marker were expressed in HeLa cells for 48 h. Immunocytochemistry was then performed (Green: ER labelled by GFP-Cb5; Blue: different Flag-Fis1 forms; Scale bar, 10 μm)
The SUMO Protease SENP3 regulates Mitochondrial Autophagy mediated by Fis1 (2021)
Figure 7

Figure 7. SUMOylatable Fis1 is essential for SENP3 regulation of mitophagy.

Expression of Flag-Fis1-SUMO-2ΔGG, but not Flag-Fis1, in HeLa cells prevents DFP-mediated induction of LC3-II. Flag-Fis1 WT or Flag-Fis1-SUMO-2ΔGG mutant was transfected into HeLa cells for 48 h and the cells were treated with DFP for a further 24 h. Lysate samples were blotted as indicated.

Expression of a SUMOylation-deficient Flag-Fis1 K149R mutant in HeLa cells induces LC3-II. Flag-Fis1 WT or Flag-Fis1 K149R mutant was transfected into HeLa cells in which endogenous Fis1 was depleted using siRNA for 48 h. Lysate samples were blotted as indicated.

Expressing a SUMOylation-deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP-induced LC3-II induction. pcDNA3, Flag-Fis1 WT or Flag-Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA (I); siRNA concentration, 20nM), and the cells were treated with DFP for a further 24 h (DFP, 1mM). Lysate samples were blotted as indicated.

SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP-Fis1 K149R in the presence of DFP. CFP-Fis1 WT or CFP-Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h, and the cells were treated with DFP for a further 24 h (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Cyan/blue: CFP-Fis1; Magenta, SENP3; Scale bar, 10 μm); Values are presented as mean ± SEM (n=21 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test). Knockdown of SENP3 was further confirmed by immunoblotting (the lower right panel).
The RNA exosome complex degrades expanded hexanucleotide repeat RNA in C9orf72 FTLD/ALS (2020)
Figure 1

Figure 1: Knockdown of EXOSC10 increases repeat RNA and poly-GA expression

A. Schematic representation of a plasmid expressing (G4C2) 80 repeats together with 5' flanking of the C9orf72 repeat (113 bp) and artificially introduced 3' 3xTAG.

B, C. siRNA-mediated knockdown of EXOSC10 (EXO10), but not of DIS3 and DIS3L, significantly increased generation of poly-GA in HeLa cells expressing (G4C2) 80 repeats. 3 independent experiments. Each experiment performed in duplicates. "ctrl" indicates a control vector which lacks the G4C2 repeat but still contains the 5' flanking region and 3xTAG.

D. Increased G4C2 repeat RNA expression upon EXOSC10 knockdown on RT-qPCR. 4 independent experiments. Each experiment performed in duplicates.

E, F. Knockdown of EXOSC10 increased RNA foci in HeLa cells expressing (G4C2) 80 repeats visualized by in situ hybridization targeting sense RNA foci. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments. Scale bar = 10 μm.

G. Knockdown of EXOSC10 increased RNA foci intensity in HeLa cells expressing (G4C2) 80 repeats. Each dot represents average cellular RNA foci intensity (Arbitrary unit) of RNA foci-positive cells in each randomly taken image. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments.

Data information: All graphs except (G) are shown as mean ± SEM. (G) is shown in box-and-whisker plot (the central band is the median. the ends of the box represent the 1st and 3rd quartile, the whiskers extend from the ends of the box to the outermost data point that falls within "3rd quartile + 1.5 × interquartile range" to "1st quartile - 1.5 × interquartile range"). *p < 0.05, ***p < 0.001; ANOVA with Dunnett's post-test vs Ct (=control) (C) or two-tailed unpaired t-test (D, F, G). A dot in graph represents a data point from single well of cell culture plate (C, D, F). Ct: non-targeting siRNA. EXO10#5: an siRNA targeting EXOSC10.
The RNA exosome complex degrades expanded hexanucleotide repeat RNA in C9orf72 FTLD/ALS (2020)
Figure 2

Figure 2: Suppression of repeat RNA and poly-GA by EXOSC10 expression

A, B. Dose-dependent suppression of poly-GA by exogenous EXOSC10 upon EXOSC10 knockdown. 3 independent experiments. Each experiment performed in duplicates.

C. Suppression of G4C2 repeat RNA by exogenous EXOSC10 on EXOSC10 knockdown. 5 independent experiments. Each experiment performed in duplicates. Concentration of transfected EXOSC10 plasmid was 0.2 ng/μl in 1 ml medium.

D. Overexpression of EXOSC10 decreases G4C2 repeat RNA. 3 independent experiments.

Data information: All graphs are shown as mean ± SEM. **p < 0.01, ***p < 0.001; ANOVA with Dunnett's post-test vs Ct (B) or with Turkey-Kramer's post-test (C), two-tailed unpaired t-test (D). A dot in graph represents a data point from single well of cell culture plate. Ct: non-targeting siRNA. EXO10#5: an siRNA targeting EXOSC10.
The RNA exosome complex degrades expanded hexanucleotide repeat RNA in C9orf72 FTLD/ALS (2020)
Figure 3

Figure 3: EXOSC10 degrades hexanucleotide repeat RNA in vivo and in vitro

A, B. Under transcription inhibition with Actinomycin D, cells with EXOSC10 knockdown (EXO10 KD) showed more stable expression of the repeat RNA compared to cells with control knockdown with non-targeting siRNA (Ct KD). 7 and 6 independent experiments were performed.

C, D. Degradation of FAM-labeled synthetic (G4C2)8 repeat RNA with recombinant human EXOSC10 (hEXO10) (Upper left). 3' blocked FAM-labeled G4C2 repeat RNA resisting 3' exoribonuclease activity is not degraded by recombinant EXOSC10 (Lower left). Lack of recombinant EXOSC10 (Upper right) or Mg2+ ion which is essential co-factor of EXOSC10 (Lower right) abolished the RNA degradation activity. 3 or 4 independent experiments. Quantification of the top bands are shown in (D).

E, F. Degradation of antisense C4G2 repeat RNA substrate with recombinant human EXOSC10 (Left). Without recombinant EXOSC10, no substrate degradation was observed (Right). 5 or 6 independent experiments. Quantification is shown in (F).

Data information: Graph (A, B) are shown as mean ± SEM. Graph (D, F) are shown as mean ± 95%CI. # p=0.1144, *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed paired t-test at each time point (A, B) or ANOVA with Dunnett's post-test vs hEXO10(-) at each time point (D, F). A dot in graph (A, B) represents a data point from single well of cell culture plate. A dot in graph (D, F) represents a data obtained from an aliquot of a reaction mixture sequentially obtained at each time point (0, 2.5, 5, 15, 30 min).
The RNA exosome complex degrades expanded hexanucleotide repeat RNA in C9orf72 FTLD/ALS (2020)
Figure 4

Figure 4: Knockdown of EXOSC10 increases RNA foci and repeat RNA transcripts in cells derived from C9orf72 mutation carriers.

A. In situ hybridization. G4C2 RNA foci were detected in a C9 patient-derived fibroblast but not in a control-derived fibroblast with non-targeting siRNA treatment (left panels, control/si Ct. middle panels, C9/si Ct). Knockdown of EXSOC10 increased RNA foci (right panels, C9/siEXOSC10#5). Nuclei were stained with DAPI (blue). White arrows indicate nuclear RNA foci. The green arrows show cytoplasmic RNA foci. Scale bar = 10 μm.

B. Quantification of %cell with G4C2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci.

C. Quantification of the number of G4C2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci.

D. Quantification of %cell with C4G2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci.

E. Quantification of the number of C4G2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci.

F, G. Increased endogenous repeat RNA expression upon EXOSC10 knockdown in strand specific RT-qPCR. 10 independent knockdown experiments (Case1 N=4, Case2 N=4, Case3 N=2) were performed.

Data information: Graph (B-E) are shown as mean ± 95%CI. Graph (F, G) are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test (B-G). Single dot in graph (B-E) represents an average value from 10 different cells from a coverslip. A dot in graph (F, G) represents a data point from single well of cell culture plate. Ct: non-targeting siRNA. EXO10#5: an siRNA targeting EXOSC10. Additional information is summarized in Appendix Figure S1.
The RNA exosome complex degrades expanded hexanucleotide repeat RNA in C9orf72 FTLD/ALS (2020)
Figure 5

Figure 5: DIS3 compensates repeat RNA degradation in the absence of EXOSC10

A, B, C. Double knockdown of EXOSC10 and DIS3 significantly increased poly-GA and repeat RNA expression compared with single knockdown of EXOSC10 in cells expressing (G4C2) 80 repeats. 3 independent experiments. Each experiment performed in duplicates.

D. Overexpression of DIS3 compensated EXOSC10 knockdown-induced repeat RNA accumulation. 3 independent experiments. Each experiment performed in duplicates.

E, F. Catalytically dead mutant EXOSC10 rescued EXOSC10 knockdown-induced GA increase. 3 independent experiments. Each experiment performed in duplicates.

Data information: All graphs are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA with Turkey-Kramer's post-test (B, C, D, F). A dot in graph represents a data point from single well of cell culture plate. Ct: non-targeting siRNA. EXO10#5: an siRNA targeting EXOSC10. DIS3#11: an siRNA targeting DIS3. "(-)" indicates mock transfection of backbone vector.
The RNA exosome complex degrades expanded hexanucleotide repeat RNA in C9orf72 FTLD/ALS (2020)
Figure 6

Figure 6: Arginine-rich DPR modulates intracellular distribution of endogenous EXOSC10

A, B. HeLa cells were transfected with one of the G4C2 repeat plasmids (A), antisense C4G2 repeat plasmid (B) or control mock plasmid. "mock" indicates a control vector which lacks the repeat but still contains the 5' flanking region and 3xTAG.

C-F. HA-tagged poly-GA, poly-GP, poly-GR or poly-PR (red) and EXOSC10 (green) were immunostained with anti-HA and anti-EXOSC10 antibodies. Nuclei were stained with DAPI (blue). Scale bar = 10 μm.

G. EXOSC10 (green) were immunostained with anti-EXOSC10 antibody in cells transfected with control mock plasmid. Nuclei were stained with DAPI (blue). Scale bar = 10 μm.

H, I. Quantification of the frequency of cells with mislocalized EXOSC10. Only DPR-positive cells were quantified (except mock transfected cells expressing no DPR. In this case, all cells in the picture was quantified). 3 independent experiments. (Total cell counts: (figure H) mock 300 cells, GA 112 cells, GP 69 cells, GR 53 cells, (figure I) mock 445 cells, PR 159 cells).

Data information: All graphs are shown as mean ± SEM. **p < 0.01, ***p < 0.001; ANOVA with Dunnett's post-test vs mock (H) or two-tailed unpaired t-test (I). A dot in graph represents a data from single well of cell culture plate.